Fabrication of glass gas cells for the HALOE and MAPS satellite experiments

NASA Technical Reports Server (NTRS)

Sullivan, E. M.; Walthall, H. G.

1984-01-01

The Halogen Occultation Experiment (HALOE) and the Measurement of Air Pollution from Satellites (MAPS) experiment are satellite-borne experiments which measure trace constituents in the Earth's atmosphere. The instruments which obtain the data for these experiments are based on the gas filter correlation radiometer measurement technique. In this technique, small samples of the gases of interest are encapsulated in glass cylinders, called gas cells, which act as very selective optical filters. This report describes the techniques employed in the fabrication of the gas cells for the HALOE and MAPS instruments. Details of the method used to fuse the sapphire windows (required for IR transmission) to the glass cell bodies are presented along with detailed descriptions of the jigs and fixtures used during the assembly process. The techniques and equipment used for window inspection and for pairing the HALOE windows are discussed. Cell body materials and the steps involved in preparing the cell bodies for the glass-to-sapphire fusion process are given.

Application of Gene Expression Trajectories Initiated from ErbB Receptor Activation Highlights the Dynamics of Divergent Promoter Usage.

PubMed

Carbajo, Daniel; Magi, Shigeyuki; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Arner, Erik; Forrest, Alistair R R; Carninci, Piero; Hayashizaki, Yoshihide; Daub, Carsten O; Okada-Hatakeyama, Mariko; Mar, Jessica C

2015-01-01

Understanding how cells use complex transcriptional programs to alter their fate in response to specific stimuli is an important question in biology. For the MCF-7 human breast cancer cell line, we applied gene expression trajectory models to identify the genes involved in driving cell fate transitions. We modified trajectory models to account for the scenario where cells were exposed to different stimuli, in this case epidermal growth factor and heregulin, to arrive at different cell fates, i.e. proliferation and differentiation respectively. Using genome-wide CAGE time series data collected from the FANTOM5 consortium, we identified the sets of promoters that were involved in the transition of MCF-7 cells to their specific fates versus those with expression changes that were generic to both stimuli. Of the 1,552 promoters identified, 1,091 had stimulus-specific expression while 461 promoters had generic expression profiles over the time course surveyed. Many of these stimulus-specific promoters mapped to key regulators of the ERK (extracellular signal-regulated kinases) signaling pathway such as FHL2 (four and a half LIM domains 2). We observed that in general, generic promoters peaked in their expression early on in the time course, while stimulus-specific promoters tended to show activation of their expression at a later stage. The genes that mapped to stimulus-specific promoters were enriched for pathways that control focal adhesion, p53 signaling and MAPK signaling while generic promoters were enriched for cell death, transcription and the cell cycle. We identified 162 genes that were controlled by an alternative promoter during the time course where a subset of 37 genes had separate promoters that were classified as stimulus-specific and generic. The results of our study highlighted the degree of complexity involved in regulating a cell fate transition where multiple promoters mapping to the same gene can demonstrate quite divergent expression profiles.

Persistent poliovirus infection of human fetal brain cells.

PubMed

Pavio, N; Buc-Caron, M H; Colbère-Garapin, F

1996-09-01

It has been suggested that poliovirus (PV), the causative agent of poliomyelitis, could persist in surviving patients. We have previously shown that PV can persistently infect some human cell lines in vitro, particularly neuroblastoma cell lines. We report here an ex vivo model in which PV can persistently infect primary cultures of human fetal brain cells. Two mutations involving capsid residues 142 of VP2 and 95 of VP1 were repeatedly selected during the persistent infections. These residues are located in capsid regions known to be involved in interactions between PV and its receptor. During the first week after infection, viral antigens were found in cells of both the neuronal and glial lineages. In contrast, 2 weeks after infection, viral antigens were detected almost exclusively in cells of the neuronal lineage. They were detected predominantly in cells expressing a marker of early commitment to the neuronal lineage, MAP-5, particularly in neuroblasts. Viral antigens were also found in immature progenitors expressing a neuroepithelium marker, nestin, and in cells expressing a marker of postmitotic neurons, MAP-2. The presence of viral antigens in postmitotic neurons suggests that PV can persist in neurons of patients who have survived poliomyelitis.

NaviCell Web Service for network-based data visualization.

PubMed

Bonnet, Eric; Viara, Eric; Kuperstein, Inna; Calzone, Laurence; Cohen, David P A; Barillot, Emmanuel; Zinovyev, Andrei

2015-07-01

Data visualization is an essential element of biological research, required for obtaining insights and formulating new hypotheses on mechanisms of health and disease. NaviCell Web Service is a tool for network-based visualization of 'omics' data which implements several data visual representation methods and utilities for combining them together. NaviCell Web Service uses Google Maps and semantic zooming to browse large biological network maps, represented in various formats, together with different types of the molecular data mapped on top of them. For achieving this, the tool provides standard heatmaps, barplots and glyphs as well as the novel map staining technique for grasping large-scale trends in numerical values (such as whole transcriptome) projected onto a pathway map. The web service provides a server mode, which allows automating visualization tasks and retrieving data from maps via RESTful (standard HTTP) calls. Bindings to different programming languages are provided (Python and R). We illustrate the purpose of the tool with several case studies using pathway maps created by different research groups, in which data visualization provides new insights into molecular mechanisms involved in systemic diseases such as cancer and neurodegenerative diseases. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

NaviCell Web Service for network-based data visualization

PubMed Central

Bonnet, Eric; Viara, Eric; Kuperstein, Inna; Calzone, Laurence; Cohen, David P. A.; Barillot, Emmanuel; Zinovyev, Andrei

2015-01-01

Data visualization is an essential element of biological research, required for obtaining insights and formulating new hypotheses on mechanisms of health and disease. NaviCell Web Service is a tool for network-based visualization of ‘omics’ data which implements several data visual representation methods and utilities for combining them together. NaviCell Web Service uses Google Maps and semantic zooming to browse large biological network maps, represented in various formats, together with different types of the molecular data mapped on top of them. For achieving this, the tool provides standard heatmaps, barplots and glyphs as well as the novel map staining technique for grasping large-scale trends in numerical values (such as whole transcriptome) projected onto a pathway map. The web service provides a server mode, which allows automating visualization tasks and retrieving data from maps via RESTful (standard HTTP) calls. Bindings to different programming languages are provided (Python and R). We illustrate the purpose of the tool with several case studies using pathway maps created by different research groups, in which data visualization provides new insights into molecular mechanisms involved in systemic diseases such as cancer and neurodegenerative diseases. PMID:25958393

The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

PubMed

Parker, Andrew; Cross, Sally H; Jackson, Ian J; Hardisty-Hughes, Rachel; Morse, Susan; Nicholson, George; Coghill, Emma; Bowl, Michael R; Brown, Steve D M

2015-12-01

Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss. © 2015. Published by The Company of Biologists Ltd.

Mapping cell surface adhesion by rotation tracking and adhesion footprinting

NASA Astrophysics Data System (ADS)

Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

2017-03-01

Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

ERK1/2 and p38 MAP kinases control prion protein fragment 90-231-induced astrocyte proliferation and microglia activation.

PubMed

Thellung, Stefano; Villa, Valentina; Corsaro, Alessandro; Pellistri, Francesca; Venezia, Valentina; Russo, Claudio; Aceto, Antonio; Robello, Mauro; Florio, Tullio

2007-11-01

Astrogliosis and microglial activation are a common feature during prion diseases, causing the release of chemoattractant and proinflammatory factors as well as reactive free radicals, involved in neuronal degeneration. The recombinant protease-resistant domain of the prion protein (PrP90-231) displays in vitro neurotoxic properties when refolded in a beta-sheet-rich conformer. Here, we report that PrP90-231 induces the secretion of several cytokines, chemokines, and nitric oxide (NO) release, in both type I astrocytes and microglial cells. PrP90-231 elicited in both cell types the activation of ERK1/2 MAP kinase that displays, in astrocytes, a rapid kinetics and a proliferative response. Conversely, in microglia, PrP90-231-dependent MAP kinase activation was delayed and long lasting, inducing functional activation and growth arrest. In microglial cells, NO release, dependent on the expression of the inducible NO synthase (iNOS), and the secretion of the chemokine CCL5 were Ca(2+) dependent and under the control of the MAP kinases ERK1/2 and p38: ERK1/2 inhibition, using PD98059, reduced iNOS expression, while p38 blockade by PD169316 inhibited CCL5 release. In summary, we demonstrate that glial cells are activated by extracellular misfolded PrP90-231 resulting in a proliferative/secretive response of astrocytes and functional activation of microglia, both dependent on MAP kinase activation. In particular, in microglia, PrP90-231 activated a complex signalling cascade involved in the regulation of NO and chemokine release. These data argue in favor of a causal role for misfolded prion protein in sustaining glial activation and, possibly, glia-mediated neuronal death.

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

Reduced transcript stabilization restricts TNF-alpha expression in RAW264.7 macrophages infected with pathogenic mycobacteria: evidence for an involvement of lipomannan.

PubMed

Basler, Tina; Holtmann, Helmut; Abel, Jens; Eckstein, Torsten; Baumer, Wolfgang; Valentin-Weigand, Peter; Goethe, Ralph

2010-01-01

Despite the critical role that TNF-alpha plays in the containment of mycobacterial infection, the mechanisms involved in regulation of its expression by mycobacteria are poorly defined. We addressed this question by studying MAP, which causes a chronic enteritis in ruminants and is linked to human Crohn's disease. We found that in MAP infected macrophages, TNF-alpha gene expression was substantially lower than in macrophages infected with nonpathogenic MS or stimulated with LPS. TNF-alpha transcriptional one could not fully explain the differential TNF-alpha mRNA expression, suggesting that there must be a substantial contribution by post-transcriptional mechanisms.Accordingly, we found reduced TNF-alpha mRNA stability in MAP-infected macrophages. Further comparison of MAP- and MS-infected macrophages revealed that lower TNF-alpha mRNA stability combined with lower mRNA and protein expression in MAP-infected macrophages correlated with lower p38 MAPK phosphorylation. These findings were independent of viability of MAP and MS. We demonstrate that the major mycobacterial cell-wall lipoglycan LM of MAP and MS induced TNF-alpha mRNA transcription,but only the MS-LM induced p38 MAPK-dependent transcript stabilization. Overall, our data suggest that pathogenic mycobacteria cause weak p38 and TNF-alpha mRNA stabilization as a result of their structural cell-wall components such as LM and thereby, restrict TNF-alpha expression in macrophages.

MAP3K8/TPL-2/COT is a potential predictive marker for MEK inhibitor treatment in high-grade serous ovarian carcinomas.

PubMed

Gruosso, Tina; Garnier, Camille; Abelanet, Sophie; Kieffer, Yann; Lemesre, Vincent; Bellanger, Dorine; Bieche, Ivan; Marangoni, Elisabetta; Sastre-Garau, Xavier; Mieulet, Virginie; Mechta-Grigoriou, Fatima

2015-10-12

Ovarian cancer is a silent disease with a poor prognosis that urgently requires new therapeutic strategies. In low-grade ovarian tumours, mutations in the MAP3K BRAF gene constitutively activate the downstream kinase MEK. Here we demonstrate that an additional MAP3K, MAP3K8 (TPL-2/COT), accumulates in high-grade serous ovarian carcinomas (HGSCs) and is a potential prognostic marker for these tumours. By combining analyses on HGSC patient cohorts, ovarian cancer cells and patient-derived xenografts, we demonstrate that MAP3K8 controls cancer cell proliferation and migration by regulating key players in G1/S transition and adhesion dynamics. In addition, we show that the MEK pathway is the main pathway involved in mediating MAP3K8 function, and that MAP3K8 exhibits a reliable predictive value for the effectiveness of MEK inhibitor treatment. Our data highlight key roles for MAP3K8 in HGSC and indicate that MEK inhibitors could be a useful treatment strategy, in combination with conventional chemotherapy, for this disease.

Systemic and mucosal immune reactivity upon Mycobacterium avium ssp. paratuberculosis infection in mice.

PubMed

Koc, Arzu; Bargen, Imke; Suwandi, Abdulhadi; Roderfeld, Martin; Tschuschner, Annette; Rath, Timo; Gerlach, Gerald F; Hornef, Mathias; Goethe, Ralph; Weiss, Siegfried; Roeb, Elke

2014-01-01

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne's disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn's disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2-/- mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4+ and CD8+ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4+ T cells. Whereby inflammation in infected and CD4+CD45RB(hi) T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8+ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4+CD45RB(lo/int) T cells. We propose, the usual non-colitogenic CD4+CD45RB(lo/int) T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne's diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired.

Systemic and Mucosal Immune Reactivity upon Mycobacterium avium ssp. paratuberculosis Infection in Mice

PubMed Central

Suwandi, Abdulhadi; Roderfeld, Martin; Tschuschner, Annette; Rath, Timo; Gerlach, Gerald F.; Hornef, Mathias; Goethe, Ralph; Weiss, Siegfried; Roeb, Elke

2014-01-01

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne's disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn's disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2 −/− mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4+ and CD8+ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4+ T cells. Whereby inflammation in infected and CD4+CD45RBhi T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8+ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4+CD45RBlo/int T cells. We propose, the usual non-colitogenic CD4+CD45RBlo/int T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne's diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired. PMID:24728142

Microtubule-associated protein 1S-related autophagy inhibits apoptosis of intestinal epithelial cells via Wnt/β-catenin signaling in Crohn's disease.

PubMed

Bai, Wenxia; Bai, Jian'an; Li, Yanhai; Tian, Delong; Shi, Ruihua

2017-04-08

Many autophagy-related genes, to our knowledge, have been identified as Crohn's disease (CD) polymorphic sites by genomic wide studies. As a novel member of the microtubule-associated protein 1 (MAP1) family, MAP1S is a microtubule-binding proteins involved in autophagy. However, its expression and potential functions in CD have not been understood. For the first time, we discovered the up-regulated MAP1S and autophagy level (indicated by LC3-Ⅱ/LC3-Ⅰ) in inflamed epithelium among CD patients. Similarly, in TNBS-induced murine colitis model, MAP1S expression was obviously increased. Meanwhile, we found the co-location of MAP1S and active-caspase 3 which acted as "apoptotic executor" which might indicate the basis of their co-efficient. At the cellular level, MAP1S silencing inhibited starvation-induced over-expression of active-caspase 3 partially via Wnt/β-catenin signaling activation in HCT-116 cells. Finally, we demonstrated that IWP-2, an inhibitor of the Wnt/β-catenin signaling, reversed the down-regulation of active-caspase 3 induced by MAP1S siRNA in HCT-116 cells. Taken together, our results suggested that MAP1S were up-regulated among CD patients and MAP1S-related autophagy inhibits apoptosis of intestinal epithelial cells (IECs) through Wnt/β-catenin signaling pathway which might play a vital role in the protection of intestinal mucosal barrier and inhibition the progression of CD. Copyright © 2017 Elsevier Inc. All rights reserved.

In Silico Identification of Epitopes in Mycobacterium avium subsp. paratuberculosis Proteins That Were Upregulated under Stress Conditions

PubMed Central

Gurung, Ratna B.; Purdie, Auriol C.; Begg, Douglas J.

2012-01-01

Johne's disease in ruminants is caused by Mycobacterium avium subsp. paratuberculosis. Diagnosis of M. avium subsp. paratuberculosis infection is difficult, especially in the early stages. To date, ideal antigen candidates are not available for efficient immunization or immunodiagnosis. This study reports the in silico selection and subsequent analysis of epitopes of M. avium subsp. paratuberculosis proteins that were found to be upregulated under stress conditions as a means to identify immunogenic candidate proteins. Previous studies have reported differential regulation of proteins when M. avium subsp. paratuberculosis is exposed to stressors which induce a response similar to dormancy. Dormancy may be involved in evading host defense mechanisms, and the host may also mount an immune response against these proteins. Twenty-five M. avium subsp. paratuberculosis proteins that were previously identified as being upregulated under in vitro stress conditions were analyzed for B and T cell epitopes by use of the prediction tools at the Immune Epitope Database and Analysis Resource. Major histocompatibility complex class I T cell epitopes were predicted using an artificial neural network method, and class II T cell epitopes were predicted using the consensus method. Conformational B cell epitopes were predicted from the relevant three-dimensional structure template for each protein. Based on the greatest number of predicted epitopes, eight proteins (MAP2698c [encoded by desA2], MAP2312c [encoded by fadE19], MAP3651c [encoded by fadE3_2], MAP2872c [encoded by fabG5_2], MAP3523c [encoded by oxcA], MAP0187c [encoded by sodA], and the hypothetical proteins MAP3567 and MAP1168c) were identified as potential candidates for study of antibody- and cell-mediated immune responses within infected hosts. PMID:22496492

MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

PubMed

Webb, R; Troyan, T; Sherman, D; Sherman, L A

1994-08-01

Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes.

Selective stabilization of tau in axons and microtubule-associated protein 2C in cell bodies and dendrites contributes to polarized localization of cytoskeletal proteins in mature neurons.

PubMed

Hirokawa, N; Funakoshi, T; Sato-Harada, R; Kanai, Y

1996-02-01

In mature neurons, tau is abundant in axons, whereas microtubule-associated protein 2 (MAP2) and MAP2C are specifically localized in dendrites. Known mechanisms involved in the compartmentalization of these cytoskeletal proteins include the differential localization of mRNA (MAP2 mRNA in dendrites, MAP2C mRNA in cell body, and Tau mRNA in proximal axon revealed by in situ hybridization) (Garner, C.C., R.P. Tucker, and A. Matus. 1988. Nature (Lond.). 336:674-677; Litman, P., J. Barg, L. Rindzooski, and I. Ginzburg. 1993. Neuron. 10:627-638), suppressed transit of MAP2 into axons (revealed by cDNA transfection into neurons) (Kanai, Y., and N. Hirokawa. 1995. Neuron. 14:421-432), and differential turnover of MAP2 in axons vs dendrites (Okabe, S., and N. Hirokawa. 1989. Proc. Natl. Acad. Sci. USA. 86:4127-4131). To investigate whether differential turnover of MAPs contributes to localization of other major MAPs in general, we microinjected biotinylated tau, MAP2C, or MAP2 into mature spinal cord neurons in culture (approximately 3 wk) and then analyzed their fates by antibiotin immunocytochemistry. Initially, each was detected in axons and dendrites, although tau persisted only in axons, whereas MAP2C and MAP2 were restricted to cell bodies and dendrites. Injected MAP2C and MAP2 bound to dendritic microtubules more firmly than to microtubules in axons, while injected tau bound to axonal microtubules more firmly than to microtubules in dendrites. Thus, beyond contributions from mRNA localization and selective axonal transport, compartmentalization of each of the three major MAPs occurs through local differential turnover.

Construction of a 780-kb PAC, BAC, and cosmid contig encompassing the minimal critical deletion involved in B cell lymphocytic leukemia at 13q14.3

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

Bouyge-Moreau, I.; Rondeau, G.; Andre, M.T.

A putative tumor suppressor gene involved in B cell chronic lymphocytic leukemia (B-CLL) was mapped to human chromosome 13q14.3 close to the genetic markers D13S25 and D13S319. We constructed a 780-kb-long contig composed of cosmids, bacterial artificial chromosomes, and bacteriophage PI-derived artificial chromosomes that provides essential information and tools for the positional cloning of this gene. The contig contains both flanking markers as well as several additional genetic markers, three ESTs, and one potential CpG island. In addition, using one B-CLL patient, we characterized a small internal deleted region of 550 kb. Comparing this deletion with other recently published deletionsmore » narrows the minimally deleted area to less than 100 kb in our physical map. This deletion core region should contain all or part of the disrupted in B cell malignancies tumor suppressor gene. 27 refs., 3 figs.« less

Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons.

PubMed

Lebeau, Geneviève; Miller, Linda C; Tartas, Maylis; McAdam, Robyn; Laplante, Isabel; Badeaux, Frédérique; DesGroseillers, Luc; Sossin, Wayne S; Lacaille, Jean-Claude

2011-01-01

The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis-dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

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

Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie

2011-10-14

Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation inmore » HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.« less

Parallel regulation of feedforward inhibition and excitation during whisker map plasticity

PubMed Central

House, David RC; Elstrott, Justin; Koh, Eileen; Chung, Jason; Feldman, Daniel E.

2011-01-01

Sensory experience drives robust plasticity of sensory maps in cerebral cortex, but the role of inhibitory circuits in this process is not fully understood. We show that classical deprivation-induced whisker map plasticity in layer 2/3 (L2/3) of rat somatosensory (S1) cortex involves robust weakening of L4-L2/3 feedforward inhibition. This weakening was caused by reduced L4 excitation onto L2/3 fast-spiking (FS) interneurons, which mediate sensitive feedforward inhibition, and was partially offset by strengthening of unitary FS to L2/3 pyramidal cell synapses. Weakening of feedforward inhibition paralleled the known weakening of feedforward excitation, so that mean excitatory-inhibitory balance and timing onto L2/3 pyramidal cells were preserved. Thus, reduced feedforward inhibition is a covert compensatory process that can maintain excitatory-inhibitory balance during classical deprivation-induced Hebbian map plasticity. PMID:22153377

Glutamate-dependent transcriptional regulation in bergmann glia cells: involvement of p38 MAP kinase.

PubMed

Zepeda, Rossana C; Barrera, Iliana; Castelán, Francisco; Soto-Cid, Abraham; Hernández-Kelly, Luisa C; López-Bayghen, Esther; Ortega, Arturo

2008-07-01

Glutamate (Glu) is the major excitatory neurotransmitter in the Central Nervous System (CNS). Ionotropic and metabotropic glutamate receptors (GluRs) are present in neurons and glial cells and are involved in gene expression regulation. Mitogen-activated proteins kinases (MAPK) are critical for all the membrane to nuclei signaling pathways described so far. In cerebellar Bergmann glial cells, glutamate-dependent transcriptional regulation is partially dependent on p42/44 MAPK activity. Another member of this kinase family, p38 MAPK is activated by non-mitogenic stimuli through its Thr180/Tyr182 phosphorylation and phosphorylates cytoplasmic and nuclear protein targets involved in translational and transcriptional events. Taking into consideration that the role of p38MAPK in glial cells is not well understood, we demonstrate here that glutamate increases p38 MAPK phosphorylation in a time and dose dependent manner in cultured chick cerebellar Bergmann glial cells (BGC). Moreover, p38 MAPK is involved in the glutamate-induced transcriptional activation in these cells. Ionotropic as well as metabotropic glutamate receptors participate in p38 MAPK activation. The present findings demonstrate the involvement of p38 MAPK in glutamate-dependent gene expression regulation in glial cells.

Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes

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

Kere, J.; Grzeschik, K.H.; Limon, J.

1993-05-01

Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids the authors mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene. 32 refs., 3 figs., 1 tab.

Two-dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus.

PubMed

Vödisch, Martin; Albrecht, Daniela; Lessing, Franziska; Schmidt, André D; Winkler, Robert; Guthke, Reinhard; Brakhage, Axel A; Kniemeyer, Olaf

2009-03-01

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. We established a 2-D reference map for A. fumigatus. Using MALDI-TOF-MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

PubMed

Zhao, Dezheng; Zhan, Yanai; Koon, Hon Wai; Zeng, Huiyan; Keates, Sarah; Moyer, Mary P; Pothoulakis, Charalabos

2004-10-15

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

PubMed Central

Salmond, G P; Lutkenhaus, J F; Donachie, W D

1980-01-01

We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. Images PMID:6998962

Bidirectional modulation of visual plasticity by cholinergic receptor subtypes in the frog optic tectum

PubMed Central

Yu, Chuan-Jiang; Butt, Christopher M.; Debski, Elizabeth A.

2008-01-01

Cholinergic input to the optic tectum is necessary for visual map maintenance. To understand why, we examined the effects of activation of the different cholinergic receptor subtypes in tectal brain slices and determined whether the retinotectal map was affected by manipulations of their activity in vivo. Both α-bungarotoxin sensitive and insensitive nicotinic receptor agonists increased spontaneous postsynaptic currents (sPSCs) in a subpopulation of patch-clamped tectal cells; application of subtype selective receptor antagonists reduced nicotine-induced increases in sPSCs. Activation of α-bungarotoxin insensitive nicotinic receptors also induced substantial inward current in some cells. Muscarinic receptor mediated outward current responses were blocked by the M2-like muscarinic receptor antagonists himbacine or AF-DX 384 and mimicked by application of the M2-like agonist oxotremorine. A less frequently observed muscarinic response involving a change in sPSC frequency appeared to be mediated by M1-like muscarinic receptors. In separate experiments, pharmacological manipulation of cholinergic receptor subtype activation led to changes in the activity-dependent visual map created in the tectum by retinal ganglion cell terminals. Chronic exposure of the tectum to either α-bungarotoxin insensitive, α-bungarotoxin sensitive or M1-like receptor antagonists resulted in map disruption. However, treatment with the M2-like receptor antagonist, AF-DX 384, compressed the map. We conclude that nicotinic or M1-like muscarinic receptors control input to tectal cells while α-bungarotoxin insensitive nicotinic receptors and M2-like muscarinic receptors change tectal cell responses to that input. Blockade of the different cholinergic receptor subtypes can have opposing effects on map topography that are consistent with expected effects on tectal cell activity levels. PMID:12670313

MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

PubMed Central

Webb, R; Troyan, T; Sherman, D; Sherman, L A

1994-01-01

Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes. Images PMID:8051004

Study of melanoma invasion by FTIR spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Y.; Sulé-Suso, J.; Sockalingum, G. D.

2008-02-01

Compared to other forms of skin cancer, a malignant melanoma has a high risk of spreading to other parts of the body. Melanoma invasion is a complex process involving changes in cell-extracellular matrix (ECM) interaction and cell-cell interactions. To fully understand the factors which control the invasion process, a human skin model system was reconstructed. HBL (a commercially available cell line) melanoma cells were seeded on a skin model with and without the presence of keratinocytes and/or fibroblasts. After 14 days culture, the skin specimens were fixed, parafin embedded and cut into 7 µm sections. The de-parafinised sections were investigated by synchrotron Fourier transformed infrared (FTIR) microspectroscopy to study skin cell invasion behaviour. The advantage of using FTIR is its ability to obtain the fingerprint information of the invading cells in terms of protein secondary structure in comparison to non-invading cells and the concentration of the enzyme (matrix-metalloproteinase) which digests protein matrix, near the invading cells. With aid of the spectral mapping images, it is possible to pinpoint the cells in non-invasion and invasion area and analyse the respective spectra. It has been observed that the protein bands in cells and matrix shifted between non-invasive and invasive cells in the reconstructed skin model. We hypothesise that by careful analysis of the FTIR data and validation by other models, FTIR studies can reveal information on which type of cells and proteins are involved in melanoma invasion. Thus, it is possible to trace the cell invasion path by mapping the spectra along the interface of cell layer and matrix body by FTIR spectroscopy.

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: Involvement of the human ERCC2 DNA repair gene

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

Flejter, W.L.; McDaniel, L.D.; Johns, D.

1992-01-01

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. The authors now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2)more » gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.« less

The t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytoid lymphoma involves the PAX-5 gene.

PubMed

Iida, S; Rao, P H; Nallasivam, P; Hibshoosh, H; Butler, M; Louie, D C; Dyomin, V; Ohno, H; Chaganti, R S; Dalla-Favera, R

1996-12-01

The t(9;14)(p13;q32) translocation is associated with approximately 50% of lymphoplasmacytoid lymphoma (LPL), a subtype of B-cell non-Hodgkin's lymphoma (NHL). We cloned the chromosomal breakpoint of der (14) from an LPL case (1052) and showed that it involved a junction between 9p13 and the switch micro region of the Ig heavy chain locus (IgH) on 14q32. Using a YAC contig spanning 1.5 megabase (Mb), we determined that the 9p13 breakpoint in one case (1052) mapped within a 270-kb restriction fragment containing two previously reported 9p breakpoints associated with a alpha-heavy chain disease case (MAL) and KI-1 positive diffuse large cell lymphoma (DLCL) cell line (KIS-1). The same fragment also contained the PAX-5 gene which encodes a B-cell specific transcription factor involved in the control of B-cell proliferation and differentiation. The breakpoints of KIS-1 and 1052 were mapped within the 5' noncoding region of PAX-5, while the 9p13 breakpoint of MAL mapped 230 to 270 kb upstream to PAX-5. In all three cases, the translocation caused the juxtaposition of the PAX-5 gene to the IgH locus in the opposite direction of transcription. When compared with six other DLCL cell lines lacking t(9;14)(p13;q32), the KIS-1 cell line showed an 11-fold overexpression of PAX-5 mRNA and a significantly reduced expression of the p53 gene, which is normally regulated by PAX-5. Moreover, metaphase and interphase fluorescence in situ hybridization (FISH) analysis using a YAC clone spanning 1 Mb including the PAX-5 as a probe identified chromosomal translocations in 5 of 7 cases carrying 9p13 translocations. These findings suggest that the PAX-5 gene is the target of the t(9;14) in LPL whereby its expression may be deregulated by juxtaposition to IgH regulatory elements, thus contributing to lymphomagenesis.

Sentinel lymph node biopsy as a prognostic factor in non-metastatic colon cancer: a prospective study.

PubMed

Estrada, O; Pulido, L; Admella, C; Hidalgo, L-A; Clavé, P; Suñol, X

2017-04-01

Around a third of node-negative patients with colon cancer experience a recurrence after surgery, suggesting poor staging. Sentinel lymph node techniques combined with immunochemistry could improve colon cancer staging. We prospectively assessed the effect of Sentinel node mapping on staging and survival in patients with non-metastatic colon cancer. An observational and prospective study was designed. 105 patients with colon cancer were selected. Patients were classified according to node involvement as: N1, with node invasion detected by the conventional techniques; up-staged, with node invasion detected only by sentinel node mapping; and N0, with negative lymph node involvement by both techniques. Five-year survival and disease-free survival rates were analysed. Multivariate regression analyses were performed to identify prognostic factors for disease-free and overall survival. Sentinel node mapping was successfully applied in 78 patients: 33 % were N1; 24.5 % were up-staged (18 patients with isolated tumour cells and 1 patient with micrometastases); and 42.5 % were N0. N1 patients had the poorest overall 5-year survival (65.4 %) and 5-year disease-free survival (69.2 %) rates compared with the other two groups. No significant 5-year survival differences were observed between N0 patients (87.9 %) and up-staged patients (84.2 %). Patients up-staged after sentinel node mapping do not have a poorer prognosis than patients without node involvement. Detection of isolated cancer cells was not a poor prognosis factor in these patients.

Detection of serum antibodies cross-reacting with Mycobacterium avium subspecies paratuberculosis and beta-cell antigen zinc transporter 8 homologous peptides in patients with high-risk proliferative diabetic retinopathy.

PubMed

Pinna, Antonio; Masala, Speranza; Blasetti, Francesco; Maiore, Irene; Cossu, Davide; Paccagnini, Daniela; Mameli, Giuseppe; Sechi, Leonardo A

2014-01-01

MAP3865c, a Mycobacterium avium subspecies paratuberculosis (MAP) cell membrane protein, has a relevant sequence homology with zinc transporter 8 (ZnT8), a beta-cell membrane protein involved in Zn++ transportation. Recently, antibodies recognizing MAP3865c epitopes have been shown to cross-react with ZnT8 in type 1 diabetes patients. The purpose of this study was to detect antibodies against MAP3865c peptides in patients with high-risk proliferative diabetic retinopathy and speculate on whether they may somehow be involved in the pathogenesis of this severe retinal disorder. Blood samples were obtained from 62 type 1 and 80 type 2 diabetes patients with high-risk proliferative diabetic retinopathy and 81 healthy controls. Antibodies against 6 highly immunogenic MAP3865c peptides were detected by indirect ELISA. Type 1 diabetes patients had significantly higher rates of positive antibodies than controls. Conversely, no statistically significant differences were found between type 2 diabetes patients and controls. After categorization of type 1 diabetes patients into two groups, one with positive, the other with negative antibodies, we found that they had similar mean visual acuity (∼ 0.6) and identical rates of vitreous hemorrhage (28.6%). Conversely, Hashimoto's thyroiditis prevalence was 4/13 (30.7%) in the positive antibody group and 1/49 (2%) in the negative antibody group, a statistically significant difference (P = 0.016). This study confirmed that type 1 diabetes patients have significantly higher rates of positive antibodies against MAP/ZnT8 peptides, but failed to find a correlation between the presence of these antibodies and the severity degree of high-risk proliferative diabetic retinopathy. The significantly higher prevalence of Hashimoto's disease among type 1 diabetes patients with positive antibodies might suggest a possible common environmental trigger for these conditions.

Peroxide accumulation and cell death in filamentous fungi induced by contact with a contestant.

PubMed

Silar, Philippe

2005-02-01

Podospora anserina and Coprinopsis cinerea (syn. Coprinus cinereus) are endowed with a defence system able to differentiate self vs. non-self and involving the generation of peroxide. Indeed, they produce peroxide when confronted with a filamentous fungus, only in non-self confrontations. Both species are not able to recognize yeasts and show a differential response to bacteria. The accumulation of peroxides in the ascomycete Podospora anserina requires an NADPH oxidase and a MAP kinase cascade, previously shown to be involved in fruit body formation, cell differentiation and cell degeneration. Confrontation is accompanied by the death of the contestant hyphae only in specific combinations of species. As in animals and plants, data suggest that peroxide is likely involved in signalling rather than playing a direct toxic role. Fungi display more complex behaviours than generally acknowledged, i.e. they are able to recognize potential contestants and built up defence reactions involving evolutionary conserved enzymes.

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

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

Arai, Roberto J.; Ogata, Fernando T.; Batista, Wagner L.

2008-12-01

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects ofmore » GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.« less

The Importance of Caveolin-1 as Key-Regulator of Three-Dimensional Growth in Thyroid Cancer Cells Cultured under Real and Simulated Microgravity Conditions

PubMed Central

Riwaldt, Stefan; Bauer, Johann; Pietsch, Jessica; Braun, Markus; Segerer, Jürgen; Schwarzwälder, Achim; Corydon, Thomas J.; Infanger, Manfred; Grimm, Daniela

2015-01-01

We recently demonstrated that the CAV1 gene was down-regulated, when poorly differentiated thyroid FTC-133 cancer cells formed spheroids under simulated microgravity conditions. Here, we present evidence that the caveolin-1 protein is involved in the inhibition of spheroid formation, when confluent monolayers are exposed to microgravity. The evidence is based on proteins detected in cells and their supernatants of the recent spaceflight experiment: “NanoRacks-CellBox-Thyroid Cancer”. The culture supernatant had been collected in a special container adjacent to the flight hardware incubation chamber and stored at low temperature until it was analyzed by Multi-Analyte Profiling (MAP) technology, while the cells remaining in the incubation chamber were fixed by RNAlater and examined by mass spectrometry. The soluble proteins identified by MAP were investigated in regard to their mutual interactions and their influence on proteins, which were associated with the cells secreting the soluble proteins and had been identified in a preceding study. A Pathway Studio v.11 analysis of the soluble and cell-associated proteins together with protein kinase C alpha (PRKCA) suggests that caveolin-1 is involved, when plasminogen enriched in the extracellular space is not activated and the vascular cellular adhesion molecule (VCAM-1) mediated cell–cell adhesion is simultaneously strengthened and activated PRKCA is recruited in caveolae, while the thyroid cancer cells do not form spheroids. PMID:26633361

Combined use of X-ray fluorescence microscopy, phase contrast imaging for high resolution quantitative iron mapping in inflamed cells

NASA Astrophysics Data System (ADS)

Gramaccioni, C.; Procopio, A.; Farruggia, G.; Malucelli, E.; Iotti, S.; Notargiacomo, A.; Fratini, M.; Yang, Y.; Pacureanu, A.; Cloetens, P.; Bohic, S.; Massimi, L.; Cutone, A.; Valenti, P.; Rosa, L.; Berlutti, F.; Lagomarsino, S.

2017-06-01

X-ray fluorescence microscopy (XRFM) is a powerful technique to detect and localize elements in cells. To derive information useful for biology and medicine, it is essential not only to localize, but also to map quantitatively the element concentration. Here we applied quantitative XRFM to iron in phagocytic cells. Iron, a primary component of living cells, can become toxic when present in excess. In human fluids, free iron is maintained at 10-18 M concentration thanks to iron binding proteins as lactoferrin (Lf). The iron homeostasis, involving the physiological ratio of iron between tissues/secretions and blood, is strictly regulated by ferroportin, the sole protein able to export iron from cells to blood. Inflammatory processes induced by lipopolysaccharide (LPS) or bacterial pathoge inhibit ferroportin synthesis in epithelial and phagocytic cells thus hindering iron export, increasing intracellular iron and bacterial multiplication. In this respect, Lf is emerging as an important regulator of both iron and inflammatory homeostasis. Here we studied phagocytic cells inflamed by bacterial LPS and untreated or treated with milk derived bovine Lf. Quantitative mapping of iron concentration and mass fraction at high spatial resolution is obtained combining X-ray fluorescence microscopy, atomic force microscopy and synchrotron phase contrast imaging.

International workshop on chromosome 3. Final report, April 15, 1991--April 14, 1992

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

Gemmill, R.M.

1992-07-01

The Second Workshop on Human Chromosome 3 was held on April 4--5, 1991 at Denver, Colorado. There were 43 participants representing 8 nations. The workshop participants reviewed the current state of the chromosome 3 map, both physical and genetic, and prepared lists of markers and cell lines to be made commonly available. These markers and cell lines should be incorporated into the mapping efforts of diverse groups to permit the integration of data and development of consensus maps at future workshops. Region specific efforts were described for sections of the chromosome harboring genes thought to be involved in certain diseasesmore » including Von Hippel-Lindau disease, 3p-syndrome, lung cancer and renal cancer. Selected papers have been processed separately for inclusion in the Energy Science and Technology Database.« less

Insights into bird wing evolution and digit specification from polarizing region fate maps.

PubMed

Towers, Matthew; Signolet, Jason; Sherman, Adrian; Sang, Helen; Tickle, Cheryll

2011-08-09

The proposal that birds descended from theropod dinosaurs with digits 2, 3 and 4 was recently given support by short-term fate maps, suggesting that the chick wing polarizing region-a group that Sonic hedgehog-expressing cells-gives rise to digit 4. Here we show using long-term fate maps that Green fluorescent protein-expressing chick wing polarizing region grafts contribute only to soft tissues along the posterior margin of digit 4, supporting fossil data that birds descended from theropods that had digits 1, 2 and 3. In contrast, digit IV of the chick leg with four digits (I-IV) arises from the polarizing region. To determine how digit identity is specified over time, we inhibited Sonic hedgehog signalling. Fate maps show that polarizing region and adjacent cells are specified in parallel through a series of anterior to posterior digit fates-a process of digit specification that we suggest is involved in patterning all vertebrate limbs with more than three digits.

Genetic control of an epigenetic cell degeneration syndrome in Podospora anserina.

PubMed

Haedens, Vicki; Malagnac, Fabienne; Silar, Philippe

2005-06-01

Filamentous fungi frequently present degenerative processes, whose molecular basis is very often unknown. Here, we present three mutant screens that result in the identification of 29 genes that directly or indirectly control Crippled Growth (CG), an epigenetic cell degeneration of the filamentous ascomycete Podospora anserina. Two of these genes were previously shown to encode a MAP kinase kinase kinase and an NADPH oxidase involved in a signal transduction cascade that participates in stationary phase differentiations, fruiting body development and defence against fungal competitors. The numerous genes identified can be incorporated in a model in which CG results from the sustained activation of the MAP kinase cascade. Our data also emphasize the complex regulatory network underlying three interconnected processes in P. anserina: sexual reproduction, defence against competitors, and cell degeneration.

Analysis of Nuclear Lamina Proteins in Myoblast Differentiation by Functional Complementation.

PubMed

Tapia, Olga; Gerace, Larry

2016-01-01

We describe straightforward methodology for structure-function mapping of nuclear lamina proteins in myoblast differentiation, using populations of C2C12 myoblasts in which the endogenous lamina components are replaced with ectopically expressed mutant versions of the proteins. The procedure involves bulk isolation of C2C12 cell populations expressing the ectopic proteins by lentiviral transduction, followed by depletion of the endogenous proteins using siRNA, and incubation of cells under myoblast differentiation conditions. Similar methodology may be applied to mouse embryo fibroblasts or to other cell types as well, for the identification and characterization of sequences of lamina proteins involved in functions that can be measured biochemically or cytologically.

Overexpression of peptide deformylase in breast, colon, and lung cancers.

PubMed

Randhawa, Harsharan; Chikara, Shireen; Gehring, Drew; Yildirim, Tuba; Menon, Jyotsana; Reindl, Katie M

2013-07-01

Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation.

Overexpression of peptide deformylase in breast, colon, and lung cancers

PubMed Central

2013-01-01

Background Human mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression. Methods The mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay. Results PDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines. Conclusions This is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation. PMID:23815882

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

Brechenmacher, Laurent; Nguyen, Tran H.; Hixson, Kim K.

Root hairs are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean root hair cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean root hairs using an accurate mass and time tag approach, establishing the most comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome database using different proteases.more » Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to root hair formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and RAB proteins). Interestingly, some of these proteins appear to be specifically expressed in root hairs and constitute very good candidates for further studies to elucidate unique features of this single cell model.« less

Mapping and proteomic analysis of albumin and globulin proteins in hexaploid wheat kernels (Triticum aestivum L.).

PubMed

Merlino, Marielle; Leroy, Philippe; Chambon, Christophe; Branlard, Gérard

2009-05-01

Albumins and globulins of wheat endosperm represent 20% of total kernel protein. They are soluble proteins, mainly enzymes and proteins involved in cell functions. Two-dimensional gel immobiline electrophoresis (2DE) (pH 4-7) x SDS-Page revealed around 2,250 spots. Ninety percent of the spots were common between the very distantly related cultivars 'Opata 85' and 'Synthetic W7984', the two parents of the International Triticeae Mapping Initiative (ITMI) progeny. 'Opata' had 130 specific spots while 'Synthetic' had 96. 2DE and image analysis of the soluble proteins present in 112 recombinant inbred lines of the F9-mapped ITMI progeny enabled 120 unbiased segregating spots to be mapped on 21 wheat (Triticum aestivum L. em. Thell) chromosomes. After trypsic digestion, mapped spots were subjected to MALDI-Tof or tandem mass spectrometry for protein identification by database mining. Among the 'Opata' and 'Synthetic' spots identified, many enzymes have already been mapped in the barley and rice genomes. Multigene families of Heat Shock Proteins, beta-amylases, UDP-glucose pyrophosphorylases, peroxydases and thioredoxins were successfully identified. Although other proteins remain to be identified, some differences were found in the number of segregating proteins involved in response to stress: 11 proteins found in the modern selected cultivar 'Opata 85' as compared to 4 in the new hexaploid ;Synthetic W7984'. In addition, 'Opata' and 'Synthetic' differed in the number of proteins involved in protein folding (2 and 10, respectively). The usefulness of the mapped enzymes for future research on seed composition and characteristics is discussed.

A multiplexed system for quantitative comparisons of chromatin landscapes

PubMed Central

van Galen, Peter; Viny, Aaron D.; Ram, Oren; Ryan, Russell J.H.; Cotton, Matthew J.; Donohue, Laura; Sievers, Cem; Drier, Yotam; Liau, Brian B.; Gillespie, Shawn M.; Carroll, Kaitlin M.; Cross, Michael B.; Levine, Ross L.; Bernstein, Bradley E.

2015-01-01

Genome-wide profiling of histone modifications can provide systematic insight into the regulatory elements and programs engaged in a given cell type. However, conventional chromatin immunoprecipitation and sequencing (ChIP-seq) does not capture quantitative information on histone modification levels, requires large amounts of starting material, and involves tedious processing of each individual sample. Here we address these limitations with a technology that leverages DNA barcoding to profile chromatin quantitatively and in multiplexed format. We concurrently map relative levels of multiple histone modifications across multiple samples, each comprising as few as a thousand cells. We demonstrate the technology by monitoring dynamic changes following inhibition of P300, EZH2 or KDM5, by linking altered epigenetic landscapes to chromatin regulator mutations, and by mapping active and repressive marks in purified human hematopoietic stem cells. Hence, this technology enables quantitative studies of chromatin state dynamics across rare cell types, genotypes, environmental conditions and drug treatments. PMID:26687680

Valproate inhibits MAP kinase signalling and cell cycle progression in S. cerevisiae.

PubMed

Desfossés-Baron, Kristelle; Hammond-Martel, Ian; Simoneau, Antoine; Sellam, Adnane; Roberts, Stephen; Wurtele, Hugo

2016-10-26

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

HapMap scanning of novel human minor histocompatibility antigens.

PubMed

Kamei, Michi; Nannya, Yasuhito; Torikai, Hiroki; Kawase, Takakazu; Taura, Kenjiro; Inamoto, Yoshihiro; Takahashi, Taro; Yazaki, Makoto; Morishima, Satoko; Tsujimura, Kunio; Miyamura, Koichi; Ito, Tetsuya; Togari, Hajime; Riddell, Stanley R; Kodera, Yoshihisa; Morishima, Yasuo; Takahashi, Toshitada; Kuzushima, Kiyotaka; Ogawa, Seishi; Akatsuka, Yoshiki

2009-05-21

Minor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

An atlas of DNA methylation in diverse bovine tissues

USDA-ARS?s Scientific Manuscript database

We launched an effort to produce a reference cattle DNA methylation resource to improve animal production. We will employ experimental pipelines built around next generation sequencing technologies to map DNA methylation in cultured cells and primary tissues systems frequently involved in animal pro...

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models.

PubMed

Colavecchia, Silvia B; Fernández, Bárbara; Jolly, Ana; Minatel, Leonardo; Hajos, Silvia E; Paolicchi, Fernando A; Mundo, Silvia L

2016-08-01

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved. Copyright © 2016 Elsevier B.V. All rights reserved.

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

PubMed

Nitti, Mariapaola; Furfaro, Anna Lisa; Cevasco, Claudia; Traverso, Nicola; Marinari, Umberto Maria; Pronzato, Maria Adelaide; Domenicotti, Cinzia

2010-05-01

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation. 2010 Elsevier Inc. All rights reserved.

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

PubMed Central

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, David G.; Smith, Richard D.; Glass, N. Louise

2014-01-01

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication. PMID:25412208

Single nucleotide-level mapping of DNA double-strand breaks in human HEK293T cells.

PubMed

Pope, Bernard J; Mahmood, Khalid; Jung, Chol-Hee; Georgeson, Peter; Park, Daniel J

2017-03-01

Constitutional biological processes involve the generation of DNA double-strand breaks (DSBs). The production of such breaks and their subsequent resolution are also highly relevant to neurodegenerative diseases and cancer, in which extensive DNA fragmentation has been described Stephens et al. (2011), Blondet et al. (2001). Tchurikov et al. Tchurikov et al. (2011, 2013) have reported previously that frequent sites of DSBs occur in chromosomal domains involved in the co-ordinated expression of genes. This group report that hot spots of DSBs in human HEK293T cells often coincide with H3K4me3 marks, associated with active transcription Kravatsky et al. (2015) and that frequent sites of DNA double-strand breakage are likely to be relevant to cancer genomics Tchurikov et al. (2013, 2016) . Recently, they applied a RAFT (rapid amplification of forum termini) protocol that selects for blunt-ended DSB sites and mapped these to the human genome within defined co-ordinate 'windows'. In this paper, we re-analyse public RAFT data to derive sites of DSBs at the single-nucleotide level across the built genome for human HEK293T cells (https://figshare.com/s/35220b2b79eaaaf64ed8). This refined mapping, combined with accessory ENCODE data tracks and ribosomal DNA-related sequence annotations, will likely be of value for the design of clinically relevant targeted assays such as those for cancer susceptibility, diagnosis, treatment-matching and prognostication.

Enhanced IL-1{beta}-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-{kappa}B signaling

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

Muselet-Charlier, Celine; Universite Pierre et Marie Curie-Paris 6, Paris, UMR-S719, F-75012; Roque, Telma

2007-06-01

Transcription nuclear factor-{kappa}B (NF-{kappa}B) is hyperactivated in cystic fibrosis (CF) lung epithelial cells, and participates in exaggerated IL-8 production in the CF lung. We recently found that rapid activation of NF-{kappa}B occurred in a CF lung epithelial IB3-1 cell line (CF cells) upon IL-1{beta} stimulation, which was not observed in its CFTR-corrected lung epithelial S9 cell line (corrected cells). To test whether other signaling pathways such as that of mitogen-activated protein kinases (MAPKs) could be involved in IL-1{beta}-induced IL-8 production of CF cells, we investigated ERK1/2, JNK, and p38MAP signaling compared to NF-{kappa}B. Within 30 min, exposure to IL-1{beta} causedmore » high activation of NF-{kappa}B, ERK1/2, p38MAP but not JNK in CF cells compared to corrected cells. Treatment of IL-1{beta}-stimulated CF cells with a series of chemical inhibitors of NF-{kappa}B, ERK1/2, and p38MAP, when used separately, reduced slightly IL-8 production. However, when used together, these inhibitors caused a blockade in IL-1{beta}-induced IL-8 production in CF cells. Understanding of the cross-talk between NF-{kappa}B and MAPKs signaling in CF lung epithelial cells may help in developing new therapeutics to reduce lung inflammation in patients with CF.« less

Genetic identification of brain cell types underlying schizophrenia.

PubMed

Skene, Nathan G; Bryois, Julien; Bakken, Trygve E; Breen, Gerome; Crowley, James J; Gaspar, Héléna A; Giusti-Rodriguez, Paola; Hodge, Rebecca D; Miller, Jeremy A; Muñoz-Manchado, Ana B; O'Donovan, Michael C; Owen, Michael J; Pardiñas, Antonio F; Ryge, Jesper; Walters, James T R; Linnarsson, Sten; Lein, Ed S; Sullivan, Patrick F; Hjerling-Leffler, Jens

2018-06-01

With few exceptions, the marked advances in knowledge about the genetic basis of schizophrenia have not converged on findings that can be confidently used for precise experimental modeling. By applying knowledge of the cellular taxonomy of the brain from single-cell RNA sequencing, we evaluated whether the genomic loci implicated in schizophrenia map onto specific brain cell types. We found that the common-variant genomic results consistently mapped to pyramidal cells, medium spiny neurons (MSNs) and certain interneurons, but far less consistently to embryonic, progenitor or glial cells. These enrichments were due to sets of genes that were specifically expressed in each of these cell types. We also found that many of the diverse gene sets previously associated with schizophrenia (genes involved in synaptic function, those encoding mRNAs that interact with FMRP, antipsychotic targets, etc.) generally implicated the same brain cell types. Our results suggest a parsimonious explanation: the common-variant genetic results for schizophrenia point at a limited set of neurons, and the gene sets point to the same cells. The genetic risk associated with MSNs did not overlap with that of glutamatergic pyramidal cells and interneurons, suggesting that different cell types have biologically distinct roles in schizophrenia.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

PubMed Central

Perrin-Terrin, Anne-Sophie; Jeton, Florine; Pichon, Aurelien; Frugière, Alain; Richalet, Jean-Paul; Bodineau, Laurence; Voituron, Nicolas

2016-01-01

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive. PMID:27167092

Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

PubMed

Lee, Jin-Sun; Wang, Tsu-Shing; Lin, Ming Cheng; Lin, Wei-Wen; Yang, Jaw-Ji

2017-10-31

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

PubMed

Yang, H; Raizada, M K

1998-09-01

Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons that is independent of the MAP kinase pathway.

APOPTOSIS OF CEREBELLAR GRANULE CELLS INDUCED BY ORGANOTIN COMPOUNDS FOUND IN DRINKING WATER: INVOLVEMENT OF MAP KINASES.

EPA Science Inventory

Mono- and dialkyl organotins have been found in drinking water in homes and businesses served by PVC pipes. Because the structurally related trialkyl organotins (eg. trimethyltin, tributyltin) are well known neurotoxicants, there is concern over the potential for the mono- and di...

Constructing Proteome Reference Map of the Porcine Jejunal Cell Line (IPEC-J2) by Label-Free Mass Spectrometry.

PubMed

Kim, Sang Hoon; Pajarillo, Edward Alain B; Balolong, Marilen P; Lee, Ji Yoon; Kang, Dae-Kyung

2016-06-28

In this study, the global proteome of the IPEC-J2 cell line was evaluated using ultra-high performance liquid chromatography coupled to a quadrupole Q Exactive™ Orbitrap mass spectrometer. Proteins were isolated from highly confluent IPEC-J2 cells in biological replicates and analyzed by label-free mass spectrometry prior to matching against a porcine genomic dataset. The results identified 1,517 proteins, accounting for 7.35% of all genes in the porcine genome. The highly abundant proteins detected, such as actin, annexin A2, and AHNAK nucleoprotein, are involved in structural integrity, signaling mechanisms, and cellular homeostasis. The high abundance of heat shock proteins indicated their significance in cellular defenses, barrier function, and gut homeostasis. Pathway analysis and annotation using the Kyoto Encyclopedia of Genes and Genomes database resulted in a putative protein network map of the regulation of immunological responses and structural integrity in the cell line. The comprehensive proteome analysis of IPEC-J2 cells provides fundamental insights into overall protein expression and pathway dynamics that might be useful in cell adhesion studies and immunological applications.

Averaged ratio between complementary profiles for evaluating shape distortions of map projections and spherical hierarchical tessellations

NASA Astrophysics Data System (ADS)

Yan, Jin; Song, Xiao; Gong, Guanghong

2016-02-01

We describe a metric named averaged ratio between complementary profiles to represent the distortion of map projections, and the shape regularity of spherical cells derived from map projections or non-map-projection methods. The properties and statistical characteristics of our metric are investigated. Our metric (1) is a variable of numerical equivalence to both scale component and angular deformation component of Tissot indicatrix, and avoids the invalidation when using Tissot indicatrix and derived differential calculus for evaluating non-map-projection based tessellations where mathematical formulae do not exist (e.g., direct spherical subdivisions), (2) exhibits simplicity (neither differential nor integral calculus) and uniformity in the form of calculations, (3) requires low computational cost, while maintaining high correlation with the results of differential calculus, (4) is a quasi-invariant under rotations, and (5) reflects the distortions of map projections, distortion of spherical cells, and the associated distortions of texels. As an indicator of quantitative evaluation, we investigated typical spherical tessellation methods, some variants of tessellation methods, and map projections. The tessellation methods we evaluated are based on map projections or direct spherical subdivisions. The evaluation involves commonly used Platonic polyhedrons, Catalan polyhedrons, etc. Quantitative analyses based on our metric of shape regularity and an essential metric of area uniformity implied that (1) Uniform Spherical Grids and its variant show good qualities in both area uniformity and shape regularity, and (2) Crusta, Unicube map, and a variant of Unicube map exhibit fairly acceptable degrees of area uniformity and shape regularity.

Mapping of the gene for the Mel{sub 1a}-melatonin receptor to human chromosome 4 (MTNR1A) and mouse chromosome 8 (Mtnr1a)

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

Slaugenhaupt, S.A.; Liebert, C.B.; Altherr, M.R.

The pineal hormone melatonin elicits potent circadian and reproductive effects in mammals. The authors report the chromosomal location of the gene for the Mel{sub 1a}-melatonin receptor that likely mediates these circadian and reproductive actions. PCR analysis of human-rodent somatic cell hybrids showed that the receptor gene (MTNR1A) maps to human chromosome 4q35.1. An interspecific backcross analysis revealed that the mouse gene (Mtnr1a) maps to the proximal portion of chromosome 8. These loci may be involved in genetically based circadian and neuroendocrine disorders. 14 refs., 1 fig.

Sharp wave/ripple network oscillations and learning-associated hippocampal maps.

PubMed

Csicsvari, Jozsef; Dupret, David

2014-02-05

Sharp wave/ripple (SWR, 150-250 Hz) hippocampal events have long been postulated to be involved in memory consolidation. However, more recent work has investigated SWRs that occur during active waking behaviour: findings that suggest that SWRs may also play a role in cell assembly strengthening or spatial working memory. Do such theories of SWR function apply to animal learning? This review discusses how general theories linking SWRs to memory-related function may explain circuit mechanisms related to rodent spatial learning and to the associated stabilization of new cognitive maps.

A Global Map of Lipid-Binding Proteins and Their Ligandability in Cells.

PubMed

Niphakis, Micah J; Lum, Kenneth M; Cognetta, Armand B; Correia, Bruno E; Ichu, Taka-Aki; Olucha, Jose; Brown, Steven J; Kundu, Soumajit; Piscitelli, Fabiana; Rosen, Hugh; Cravatt, Benjamin F

2015-06-18

Lipids play central roles in physiology and disease, where their structural, metabolic, and signaling functions often arise from interactions with proteins. Here, we describe a set of lipid-based chemical proteomic probes and their global interaction map in mammalian cells. These interactions involve hundreds of proteins from diverse functional classes and frequently occur at sites of drug action. We determine the target profiles for several drugs across the lipid-interaction proteome, revealing that its ligandable content extends far beyond traditionally defined categories of druggable proteins. In further support of this finding, we describe a selective ligand for the lipid-binding protein nucleobindin-1 (NUCB1) and show that this compound perturbs the hydrolytic and oxidative metabolism of endocannabinoids in cells. The described chemical proteomic platform thus provides an integrated path to both discover and pharmacologically characterize a wide range of proteins that participate in lipid pathways in cells. Copyright © 2015 Elsevier Inc. All rights reserved.

The TAM-family receptor Mer mediates production of HGF through the RhoA-dependent pathway in response to apoptotic cells.

PubMed

Park, Hyun-Jung; Baen, Ji-Yeon; Lee, Ye-Ji; Choi, Youn-Hee; Kang, Jihee Lee

2012-08-01

The TAM receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. We investigated the roles of the TAM receptors in mediating the induction of hepatocyte growth factor (HGF) during the interaction of macrophages with apoptotic cells. Mer-specific neutralizing antibody, small interfering RNA (siRNA), and a recombinant Mer protein (Mer/Fc) inhibited HGF mRNA and protein expression, as well as activation of RhoA, Akt, and specific mitogen-activated protein (MAP) kinases in response to apoptotic cells. Inhibition of Axl or Tyro3 with specific antibodies, siRNA, or Fc-fusion proteins did not prevent apoptotic cell-induced HGF mRNA and protein expression and did not inhibit activation of the postreceptor signaling molecules RhoA and certain MAP kinases, including extracellular signal-regulated protein kinase and c-Jun NH(2)-terminal kinase. However, Axl- and Tyro3-specific blockers did inhibit the activation of Akt and p38 MAP kinase in response to apoptotic cells. In addition, none of the TAM receptors mediated the effects of apoptotic cells on transforming growth factor-β or epidermal growth factor mRNA expression. However, they were involved in the induction of vascular endothelial growth factor mRNA expression. Our data provide evidence that when macrophages interact with apoptotic cells, only Mer of the TAM-family receptors is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.

A highly efficient approach to protein interactome mapping based on collaborative filtering framework.

PubMed

Luo, Xin; You, Zhuhong; Zhou, Mengchu; Li, Shuai; Leung, Hareton; Xia, Yunni; Zhu, Qingsheng

2015-01-09

The comprehensive mapping of protein-protein interactions (PPIs) is highly desired for one to gain deep insights into both fundamental cell biology processes and the pathology of diseases. Finely-set small-scale experiments are not only very expensive but also inefficient to identify numerous interactomes despite their high accuracy. High-throughput screening techniques enable efficient identification of PPIs; yet the desire to further extract useful knowledge from these data leads to the problem of binary interactome mapping. Network topology-based approaches prove to be highly efficient in addressing this problem; however, their performance deteriorates significantly on sparse putative PPI networks. Motivated by the success of collaborative filtering (CF)-based approaches to the problem of personalized-recommendation on large, sparse rating matrices, this work aims at implementing a highly efficient CF-based approach to binary interactome mapping. To achieve this, we first propose a CF framework for it. Under this framework, we model the given data into an interactome weight matrix, where the feature-vectors of involved proteins are extracted. With them, we design the rescaled cosine coefficient to model the inter-neighborhood similarity among involved proteins, for taking the mapping process. Experimental results on three large, sparse datasets demonstrate that the proposed approach outperforms several sophisticated topology-based approaches significantly.

A Highly Efficient Approach to Protein Interactome Mapping Based on Collaborative Filtering Framework

PubMed Central

Luo, Xin; You, Zhuhong; Zhou, Mengchu; Li, Shuai; Leung, Hareton; Xia, Yunni; Zhu, Qingsheng

2015-01-01

The comprehensive mapping of protein-protein interactions (PPIs) is highly desired for one to gain deep insights into both fundamental cell biology processes and the pathology of diseases. Finely-set small-scale experiments are not only very expensive but also inefficient to identify numerous interactomes despite their high accuracy. High-throughput screening techniques enable efficient identification of PPIs; yet the desire to further extract useful knowledge from these data leads to the problem of binary interactome mapping. Network topology-based approaches prove to be highly efficient in addressing this problem; however, their performance deteriorates significantly on sparse putative PPI networks. Motivated by the success of collaborative filtering (CF)-based approaches to the problem of personalized-recommendation on large, sparse rating matrices, this work aims at implementing a highly efficient CF-based approach to binary interactome mapping. To achieve this, we first propose a CF framework for it. Under this framework, we model the given data into an interactome weight matrix, where the feature-vectors of involved proteins are extracted. With them, we design the rescaled cosine coefficient to model the inter-neighborhood similarity among involved proteins, for taking the mapping process. Experimental results on three large, sparse datasets demonstrate that the proposed approach outperforms several sophisticated topology-based approaches significantly. PMID:25572661

A Highly Efficient Approach to Protein Interactome Mapping Based on Collaborative Filtering Framework

NASA Astrophysics Data System (ADS)

Luo, Xin; You, Zhuhong; Zhou, Mengchu; Li, Shuai; Leung, Hareton; Xia, Yunni; Zhu, Qingsheng

2015-01-01

The comprehensive mapping of protein-protein interactions (PPIs) is highly desired for one to gain deep insights into both fundamental cell biology processes and the pathology of diseases. Finely-set small-scale experiments are not only very expensive but also inefficient to identify numerous interactomes despite their high accuracy. High-throughput screening techniques enable efficient identification of PPIs; yet the desire to further extract useful knowledge from these data leads to the problem of binary interactome mapping. Network topology-based approaches prove to be highly efficient in addressing this problem; however, their performance deteriorates significantly on sparse putative PPI networks. Motivated by the success of collaborative filtering (CF)-based approaches to the problem of personalized-recommendation on large, sparse rating matrices, this work aims at implementing a highly efficient CF-based approach to binary interactome mapping. To achieve this, we first propose a CF framework for it. Under this framework, we model the given data into an interactome weight matrix, where the feature-vectors of involved proteins are extracted. With them, we design the rescaled cosine coefficient to model the inter-neighborhood similarity among involved proteins, for taking the mapping process. Experimental results on three large, sparse datasets demonstrate that the proposed approach outperforms several sophisticated topology-based approaches significantly.

Differentiation of neuroblastoma cell line N1E-115 involves several signaling cascades.

PubMed

Oh, Ji-eun; Karlmark, Karlin Raja; Shin, Joo-ho; Pollak, Arnold; Freilinger, Angelika; Hengstschläger, Markus; Lubec, Gert

2005-03-01

No systematic searches for differential expression of signaling proteins (SP) in undifferentiated vs. differentiated cell lineages were published and herein we used protein profiling for this purpose. The NIE-115 cell line was cultivated and an aliquot was differentiated with dimethylsulfoxide (DMSO), that is known to lead to a neuronal phenotype. Cell lysates were prepared, run on two-dimensional gel electrophoresis followed by MALDI-TOF-TOF identification of proteins and maps of identified SPs were generated. Seven SPs were comparable, 27 SPs: GTP-binding/Ras-related proteins, kinases, growth factors, calcium binding proteins, phosphatase-related proteins were observed in differentiated NIE-115 cells and eight SPs of the groups mentioned above were observed in undifferentiated cells only. Switching-on/off of several individual SPs from different signaling cascades during the differentiation process is a key to understand mechanisms involved. The findings reported herein are challenging in vitro and in vivo studies to confirm a functional role for deranged SPs.

Alterations in the nuclear proteome of HIV-1 infected T-cells

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

DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified fourmore » clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.« less

Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

PubMed

Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

2001-03-15

Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

PubMed

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq

PubMed Central

Loeffler-Wirth, Henry; Hopp, Lydia; Schadendorf, Dirk; Schartl, Manfred; Anderegg, Ulf; Camp, Gray; Treutlein, Barbara; Binder, Hans; Kunz, Manfred

2017-01-01

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy. PMID:27903987

Fungal communication requires the MAK-2 pathway elements STE-20 and RAS-2, the NRC-1 adapter STE-50 and the MAP kinase scaffold HAM-5.

PubMed

Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

2014-11-01

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell-cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell-cell communication in fungi and higher eukaryotes.

Genesis of Prolactinomas: Studies Using Estrogen-Treated Animals

PubMed Central

Sarkar, Dipak K.

2010-01-01

Prolactin-secreting adenomas (prolactinomas) are the most prevalent form of pituitary tumors in humans. Our knowledge of the formation of these tumors is limited. Experimental work in animal has uncovered that estradiol exposure leads to prolactinoma formation via orchestrated events involving dopamine D2 receptors, transforming growth factor-β (TGF-β) isoforms and their receptors, as well as factors secondary to TGF-β action. Additionally, these studies determined that TGF-β and b-FGF interact to facilitate the communication between lactotropes and folliculo-stellate cells that is necessary for the mitogenic action of estradiol. The downstream signaling that governs lactotropic cell proliferation involves activation of the MAP kinase p44/42-dependent pathway. PMID:16809921

The combination of two Sle2 lupus-susceptibility loci and Cdkn2c deficiency leads to T cell-mediated pathology in B6.Faslpr mice

PubMed Central

Xu, Zhiwei; Croker, Byron P.; Morel, Laurence

2013-01-01

The NZM2410 Sle2c1 lupus susceptibility locus is responsible for the expansion of the B1a cell compartment and for the induction of T-cell induced renal and skin pathology on a CD95 deficient (Faslpr)-background. We have previously shown that deficiency in cyclin-dependent kinase inhibitor p18INK4c (p18) was responsible for the B1a cell expansion but was not sufficient to account for the pathology in B6.lpr mice. This study was designed to map the additional Sle2c1 loci responsible for autoimmune pathology when co-expressed with CD95 deficiency. The production, fine-mapping and phenotypic characterization of five recombinant intervals indicated that three interacting sub-loci were responsive for inducting autoimmune pathogenesis in B6.lpr mice. One of these sub-loci corresponds most likely to p18-deficiency. Another major locus mapping to a 2 Mb region at the telomeric end of Sle2c1 is necessary to both renal and skin pathology. Finally, a third locus centromeric to p18 enhances the severity of lupus nephritis. These results provide new insights into the genetic interactions leading to SLE disease presentation, and represent a major step towards the identification of novel susceptibility genes involved in T-cell mediated organ damage. PMID:23698709

A grid matrix-based Raman spectroscopic method to characterize different cell milieu in biopsied axillary sentinel lymph nodes of breast cancer patients.

PubMed

Som, Dipasree; Tak, Megha; Setia, Mohit; Patil, Asawari; Sengupta, Amit; Chilakapati, C Murali Krishna; Srivastava, Anurag; Parmar, Vani; Nair, Nita; Sarin, Rajiv; Badwe, R

2016-01-01

Raman spectroscopy which is based upon inelastic scattering of photons has a potential to emerge as a noninvasive bedside in vivo or ex vivo molecular diagnostic tool. There is a need to improve the sensitivity and predictability of Raman spectroscopy. We developed a grid matrix-based tissue mapping protocol to acquire cellular-specific spectra that also involved digital microscopy for localizing malignant and lymphocytic cells in sentinel lymph node biopsy sample. Biosignals acquired from specific cellular milieu were subjected to an advanced supervised analytical method, i.e., cross-correlation and peak-to-peak ratio in addition to PCA and PC-LDA. We observed decreased spectral intensity as well as shift in the spectral peaks of amides and lipid bands in the completely metastatic (cancer cells) lymph nodes with high cellular density. Spectral library of normal lymphocytes and metastatic cancer cells created using the cellular specific mapping technique can be utilized to create an automated smart diagnostic tool for bench side screening of sampled lymph nodes. Spectral library of normal lymphocytes and metastatic cancer cells created using the cellular specific mapping technique can be utilized to develop an automated smart diagnostic tool for bench side screening of sampled lymph nodes supported by ongoing global research in developing better technology and signal and big data processing algorithms.

Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

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

Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

1995-04-10

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, althoughmore » its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.« less

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE PAGES

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; ...

2014-11-20

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

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

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

Topographical maps as complex networks

NASA Astrophysics Data System (ADS)

da Fontoura Costa, Luciano; Diambra, Luis

2005-02-01

The neuronal networks in the mammalian cortex are characterized by the coexistence of hierarchy, modularity, short and long range interactions, spatial correlations, and topographical connections. Particularly interesting, the latter type of organization implies special demands on developing systems in order to achieve precise maps preserving spatial adjacencies, even at the expense of isometry. Although the object of intensive biological research, the elucidation of the main anatomic-functional purposes of the ubiquitous topographical connections in the mammalian brain remains an elusive issue. The present work reports on how recent results from complex network formalism can be used to quantify and model the effect of topographical connections between neuronal cells over the connectivity of the network. While the topographical mapping between two cortical modules is achieved by connecting nearest cells from each module, four kinds of network models are adopted for implementing intramodular connections, including random, preferential-attachment, short-range, and long-range networks. It is shown that, though spatially uniform and simple, topographical connections between modules can lead to major changes in the network properties in some specific cases, depending on intramodular connections schemes, fostering more effective intercommunication between the involved neuronal cells and modules. The possible implications of such effects on cortical operation are discussed.

Neurotensin protects pancreatic beta cells from apoptosis.

PubMed

Coppola, Thierry; Béraud-Dufour, Sophie; Antoine, Aurélie; Vincent, Jean-Pierre; Mazella, Jean

2008-01-01

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.

Neuroprotective and Blood-Retinal Barrier-Preserving Effects of Cannabidiol in Experimental Diabetes

PubMed Central

El-Remessy, Azza B.; Al-Shabrawey, Mohamed; Khalifa, Yousuf; Tsai, Nai-Tse; Caldwell, Ruth B.; Liou, Gregory I.

2006-01-01

Diabetic retinopathy is characterized by blood-retinal barrier (BRB) breakdown and neurotoxicity. These pathologies have been associated with oxidative stress and proinflammatory cytokines, which may operate by activating their downstream target p38 MAP kinase. In the present study, the protective effects of a nonpsychotropic cannabinoid, cannabidiol (CBD), were examined in streptozotocin-induced diabetic rats after 1, 2, or 4 weeks. Retinal cell death was determined by terminal dUTP nick-end labeling assay; BRB function by quantifying extravasation of bovine serum albumin-fluorescein; and oxidative stress by assays for lipid peroxidation, dichlorofluorescein fluorescence, and tyrosine nitration. Experimental diabetes induced significant increases in oxidative stress, retinal neuronal cell death, and vascular permeability. These effects were associated with increased levels of tumor necrosis factor-α, vascular endothelial growth factor, and intercellular adhesion molecule-1 and activation of p38 MAP kinase, as assessed by enzyme-linked immunosorbent assay, immunohistochemistry, and/or Western blot. CBD treatment significantly reduced oxidative stress; decreased the levels of tumor necrosis factor-α, vascular endothelial growth factor, and intercellular adhesion molecule-1; and prevented retinal cell death and vascular hyperpermeability in the diabetic retina. Consistent with these effects, CBD treatment also significantly inhibited p38 MAP kinase in the diabetic retina. These results demonstrate that CBD treatment reduces neurotoxicity, inflammation, and BRB breakdown in diabetic animals through activities that may involve inhibition of p38 MAP kinase. PMID:16400026

Systems Biology Analysis of Gene Expression during In Vivo Mycobacterium avium paratuberculosis Enteric Colonization Reveals Role for Immune Tolerance

PubMed Central

Khare, Sangeeta; Lawhon, Sara D.; Drake, Kenneth L.; Nunes, Jairo E. S.; Figueiredo, Josely F.; Rossetti, Carlos A.; Gull, Tamara; Everts, Robin E.; Lewin, Harris A.; Galindo, Cristi L.; Garner, Harold R.; Adams, Leslie Garry

2012-01-01

Survival and persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in the intestinal mucosa is associated with host immune tolerance. However, the initial events during MAP interaction with its host that lead to pathogen survival, granulomatous inflammation, and clinical disease progression are poorly defined. We hypothesize that immune tolerance is initiated upon initial contact of MAP with the intestinal Peyer's patch. To test our hypothesis, ligated ileal loops in neonatal calves were infected with MAP. Intestinal tissue RNAs were collected (0.5, 1, 2, 4, 8 and 12 hrs post-infection), processed, and hybridized to bovine gene expression microarrays. By comparing the gene transcription responses of calves infected with the MAP, informative complex patterns of expression were clearly visible. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis, and genes were grouped into the specific pathways and gene ontology categories to create a holistic model. This model revealed three different phases of responses: i) early (30 min and 1 hr post-infection), ii) intermediate (2, 4 and 8 hrs post-infection), and iii) late (12 hrs post-infection). We describe here the data that include expression profiles for perturbed pathways, as well as, mechanistic genes (genes predicted to have regulatory influence) that are associated with immune tolerance. In the Early Phase of MAP infection, multiple pathways were initiated in response to MAP invasion via receptor mediated endocytosis and changes in intestinal permeability. During the Intermediate Phase, perturbed pathways involved the inflammatory responses, cytokine-cytokine receptor interaction, and cell-cell signaling. During the Late Phase of infection, gene responses associated with immune tolerance were initiated at the level of T-cell signaling. Our study provides evidence that MAP infection resulted in differentially regulated genes, perturbed pathways and specifically modified mechanistic genes contributing to the colonization of Peyer's patch. PMID:22912686

Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

PubMed

Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

2015-05-01

The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

Pharmacological activity and protein phosphorylation caused by nitric oxide-releasing microparticles.

PubMed

Yoo, Jin-Wook; Choe, Eun-Sang; Ahn, Sung-Min; Lee, Chi H

2010-01-01

Nitric oxide (NO)-releasing microparticles were developed as a potential treatment option against various blood flow irregulations including sexual dysfunction, atherosclerosis and metal stent-induced restenosis. Polymeric microparticles containing diethylenetriamine diazeniumdiolate (DETA NONOate), a NO donor, were prepared using modified double-emulsion solvent evaporation method to maximize the loading efficacy and stability of DETA NONOate. The pharmacological effects of the NO-releasing microparticles were evaluated by examining the changes in the vaginal blood flow in rats. The effects of NO on the phosphorylation of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in excised vaginal mucosa, such as extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38, were examined using immunoblotting technique to determine whether NO activates PKC, which subsequently plays an integral role in the formation of PKC-MAP kinase modules. The viability of vagina cells (VK2E6E7) upon exposure to NO-releasing microparticles was examined for cytotoxicity assessment. In contrast to rapid and short-term effects of non-formulated DETA NONOate, microparticles containing DETA NONOate exerted beneficial effects on the blood flow (148+/-13%) for an extended period of time, inducing a significant change at 5 min after its application and the maximum blood flow of 172+/-23% at 120 min. The enhanced vaginal blood flow was maintained for up to 210 min and gradually returned to the baseline afterward. The results of Western immunoblotting study displayed differential expression of MAP kinases (ERK1/2 and JNK) upon NO treatment, clearly demonstrating that PKC is involved in the blood flow regulation process. There were no significant changes in cell viability in vaginal cells upon exposure to NO-releasing microparticles as compared with the control. The results of this work supported that NO-releasing microparticles could improve the vaginal blood flow without causing cytotoxic effects and PKC-MAP kinase modules are involved in the NO-induced blood flow regulation process.

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors.

PubMed

Ratajczak-Wrona, W; Jablonska, E; Garley, M; Jablonski, J; Radziwon, P; Iwaniuk, A

2013-01-01

The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.

Osmotic Stress Signaling and Osmoadaptation in Yeasts

PubMed Central

Hohmann, Stefan

2002-01-01

The ability to adapt to altered availability of free water is a fundamental property of living cells. The principles underlying osmoadaptation are well conserved. The yeast Saccharomyces cerevisiae is an excellent model system with which to study the molecular biology and physiology of osmoadaptation. Upon a shift to high osmolarity, yeast cells rapidly stimulate a mitogen-activated protein (MAP) kinase cascade, the high-osmolarity glycerol (HOG) pathway, which orchestrates part of the transcriptional response. The dynamic operation of the HOG pathway has been well studied, and similar osmosensing pathways exist in other eukaryotes. Protein kinase A, which seems to mediate a response to diverse stress conditions, is also involved in the transcriptional response program. Expression changes after a shift to high osmolarity aim at adjusting metabolism and the production of cellular protectants. Accumulation of the osmolyte glycerol, which is also controlled by altering transmembrane glycerol transport, is of central importance. Upon a shift from high to low osmolarity, yeast cells stimulate a different MAP kinase cascade, the cell integrity pathway. The transcriptional program upon hypo-osmotic shock seems to aim at adjusting cell surface properties. Rapid export of glycerol is an important event in adaptation to low osmolarity. Osmoadaptation, adjustment of cell surface properties, and the control of cell morphogenesis, growth, and proliferation are highly coordinated processes. The Skn7p response regulator may be involved in coordinating these events. An integrated understanding of osmoadaptation requires not only knowledge of the function of many uncharacterized genes but also further insight into the time line of events, their interdependence, their dynamics, and their spatial organization as well as the importance of subtle effects. PMID:12040128

A putative homologue of CDC20/CDH1 in the malaria parasite is essential for male gamete development.

PubMed

Guttery, David S; Ferguson, David J P; Poulin, Benoit; Xu, Zhengyao; Straschil, Ursula; Klop, Onny; Solyakov, Lev; Sandrini, Sara M; Brady, Declan; Nieduszynski, Conrad A; Janse, Chris J; Holder, Anthony A; Tobin, Andrew B; Tewari, Rita

2012-02-01

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis.

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

PubMed Central

Guttery, David S.; Ferguson, David J. P.; Poulin, Benoit; Xu, Zhengyao; Straschil, Ursula; Klop, Onny; Solyakov, Lev; Sandrini, Sara M.; Brady, Declan; Nieduszynski, Conrad A.; Janse, Chris J.; Holder, Anthony A.; Tobin, Andrew B.; Tewari, Rita

2012-01-01

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis. PMID:22383885

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

PubMed

Singh, Raksha; Lee, Jae-Eun; Dangol, Sarmina; Choi, Jihyun; Yoo, Ran Hee; Moon, Jae Sun; Shim, Jae-Kyung; Rakwal, Randeep; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

2014-01-01

The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Serum-induced neurite retraction in CAD cells--involvement of an ATP-actin retractile system and the lack of microtubule-associated proteins.

PubMed

Chesta, María E; Carbajal, Agustín; Arce, Carlos A; Bisig, Carlos G

2014-11-01

Cultured catecholamine-differentiated cells [which lack the microtubule-associated proteins (MAPs): MAP1B, MAP2, Tau, STOP, and Doublecortin] proliferate in the presence of fetal bovine serum, and, in its absence, cease dividing and generate processes similar to the neurites of normal neurons. The reintroduction of serum induces neurite retraction, and proliferation resumes. The neurite retraction process in catecholamine-differentiated cells was partially characterized in this study. Microtubules in the cells were found to be in a highly dynamic state, and tubulin in the microtubules consisted primarily of the tyrosinated and deacetylated isotypes. Increased levels of acetylated or Δ2-tubulin (which are normally absent) did not prevent serum-induced neurite retraction. Treatment of differentiated cells with lysophosphatidic acid or adenosine deaminase induced neurite retraction. Inhibition of Rho-associated protein kinase, ATP depletion and microfilament disruption each (individually) blocked serum-induced neurite retraction, suggesting that an ATP-dependent actomyosin system underlies the mechanism of neurite retraction. Nocodazole treatment induced neurite retraction, but this effect was blocked by pretreatment with the microtubule-stabilizing drug paclitaxel (Taxol). Paclitaxel did not prevent serum-induced or lysophosphatidic acid-induced retraction, suggesting that integrity of microtubules (despite their dynamic state) is necessary to maintain neurite elongation, and that paclitaxel-induced stabilization alone is not sufficient to resist the retraction force induced by serum. Transfection with green fluorescent protein-Tau conferred resistance to retraction caused by serum. We hypothesize that, in normal neurons (cultured or in vivo), MAPs are necessary not only to stabilize microtubules, but also to establish interactions with other cytoskeletal or membrane components to form a stable structure capable of resisting the retraction force. © 2014 FEBS.

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

PubMed

Nim, Hieu T; Furtado, Milena B; Costa, Mauro W; Kitano, Hiroaki; Rosenthal, Nadia A; Boyd, Sarah E

2015-01-01

The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.

Transcriptional Profiling of Ileocecal Valve of Holstein Dairy Cows Infected with Mycobacterium avium subsp. Paratuberculosis

PubMed Central

Hempel, Randy J.; Bannantine, John P.

2016-01-01

Johne’s disease is a chronic infection of the small intestine caused by Mycobacterium avium subspecies paratuberculosis (MAP), an intracellular bacterium. The events of pathogen survival within the host cell(s), chronic inflammation and the progression from asymptomatic subclinical stage to an advanced clinical stage of infection, are poorly understood. This study examines gene expression in the ileocecal valve (ICV) of Holstein dairy cows at different stages of MAP infection. The ICV is known to be a primary site of MAP colonization and provides an ideal location to identify genes that are relevant to the progression of this disease. RNA was prepared from ICV tissues and RNA-Seq was used to compare gene transcription between clinical, subclinical, and uninfected control animals. Interpretation of the gene expression data was performed using pathway analysis and gene ontology categories containing multiple differentially expressed genes. Results demonstrated that many of the pathways that had strong differential gene expression between uninfected control and clinical cows were related to the immune system, such as the T- and B-cell receptor signaling, apoptosis, NOD-like receptor signaling, and leukocyte transendothelial migration pathways. In contrast, the comparison of gene transcription between control and subclinical cows identified pathways that were primarily involved in metabolism. The results from the comparison between clinical and subclinical animals indicate recruitment of neutrophils, up regulation of lysosomal peptidases, increase in immune cell transendothelial migration, and modifications of the extracelluar matrix. This study provides important insight into how cattle respond to a natural MAP infection at the gene transcription level within a key target tissue for infection. PMID:27093613

Standardized extract of Bacopa monnieri (CDRI-08): Effect on germ cell dynamics and possible mechanisms of its beneficial action on spermatogenesis and sperm quality in male mice.

PubMed

Patel, Shishir Kumar; Singh, Shilpi; Singh, Shio Kumar

2017-12-09

Bacopa monnieri (BM) is used in traditional medicine as nerve tonic. We have recently shown that CDRI-08, a standardized extract of BM, improves testicular functions and epididymal sperm quality in Parkes (P) mice. The aim of the present study was to investigate the effect of CDRI-08 on germ cell dynamics and mechanisms of its action on spermatogenesis and sperm quality in P mice, and to determine the chemical profile of the extract. CDRI-08 (40 and 80 mg/kg body weight) was orally administered to male mice for 28 days. Germ cell dynamics, oxidative stress parameters in testis and sperm, and expressions of nuclear factor-erythroid-2-related factor-2 (Nrf2), phosphorylated protein kinase B (p-Akt) and upstream kinases in mitogen-activated protein kinase (MAPK) pathway namely MAP2K1, MAP2K2 and MKK4 in the testis were evaluated. The treatment potentiated germ cell dynamics and improved sperm quality by enhancing antioxidant enzymes activities. The beneficial effects of CDRI-08 in the testis involve p-Akt-mediated activation of Nrf2, thereby enhancing antioxidant enzymes activities; upregulation of MAP2K1 and MAP2K2 and suppression of MKK4 are also implicated in this action. A total of 26 phytocomponents were identified in CDRI-08 by GC-MS. The results suggest that CDRI-08 also may prove useful in improving reproductive health in males. Copyright © 2017 Elsevier Inc. All rights reserved.

The high-osmolarity glycerol- and cell wall integrity-MAP kinase pathways of Saccharomyces cerevisiae are involved in adaptation to the action of killer toxin HM-1.

PubMed

Miyamoto, Masahiko; Furuichi, Yasuhiro; Komiyama, Tadazumi

2012-11-01

Fps1p is an aquaglyceroporin important for turgor regulation of Saccharomyces cerevisiae. Previously we reported the involvement of Fps1p in the yeast-killing action of killer toxin HM-1. The fps1 cells showed a high HM-1-resistant phenotype in hypotonic medium and an HM-1-susceptible phenotype in hypertonic medium. This osmotic dependency in HM-1 susceptibility was similar to those observed in Congo red, but different from those observed in other cell wall-disturbing agents. These results indicate that HM-1 exerts fungicidal activity mainly by binding and inserting into the yeast cell wall structure, rather than by inhibiting 1,3-β-glucan synthase. We next determined HM-1-susceptibility and diphospho-MAP kinase inductions in S. cerevisiae. In the wild-type cell, expressions of diphospho-Hog1p and -Slt2p, and mRNA transcription of CWP1 and HOR2, were induced within 1 h after an addition of HM-1. ssk1 and pbs2 cells, but not sho1 and hkr1 cells, showed HM-1-sensitive phenotypes and lacked inductions of phospho-Hog1p in response to HM-1. mid2, rom2 and bck1 cells showed HM-1-sensitive phenotypes and decreased inductions of phospho-Slt2p in response to HM-1. From these results, we postulated that the Sln1-Ypd1-Ssk1 branch of the high-osmolality glycerol (HOG) pathway and plasma membrane sensors of the cell wall integrity (CWI) pathway detect cell wall stresses caused by HM-1. We further suggested that activations of both HOG and CWI pathways have an important role in the adaptive response to HM-1 toxicity. Copyright © 2012 John Wiley & Sons, Ltd.

Heat shock instructs hESCs to exit from the self-renewal program through negative regulation of OCT4 by SAPK/JNK and HSF1 pathway.

PubMed

Byun, Kyunghee; Kim, Taek-Kyun; Oh, Jeehyun; Bayarsaikhan, Enkhjargal; Kim, Daesik; Lee, Min Young; Pack, Chan-Gi; Hwang, Daehee; Lee, Bonghee

2013-11-01

Environmental factors affect self-renewal of stem cells by modulating the components of self-renewal networks. Heat shock, an environmental factor, induces heat shock factors (HSFs), which up-regulate stress response-related genes. However, the link of heat shock to self-renewal of stem cells has not been elucidated yet. Here, we present the direct link of heat shock to a core stem cell regulator, OCT4, in the self-renewal network through SAPK/JNK and HSF1 pathway. We first showed that heat shock initiated differentiation of human embryonic stem cells (hESCs). Gene expression analysis revealed that heat shock increased the expression of many genes involved in cellular processes related to differentiation of stem cells. We then examined the effects of HSFs induced by heat shock on core self-renewal factors. Among HSFs, heat shock induced mainly HSF1 in hESCs. The HSF1 repressed the expression of OCT4, leading to the differentiation of hESCs and the above differentiation-related gene expression change. We further examined the effects of the upstream MAP (mitogen-activated protein) kinases of HSF1 on the repression of OCT4 expression by HSF1. Among the MAP kinases, SAPK/JNK controlled predominantly the repression of the OCT4 expression by HSF1. The direct link of heat shock to the core self-renewal regulator through SAPK/JNK and HSF1 provides a fundamental basis for understanding the effect of heat and other stresses involving activation of HSF1 on the self-renewal program and further controlling differentiation of hESCs in a broad spectrum of stem cell applications using these stresses. © 2013.

Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

PubMed Central

Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

2014-01-01

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. PMID:25411845

Immune response induced by Epstein-Barr virus and Mycobacterium avium subsp. paratuberculosis peptides in current and past infectious mononucleosis: a risk for multiple sclerosis?

PubMed

Mameli, G; Madeddu, G; Cossu, D; Galleri, G; Manetti, R; Babudieri, S; Mura, M Stella; Sechi, L A

2016-01-01

Infectious mononucleosis (IM) caused by Epstein-Barr virus (EBV) has been associated with increased risk of multiple sclerosis (MS). However, the mechanism linking these pathologies is unclear. Different reports indicate the association of EBV, and recently Mycobacterium avium subsp. paratuberculosis (MAP), with MS. For a better understanding of the role of these pathogens, the host response induced by selected antigenic peptides in subjects with a history of IM that significantly increases the risk of MS was investigated. Both humoral and cell-mediated response against peptides able to induce a specific immune activation in MS patients deriving from lytic and latent EBV antigens BOLF1(305-320), EBNA1(400-413), from MAP MAP_4027(18-32), MAP_0106c(121-132) and from human proteins IRF5(424-434) and MBP(85-98) in subjects with current and past IM were examined. EBNA1 and MAP_0106c peptides were able to induce a humoral immune response in subjects with a history of clinical IM in an independent manner. Moreover, these peptides were capable of inducing pro-inflammatory cytokine interferon γ by CD4+ and CD8+ T lymphocytes and interleukin 6 and tumour necrosis factor α by CD14+ monocyte cells. Our results highlight that EBV and MAP may be involved independently in the same causal process leading to MS in subjects with a history of IM. © 2015 EAN.

A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

PubMed Central

Escobedo, Galileo; Soldevila, Gloria; Ortega-Pierres, Guadalupe; Chávez-Ríos, Jesús Ramsés; Nava, Karen; Fonseca-Liñán, Rocío; López-Griego, Lorena; Hallal-Calleros, Claudia; Ostoa-Saloma, Pedro; Morales-Montor, Jorge

2010-01-01

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design. PMID:20145710

Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

PubMed Central

Johard, Helena; Mahdessian, Diana; Fedr, Radek; Marks, Carolyn; Medalová, Jiřina; Souček, Karel; Lundberg, Emma; Linnarsson, Sten; Bryja, Vítězslav; Sekyrova, Petra; Altun, Mikael; Andäng, Michael

2017-01-01

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development. PMID:29228002

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

Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize themore » cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.« less

TPL-2 restricts Ccl24-dependent immunity to Heligmosomoides polygyrus

PubMed Central

Kannan, Yashaswini; Entwistle, Lewis J.; Pelly, Victoria S.; Perez-Lloret, Jimena; Ley, Steven C.

2017-01-01

TPL-2 (COT, MAP3K8) kinase activates the MEK1/2-ERK1/2 MAPK signaling pathway in innate immune responses following TLR, TNFR1 and IL-1R stimulation. TPL-2 contributes to type-1/Th17-mediated autoimmunity and control of intracellular pathogens. We recently demonstrated TPL-2 reduces severe airway allergy to house dust mite by negatively regulating type-2 responses. In the present study, we found that TPL-2 deficiency resulted in resistance to Heligmosomoides polygyrus infection, with accelerated worm expulsion, reduced fecal egg burden and reduced worm fitness. Using co-housing experiments, we found resistance to infection in TPL-2 deficient mice (Map3k8–/–) was independent of microbiota alterations in H. polygyrus infected WT and Map3k8–/–mice. Additionally, our data demonstrated immunity to H. polygyrus infection in TPL-2 deficient mice was not due to dysregulated type-2 immune responses. Genome-wide analysis of intestinal tissue from infected TPL-2-deficient mice identified elevated expression of genes involved in chemotaxis and homing of leukocytes and cells, including Ccl24 and alternatively activated genes. Indeed, Map3k8–/–mice had a significant influx of eosinophils, neutrophils, monocytes and Il4GFP+ T cells. Conditional knockout experiments demonstrated that specific deletion of TPL-2 in CD11c+ cells, but not Villin+ epithelial cells, LysM+ myeloid cells or CD4+ T cells, led to accelerated resistance to H. polygyrus. In line with a central role of CD11c+ cells, CD11c+ CD11b+ cells isolated from TPL-2-deficient mice had elevated Ccl24. Finally, Ccl24 neutralization in TPL-2 deficient mice significantly decreased the expression of Arg1, Retnla, Chil3 and Ear11 correlating with a loss of resistance to H. polygyrus. These observations suggest that TPL-2-regulated Ccl24 in CD11c+CD11b+ cells prevents accelerated type-2 mediated immunity to H. polygyrus. Collectively, this study identifies a previously unappreciated role for TPL-2 controlling immune responses to H. polygyrus infection by restricting Ccl24 production. PMID:28759611

Innate Immune Responses to Bacterial Ligands in the Peripheral Human Lung – Role of Alveolar Epithelial TLR Expression and Signalling

PubMed Central

Thorley, Andrew J.; Grandolfo, Davide; Lim, Eric; Goldstraw, Peter; Young, Alan; Tetley, Teresa D.

2011-01-01

It is widely believed that the alveolar epithelium is unresponsive to LPS, in the absence of serum, due to low expression of TLR4 and CD14. Furthermore, the responsiveness of the epithelium to TLR-2 ligands is also poorly understood. We hypothesised that human alveolar type I (ATI) and type II (ATII) epithelial cells were responsive to TLR2 and TLR4 ligands (MALP-2 and LPS respectively), expressed the necessary TLRs and co-receptors (CD14 and MD2) and released distinct profiles of cytokines via differential activation of MAP kinases. Primary ATII cells and alveolar macrophages and an immortalised ATI cell line (TT1) elicited CD14 and MD2-dependent responses to LPS which did not require the addition of exogenous soluble CD14. TT1 and primary ATII cells expressed CD14 whereas A549 cells did not, as confirmed by flow cytometry. Following LPS and MALP-2 exposure, macrophages and ATII cells released significant amounts of TNFα, IL-8 and MCP-1 whereas TT1 cells only released IL-8 and MCP-1. P38, ERK and JNK were involved in MALP-2 and LPS-induced cytokine release from all three cell types. However, ERK and JNK were significantly more important than p38 in cytokine release from macrophages whereas all three were similarly involved in LPS-induced mediator release from TT1 cells. In ATII cells, JNK was significantly more important than p38 and ERK in LPS-induced MCP-1 release. MALP-2 and LPS exposure stimulated TLR4 protein expression in all three cell types; significantly more so in ATII cells than macrophages and TT1 cells. In conclusion, this is the first study describing the expression of CD14 on, and TLR2 and 4 signalling in, primary human ATII cells and ATI cells; suggesting that differential activation of MAP kinases, cytokine secretion and TLR4 expression by the alveolar epithelium and macrophages is important in orchestrating a co-ordinated response to inhaled pathogens. PMID:21789185

Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

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

Wang, Lu; Aryal, Uma K.; Dai, Ziyu

2012-01-01

Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 uniquemore » N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.« less

The Cek1‑mediated MAP kinase pathway regulates exposure of α‑1,2 and β‑1,2‑mannosides in the cell wall of Candida albicans modulating immune recognition.

PubMed

Román, E; Correia, I; Salazin, A; Fradin, C; Jouault, T; Poulain, D; Liu, F-T; Pla, J

2016-07-03

The Cek1 MAP kinase (MAPK) mediates vegetative growth and cell wall biogenesis in the fungal pathogen Candida albicans. Alterations in the fungal cell wall caused by a defective Cek1‑mediated signaling pathway leads to increased β‑1,3‑glucan exposure influencing dectin‑1 fungal recognition by immune cells. We show here that cek1 cells also display an increased exposure of α‑1,2 and β‑1,2‑mannosides (α‑M and β‑M), a phenotype shared by strains defective in the activating MAPKK Hst7, suggesting a general defect in cell wall assembly. cek1 cells display walls with loosely bound material as revealed by transmission electron microscopy and are sensitive to tunicamycin, an inhibitor of N‑glycosylation. Transcriptomal analysis of tunicamycin treated cells revealed a differential pattern between cek1 and wild type cells which involved mainly cell wall and stress related genes. Mapping α‑M and β‑M epitopes in the mannoproteins of different cell wall fractions (CWMP) revealed an important shift in the molecular weight of the mannan derived from mutants defective in this MAPK pathway. We have also assessed the role of galectin‑3, a member of a β‑galactoside‑binding protein family shown to bind to and kill C. albicans through β‑M recognition, in the infection caused by cek1 mutants. Increased binding of cek1 to murine macrophages was shown to be partially blocked by lactose. Galectin-3(-/-) mice showed increased resistance to fungal infection, although galectin-3 did not account for the reduced virulence of cek1 mutants in a mouse model of systemic infection. All these data support a role for the Cek1‑mediated pathway in fungal cell wall maintenance, virulence and antifungal discovery.

NaviCell: a web-based environment for navigation, curation and maintenance of large molecular interaction maps

PubMed Central

2013-01-01

Background Molecular biology knowledge can be formalized and systematically represented in a computer-readable form as a comprehensive map of molecular interactions. There exist an increasing number of maps of molecular interactions containing detailed and step-wise description of various cell mechanisms. It is difficult to explore these large maps, to organize discussion of their content and to maintain them. Several efforts were recently made to combine these capabilities together in one environment, and NaviCell is one of them. Results NaviCell is a web-based environment for exploiting large maps of molecular interactions, created in CellDesigner, allowing their easy exploration, curation and maintenance. It is characterized by a combination of three essential features: (1) efficient map browsing based on Google Maps; (2) semantic zooming for viewing different levels of details or of abstraction of the map and (3) integrated web-based blog for collecting community feedback. NaviCell can be easily used by experts in the field of molecular biology for studying molecular entities of interest in the context of signaling pathways and crosstalk between pathways within a global signaling network. NaviCell allows both exploration of detailed molecular mechanisms represented on the map and a more abstract view of the map up to a top-level modular representation. NaviCell greatly facilitates curation, maintenance and updating the comprehensive maps of molecular interactions in an interactive and user-friendly fashion due to an imbedded blogging system. Conclusions NaviCell provides user-friendly exploration of large-scale maps of molecular interactions, thanks to Google Maps and WordPress interfaces, with which many users are already familiar. Semantic zooming which is used for navigating geographical maps is adopted for molecular maps in NaviCell, making any level of visualization readable. In addition, NaviCell provides a framework for community-based curation of maps. PMID:24099179

NaviCell: a web-based environment for navigation, curation and maintenance of large molecular interaction maps.

PubMed

Kuperstein, Inna; Cohen, David P A; Pook, Stuart; Viara, Eric; Calzone, Laurence; Barillot, Emmanuel; Zinovyev, Andrei

2013-10-07

Molecular biology knowledge can be formalized and systematically represented in a computer-readable form as a comprehensive map of molecular interactions. There exist an increasing number of maps of molecular interactions containing detailed and step-wise description of various cell mechanisms. It is difficult to explore these large maps, to organize discussion of their content and to maintain them. Several efforts were recently made to combine these capabilities together in one environment, and NaviCell is one of them. NaviCell is a web-based environment for exploiting large maps of molecular interactions, created in CellDesigner, allowing their easy exploration, curation and maintenance. It is characterized by a combination of three essential features: (1) efficient map browsing based on Google Maps; (2) semantic zooming for viewing different levels of details or of abstraction of the map and (3) integrated web-based blog for collecting community feedback. NaviCell can be easily used by experts in the field of molecular biology for studying molecular entities of interest in the context of signaling pathways and crosstalk between pathways within a global signaling network. NaviCell allows both exploration of detailed molecular mechanisms represented on the map and a more abstract view of the map up to a top-level modular representation. NaviCell greatly facilitates curation, maintenance and updating the comprehensive maps of molecular interactions in an interactive and user-friendly fashion due to an imbedded blogging system. NaviCell provides user-friendly exploration of large-scale maps of molecular interactions, thanks to Google Maps and WordPress interfaces, with which many users are already familiar. Semantic zooming which is used for navigating geographical maps is adopted for molecular maps in NaviCell, making any level of visualization readable. In addition, NaviCell provides a framework for community-based curation of maps.

Regulatory T cells in cattle and their potential role in bovine paratuberculosis.

PubMed

Coussens, Paul M; Sipkovsky, Sue; Murphy, Brooke; Roussey, Jon; Colvin, Christopher J

2012-05-01

The intracellular bacterium Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in wild and domestic ruminants. Johne's disease presents as a chronic enteritis with severe inflammation of intestinal tissues, characterized by widespread infiltration of macrophages, the target cell of MAP. Clinical signs of Johne's disease are typically accompanied by a loss of peripheral CD4+ T cell responses to MAP antigens and an increase in anti-MAP serum IgG levels. Recently, it was proposed that regulatory T cells might develop over the lengthy course of subclinical MAP infection. In the past five years, significant progress in defining bovine regulatory T cells has been made. These studies grew out of observations that IL-10 is produced by PBMCs in response to MAP antigen stimulation and that neutralization of this IL-10 could enhance IFN-γ production from MAP-antigen reactive effector T cells. Depletion studies revealed that MAP responsive cell populations producing IL-10 were largely CD4+ and CD25+, although monocytes have also been shown to produce IL-10 in response to MAP. In addition, evidence for a regulatory population of γδ T cells has also begun to accumulate. We summarize current thinking regarding regulatory T cells in MAP infection and provide data suggesting a potential link between regulatory T cells, bovine leukemia virus, and MAP. Copyright © 2012 Elsevier Ltd. All rights reserved.

Membrane androgen receptor characteristics of human ZIP9 (SLC39A) zinc transporter in prostate cancer cells: Androgen-specific activation and involvement of an inhibitory G protein in zinc and MAP kinase signaling.

PubMed

Thomas, Peter; Pang, Yefei; Dong, Jing

2017-05-15

Characteristics of novel human membrane androgen receptor (mAR), ZIP9 (SLC39A9), were investigated in ZIP9-transfected PC-3 cells (PC3-ZIP9). Ligand blot analysis showed plasma membrane [ 3 H]-T binding corresponds to the position of ZIP9 on Western blots which suggests ZIP9 can bind [ 3 H]-T alone, without a protein partner. Progesterone antagonized testosterone actions, blocking increases in zinc, Erk phosphorylation and apoptosis, further evidence that ZIP9 is specifically activated by androgens. Pre-treatment with GTPγS and pertussis toxin decreased plasma membrane [ 3 H]-T binding and blocked testosterone-induced increases in Erk phosphorylation and intracellular zinc, indicating ZIP9 is coupled to an inhibitory G protein (Gi) that mediates both MAP kinase and zinc signaling. Testosterone treatment of nuclei and mitochondria which express ZIP9 decreased their zinc contents, suggesting ZIP9 also regulates free zinc through releasing it from these intracellular organelles. The results show ZIP9 is a specific Gi coupled-mAR mediating testosterone-induced MAP kinase and zinc signaling in PC3-ZIP9 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Pegylated G-CSF Inhibits Blood Cell Depletion, Increases Platelets, Blocks Splenomegaly, and Improves Survival after Whole-Body Ionizing Irradiation but Not after Irradiation Combined with Burn

DTIC Science & Technology

2014-03-05

increased granulocyte colony stim- ulating factor (G-CSF) in mouse blood for more than 7 days [7]. The increase was initially believed to be a self ...hematopoietic stem cell mobilization from the bone marrow into the bloodstream. It is involved in recovery from infection [11, 12] and wound healing [13]. Peg-G...mapping data; corrections for the 60Co decay and the small differences in the mass energy absorption coefficients for water and soft tissue were

Occupancy change detection system and method

DOEpatents

Bruemmer, David J [Idaho Falls, ID; Few, Douglas A [Idaho Falls, ID

2009-09-01

A robot platform includes perceptors, locomotors, and a system controller. The system controller executes instructions for producing an occupancy grid map of an environment around the robot, scanning the environment to generate a current obstacle map relative to a current robot position, and converting the current obstacle map to a current occupancy grid map. The instructions also include processing each grid cell in the occupancy grid map. Within the processing of each grid cell, the instructions include comparing each grid cell in the occupancy grid map to a corresponding grid cell in the current occupancy grid map. For grid cells with a difference, the instructions include defining a change vector for each changed grid cell, wherein the change vector includes a direction from the robot to the changed grid cell and a range from the robot to the changed grid cell.

The kinases MEKK2 and MEKK3 regulate transforming growth factor-β-mediated helper T cell differentiation.

PubMed

Chang, Xing; Liu, Fang; Wang, Xiaofang; Lin, Aiping; Zhao, Hongyu; Su, Bing

2011-02-25

Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3) negatively regulated transforming growth factor-β (TGF-β)-mediated Th cell differentiation. Map3k2(-/-)Map3k3(Lck-Cre/-) mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2(-/-)Map3k3(Lck-Cre/-) naive CD4(+) T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-β stimulation in vitro. In addition, Map3k2(-/-)Map3k3(Lck-Cre/-) mice developed more severe experimental autoimmune encephalomyelitis. Map3k2(-/-)Map3k3(Lck-Cre/-) T cells exhibited impaired phosphorylation of SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-β responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-β signaling pathways is important in regulating Th cell differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.

Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

PubMed Central

Jukam, David; Teran, Nicole A; Risca, Viviana I; Smith, Owen K; Johnson, Whitney L; Skotheim, Jan M; Greenleaf, William James

2018-01-01

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing. PMID:29648534

The role of human and mouse Y chromosome genes in male infertility.

PubMed

Affara, N A; Mitchell, M J

2000-11-01

It was suggested by Ronald Fisher in 1931 that genes involved in benefit to the male (including spermatogenesis genes) would accumulate on the Y chromosome. The analysis of mouse Y chromosome deletions and the discovery of microdeletions of the human Y chromosome associated with diverse defective spermatogenic phenotypes has revealed the presence of intervals containing one or more genes controlling male germ cell differentiation. These intervals have been mapped, cloned and examined in detail for functional genes. This review discusses the genes mapping to critical spermatogenesis intervals and the evidence indicating which are the most likely candidates underlying Y-linked male infertility.

Porcine circovirus-2 capsid protein induces cell death in PK15 cells

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

Walia, Rupali; Dardari, Rkia, E-mail: rdardari@ucalgary.ca; Chaiyakul, Mark

Studies have shown that Porcine circovirus (PCV)-2 induces apoptosis in PK15 cells. Here we report that cell death is induced in PCV2b-infected PK15 cells that express Capsid (Cap) protein and this effect is enhanced in interferon gamma (IFN-γ)-treated cells. We further show that transient PCV2a and 2b-Cap protein expression induces cell death in PK15 cells at rate similar to PCV2 infection, regardless of Cap protein localization. These data suggest that Cap protein may have the capacity to trigger different signaling pathways involved in cell death. Although further investigation is needed to gain deeper insights into the nature of the pathwaysmore » involved in Cap-induced cell death, this study provides evidence that PCV2-induced cell death in kidney epithelial PK15 cells can be mapped to the Cap protein and establishes the need for future research regarding the role of Cap-induced cell death in PCV2 pathogenesis. - Highlights: • IFN-γ enhances PCV2 replication that leads to cell death in PK15 cells. • IFN-γ enhances nuclear localization of the PCV2 Capsid protein. • Transient PCV2a and 2b-Capsid protein expression induces cell death. • Cell death is not dictated by specific Capsid protein sub-localization.« less

Cell type-selective disease-association of genes under high regulatory load

PubMed Central

Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

2015-01-01

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3′ UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. PMID:26338775

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

PubMed

Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

PubMed Central

Navarro-García, F; Sánchez, M; Pla, J; Nombela, C

1995-01-01

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi. PMID:7891715

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

PubMed Central

Peckys, Diana B.; Veith, Gabriel M.; Joy, David C.; de Jonge, Niels

2009-01-01

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory. PMID:20020038

Midazolam suppresses interleukin-1β-induced interleukin-6 release from rat glial cells

PubMed Central

2011-01-01

Background Peripheral-type benzodiazepine receptor (PBR) expression levels are low in normal human brain, but their levels increase in inflammation, brain injury, neurodegenerative states and gliomas. It has been reported that PBR functions as an immunomodulator. The mechanisms of action of midazolam, a benzodiazepine, in the immune system in the CNS remain to be fully elucidated. We previously reported that interleukin (IL)-1β stimulates IL-6 synthesis from rat C6 glioma cells and that IL-1β induces phosphorylation of inhibitory kappa B (IκB), p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription (STAT)3. It has been shown that p38 MAP kinase is involved in IL-1β-induced IL-6 release from these cells. In the present study, we investigated the effect of midazolam on IL-1β-induced IL-6 release from C6 cells, and the mechanisms of this effect. Methods Cultured C6 cells were stimulated by IL-1β. IL-6 release from C6 cells was measured using an enzyme-linked immunosorbent assay, and phosphorylation of IκB, the MAP kinase superfamily, and STAT3 was analyzed by Western blotting. Results Midazolam, but not propofol, inhibited IL-1β-stimulated IL-6 release from C6 cells. The IL-1β-stimulated levels of IL-6 were suppressed by wedelolactone (an inhibitor of IκB kinase), SP600125 (an inhibitor of SAPK/JNK), and JAK inhibitor I (an inhibitor of JAK 1, 2 and 3). However, IL-6 levels were not affected by PD98059 (an inhibitor of MEK1/2). Midazolam markedly suppressed IL-1β-stimulated STAT3 phosphorylation without affecting the phosphorylation of p38 MAP kinase, SAPK/JNK or IκB. Conclusion These results strongly suggest that midazolam inhibits IL-1β-induced IL-6 release in rat C6 glioma cells via suppression of STAT3 activation. Midazolam may affect immune system function in the CNS. PMID:21682888

Murine Lupus Susceptibility Locus Sle2 Activates DNA-Reactive B Cells through Two Sub-Loci with Distinct Phenotypes

PubMed Central

Zeumer, Leilani; Sang, Allison; Niu, Haitao; Morel, Laurence

2010-01-01

The NZM2410-derived Sle2 lupus susceptibility locus induces an abnormal B cell differentiation which most prominently leads to the expansion of autoreactive B1a cells. We have mapped the expansion of B1a cells to three Sle2 sub-loci, Sle2a, Sle2b, and Sle2c. Sle2 also enhances the breach of B cell tolerance to nuclear antigens in the 56R anti-DNA immunoglobulin transgenic (Tg) model. This study used the Sle2 sub-congenic strains to map the activation of 56R Tg B cells. Sle2c strongly sustained the breach of tolerance and the activation of anti-DNA B cells. The production of Tg-encoded anti-DNA antibodies was more modest in Sle2a expressing mice, but Sle2a was responsible for the recruitment for Tg B cells to the marginal zone, a phenotype that has been found for 56R Tg B cells in mice expressing the whole Sle2 interval. In addition, Sle2a promoted the production of endogenously encoded anti-DNA antibodies. Overall, this study showed that at least two Sle2 genes are involved in the activation of anti-DNA B cells, and excluded more than two-thirds of the Sle2 interval from contributing to this phenotype. This constitutes an important step toward the identification of novel genes that play a critical role in B cell tolerance. PMID:21270826

Theory of Epithelial Cell Shape Transitions Induced by Mechanoactive Chemical Gradients.

PubMed

Dasbiswas, Kinjal; Hannezo, Edouard; Gov, Nir S

2018-02-27

Cell shape is determined by a balance of intrinsic properties of the cell as well as its mechanochemical environment. Inhomogeneous shape changes underlie many morphogenetic events and involve spatial gradients in active cellular forces induced by complex chemical signaling. Here, we introduce a mechanochemical model based on the notion that cell shape changes may be induced by external diffusible biomolecules that influence cellular contractility (or equivalently, adhesions) in a concentration-dependent manner-and whose spatial profile in turn is affected by cell shape. We map out theoretically the possible interplay between chemical concentration and cellular structure. Besides providing a direct route to spatial gradients in cell shape profiles in tissues, we show that the dependence on cell shape helps create robust mechanochemical gradients. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Involvement of the mitogen activated protein kinase Hog1p in the response of Candida albicans to iron availability

PubMed Central

2013-01-01

Background Iron is an essential nutrient for almost all organisms, and generating iron limiting conditions for pathogens is one of the host defense strategies against microbial infections. Excess of iron can be toxic; therefore, iron uptake is tightly controlled. The high affinity iron uptake system of the opportunistic pathogenic yeast Candida albicans has been shown to be essential for virulence. Several transcription factors and regulators of iron uptake genes were identified, but the knowledge of signaling pathways is still limited. Gene expression profiling of the Δhog1 deletion mutant indicated an involvement of the mitogen activated protein (MAP) kinase Hog1p. However, the function of Hog1p in the response of C. albicans to iron availability was not studied in detail. Thus, we analyzed phenotypic and molecular responses of C. albicans to different iron concentrations particularly with respect to the activity of the Hog1p MAP kinase module. Results We observed flocculation of yeast cells, when the iron ion concentration was equal to or higher than 5 μM. This phenotype was dependent on the MAP kinase Hog1p and the corresponding MAP kinase kinase Pbs2p. Moreover, high extracellular iron ion concentrations led to hyper-phosphorylation of Hog1p. We determined lower amounts of multicopper ferroxidase (MCFO) proteins and lower ferric reductase activity, when the iron ion concentration in the medium was increased. This effect was also observed for the Δhog1 mutant. However, the amounts of MCFO proteins and the cell surface ferric reductase activity were increased in the Δhog1 in comparison to wild type cells. This effect was independent of iron availability in growth media. Conclusions In C. albicans, the MAP kinase Hog1p is part of the network regulating the response of the organism to iron availability. Hog1p was transiently phosphorylated under high iron concentrations and was essential for a flocculent phenotype. Furthermore, deletion of HOG1 led to increased levels of components of the reductive iron uptake system in comparison to the wild-type, independent of iron concentrations in the media. However, the additional induction of this system by low iron concentrations was independent of HOG1. PMID:23347662

Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

PubMed Central

Waldron, Anna M.; Begg, Douglas J.; de Silva, Kumudika; Purdie, Auriol C.; Whittington, Richard J.

2015-01-01

Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 104-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n = 54) and sheep fecal and tissue (n = 90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis. PMID:25609725

A radiation hybrid map of the proximal long arm of human chromosome 11 containing the multiple endocrine neoplasia type 1 (MEN-1) and bcl-1 disease loci.

PubMed Central

Richard, C W; Withers, D A; Meeker, T C; Maurer, S; Evans, G A; Myers, R M; Cox, D R

1991-01-01

We describe a high-resolution radiation hybrid map of the proximal long arm of human chromosome 11 containing the bcl-1 and multiple endocrine neoplasia type 1 (MEN-1) disease gene loci. We used X-ray irradiation and cell fusion to generate a panel of 102 hamster-human somatic cell hybrids containing fragments of human chromosome 11. Sixteen human loci in the 11q12-13 region were mapped by statistical analysis of the cosegregation of markers in these radiation hybrids. The most likely order for these loci is C1NH-OSBP-(CD5/CD20)-PGA-FTH1-COX8-PYGM -SEA-KRN1-(MTC/P11EH/HSTF1/INT2)-GST3- PPP1A. Our localization of the human protooncogene SEA between PYGM and INT2, two markers that flank MEN-1, suggests SEA as a potential candidate for the MEN-1 locus. We map two mitogenic fibroblast growth factor genes, HSTF1 and INT2, close to bcl-1, a mapping that is consistent with previously published data. Our map places the human leukocyte antigen genes CD5 and CD20 far from the bcl-1 locus, indicating that CD5 and CD20 expression is unlikely to be altered by bcl-1 rearrangements. PPP1A, which has been postulated as a MEN-1 candidate tumor suppressor gene, and GST3, a gene transcriptionally active in many human cancers, both map distal to the bcl-1 translocation cluster and the region containing MEN-1, and therefore are unlikely to be directly involved in bcl-1 or MEN-1. PMID:1684084

A radiation hybrid map of the proximal long arm of human chromosome 11 containing the multiple endocrine neoplasia type 1 (MEN-1) and bcl-1 disease loci.

PubMed

Richard, C W; Withers, D A; Meeker, T C; Maurer, S; Evans, G A; Myers, R M; Cox, D R

1991-12-01

We describe a high-resolution radiation hybrid map of the proximal long arm of human chromosome 11 containing the bcl-1 and multiple endocrine neoplasia type 1 (MEN-1) disease gene loci. We used X-ray irradiation and cell fusion to generate a panel of 102 hamster-human somatic cell hybrids containing fragments of human chromosome 11. Sixteen human loci in the 11q12-13 region were mapped by statistical analysis of the cosegregation of markers in these radiation hybrids. The most likely order for these loci is C1NH-OSBP-(CD5/CD20)-PGA-FTH1-COX8-PYGM -SEA-KRN1-(MTC/P11EH/HSTF1/INT2)-GST3- PPP1A. Our localization of the human protooncogene SEA between PYGM and INT2, two markers that flank MEN-1, suggests SEA as a potential candidate for the MEN-1 locus. We map two mitogenic fibroblast growth factor genes, HSTF1 and INT2, close to bcl-1, a mapping that is consistent with previously published data. Our map places the human leukocyte antigen genes CD5 and CD20 far from the bcl-1 locus, indicating that CD5 and CD20 expression is unlikely to be altered by bcl-1 rearrangements. PPP1A, which has been postulated as a MEN-1 candidate tumor suppressor gene, and GST3, a gene transcriptionally active in many human cancers, both map distal to the bcl-1 translocation cluster and the region containing MEN-1, and therefore are unlikely to be directly involved in bcl-1 or MEN-1.

Efficient, validated method for detection of mycobacterial growth in liquid culture media by use of bead beating, magnetic-particle-based nucleic acid isolation, and quantitative PCR.

PubMed

Plain, Karren M; Waldron, Anna M; Begg, Douglas J; de Silva, Kumudika; Purdie, Auriol C; Whittington, Richard J

2015-04-01

Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10(4)-fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n=54) and sheep fecal and tissue (n=90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Therapeutical relevance of MAP-kinase inhibitors in renal diseases: current knowledge and future clinical perspectives.

PubMed

Grande, M Teresa; López-Novoa, José M

2008-01-01

Renal failure, both acute and chronic, represents an important health problem by its social, sanitary and economic aspects. Mitogen-activated protein kinases (MAPK) are a family of mediators involved in the transduction of extracellular stimuli to intracellular responses. The best studied members of this family are extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), Jun NH(2)-terminal kinase (JNK), p38 kinase and extracellular signal regulated kinases 5 (ERK5) also known as big MAP Kinase 1 (BMK1). MAPKs plays a role in regulating renal function and all these pathways have been demonstrated to be activated in many "in vivo" and cellular models or renal failure. As MAP kinases are key regulators in the control of cell proliferation and cell death, many more or less specific inhibitors of these pathways are being developed for the treatment of tumors. The purpose of this review is to examine the data available on the role of MAPKs activation in "in vivo" models of renal failure, as well as in different renal cell types (especially in mesangial cells, podocytes, tubular epithelial cells and fibroblasts) subjected to stress or damage. We have also reviewed the effect of MAPKs inhibition on renal damage, both "in vivo" and "in vitro". Data collected allow to suggest that therapy of chronic and acute renal disease with MAPKs inhibitors is a promising therapeutic area, although much more basic and clinical studies are necessary before this kind of therapy can be used in the everyday clinic.

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri

PubMed Central

MATSUO, Yosuke; MIYOSHI, Yukihiro; OKADA, Sanae; SATOH, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion. PMID:24936355

Vascular biology in altered gravity conditions

NASA Astrophysics Data System (ADS)

Bradamante, Silvia; Maier, Janette A. M.; Duncker, Dirk J.

2005-10-01

The physical environment of Endothelial Cells profoundly affects their gene expression, structure, function, growth differentiation and apoptosis. However, the mechanisms by which the genetic and local growth determinants driving morphogenesis are established and maintained remain unknown. Understanding how gravity affects vascular cells will offer new insights for novel therapeutical approaches for cardiovascular disease in general. In terms of tissue engineering and stem-cell therapy, significant future developments will depend on a profound understanding of the cellular and molecular basis of angiogenesis and of the biology of circulating Endothelial Precursor Cells. this MAP project has demonstrated how modelled microgravity influences endothelial proliferation and differentiation with the involvement of anti-angiogenic factors that may be responsible for the non-spontaneous formation of blood vessels.

Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs

NASA Astrophysics Data System (ADS)

Carroll, David J.; Hua, Wei

The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 μm) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

Genetic and physical map of the von Recklinghausen neurofibromatosis (NF1) region on chromosome 17

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

Yagle, M.K.; Parruti, G.; Xu, W.

The von Recklinghausen neurofibromatosis 1 (NF1) locus has been previously assigned to the proximal long arm of chromosome 17, and two NF1 patients have been identified who have constitutional balanced translocations involving 17q11.2. The authors have constructed a cosmid library from a chromosome-mediated gene transfectant, KLT8, that contains approximately 10% of chromosome 17, including 17q11.2. Cosmids isolated from this library have been mapped across a panel of somatic cell hybrids, including the hybrids from the two patients, and have been localized to seven small regions of proximal 17q. They have 5 cosmids that map directly above the two NF1 translocations,more » and 11 cosmids that map directly below. Of these, 2 cosmids in each region are linked to the disease locus and 3 of these cosmids show no recombination. One distal cosmid, 2B/B35, detects the two NF1 translocations by pulsed-field gel analysis and has been used to produce a long-range restriction map that covers the translocations.« less

Effects of Cot expression on the nuclear translocation of NF-kappaB in RBL-2H3 cells.

PubMed

Chikamatsu, Satomi; Furuno, Tadahide; Kinoshita, Yosuke; Inoh, Yoshikazu; Hirashima, Naohide; Teshima, Reiko; Nakanishi, Mamoru

2007-03-01

Cot is a serine/threonine protein kinase and is classified as a mitogen-activated protein (MAP) kinase kinase kinase. Overexpression of this protein has been shown to activate the extracellular signal-regulated kinase, the c-Jun N-terminal kinase, and the p38 MAP kinase pathways and to stimulate NF-AT and NF-kappaB-dependent transcription. Here we have shown that Cot kinase activity is intimately involved in the high affinity receptor for IgE (FcvarepsilonRI)-mediated nuclear translocation of NF-kappaB1 independent of NF-kappaB-inducing kinase (NIK) in rat basophilic leukemia (RBL-2H3) cells. A transfected green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1) resided in the cytoplasm in RBL-2H3 cells and it remained in the cytoplasm even when Cot tagged with red fluorescent protein (Cot-RFP) was co-expressed. Western blotting analysis showed that IkappaB kinases (IKKs) were expressed in RBL-2H3 cells but NIK was not. GFP-NF-kappaB1 translocated from the cytoplasm to the nucleus after the aggregation of FcvarepsilonRI in Cot-transfected cells but not in kinase-deficient Cot-transfected cells. This finding gives a new insight into the role of Cot in the FcvarepsilonRI-mediated NF-kappaB activation in mast cells.

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid.

PubMed

Zhang, Xinzheng; Xiang, Ye; Dunigan, David D; Klose, Thomas; Chipman, Paul R; Van Etten, James L; Rossmann, Michael G

2011-09-06

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the release of the internal viral membrane that was linked to the capsid by many copies of a viral membrane protein in the mature infectous virus. Simultaneously, part of the trisymmetrons around the unique vertex disassemble, probably in part because two minor capsid proteins are absent, causing Paramecium bursaria chlorella virus and the cellular contents to merge, possibly as a result of enzyme(s) within the spike assembly. This may be one of only a few recordings of successive stages of a virus while infecting a eukaryotic host in pseudoatomic detail in three dimensions.

Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid

PubMed Central

Zhang, Xinzheng; Xiang, Ye; Dunigan, David D.; Klose, Thomas; Chipman, Paul R.; Van Etten, James L.; Rossmann, Michael G.

2011-01-01

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the release of the internal viral membrane that was linked to the capsid by many copies of a viral membrane protein in the mature infectous virus. Simultaneously, part of the trisymmetrons around the unique vertex disassemble, probably in part because two minor capsid proteins are absent, causing Paramecium bursaria chlorella virus and the cellular contents to merge, possibly as a result of enzyme(s) within the spike assembly. This may be one of only a few recordings of successive stages of a virus while infecting a eukaryotic host in pseudoatomic detail in three dimensions. PMID:21873222

Cloning of the anhidrotic ectodermal dysplasia gene: Identification of cDNAs associated with CpG islands mapped near translocation breakpoint in two female patients

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

Srivastava, A.K.; Schlessinger, D.; Kere, J.

1994-09-01

The gene for the X chromosomal developmental disorder anhidrotic ectodermal dysplasia (EDA) has been mapped to Xq12-q13 by linkage analysis and is expressed in a few females with chromosomal translocations involving band Xq12-q13. A yeast artificial chromosome (YAC) contig (2.0 Mb) spanning two translocation breakpoints has been assembled by sequence-tagged site (STS)-based chromosomal walking. The two translocation breakpoints (X:autosome translocations from the affected female patients) have been mapped less than 60 kb apart within a YAC contig. Unique probes and intragenic STSs (mapped between the two translocations) have been developed and a somatic cell hybrid carrying the translocated X chromosomemore » from the AK patient has been analyzed by isolating unique probes that span the breakpoint. Several STSs made from intragenic sequences have been found to be conserved in mouse, hamster and monkey, but we have detected no mRNAs in a number of tissues tested. However, a probe and STS developed from the DNA spanning the AK breakpoint is conserved in mouse, hamster and monkey, and we have detected expressed sequences in skin cells and cDNA libraries. In addition, unique sequences have been obtained from two CpG islands in the region that maps proximal to the breakpoints. cDNAs containing these sequences are being studied as candidates for the gene affected in the etiology of EDA.« less

A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control.

PubMed

Devoghalaere, Fanny; Doucen, Thomas; Guitton, Baptiste; Keeling, Jeannette; Payne, Wendy; Ling, Toby John; Ross, John James; Hallett, Ian Charles; Gunaseelan, Kularajathevan; Dayatilake, G A; Diak, Robert; Breen, Ken C; Tustin, D Stuart; Costes, Evelyne; Chagné, David; Schaffer, Robert James; David, Karine Myriam

2012-01-13

Auxin is an important phytohormone for fleshy fruit development, having been shown to be involved in the initial signal for fertilisation, fruit size through the control of cell division and cell expansion, and ripening related events. There is considerable knowledge of auxin-related genes, mostly from work in model species. With the apple genome now available, it is possible to carry out genomics studies on auxin-related genes to identify genes that may play roles in specific stages of apple fruit development. High amounts of auxin in the seed compared with the fruit cortex were observed in 'Royal Gala' apples, with amounts increasing through fruit development. Injection of exogenous auxin into developing apples at the start of cell expansion caused an increase in cell size. An expression analysis screen of auxin-related genes involved in auxin reception, homeostasis, and transcriptional regulation showed complex patterns of expression in each class of gene. Two mapping populations were phenotyped for fruit size over multiple seasons, and multiple quantitative trait loci (QTLs) were observed. One QTL mapped to a region containing an Auxin Response Factor (ARF106). This gene is expressed during cell division and cell expansion stages, consistent with a potential role in the control of fruit size. The application of exogenous auxin to apples increased cell expansion, suggesting that endogenous auxin concentrations are at least one of the limiting factors controlling fruit size. The expression analysis of ARF106 linked to a strong QTL for fruit weight suggests that the auxin signal regulating fruit size could partially be modulated through the function of this gene. One class of gene (GH3) removes free auxin by conjugation to amino acids. The lower expression of these GH3 genes during rapid fruit expansion is consistent with the apple maximising auxin concentrations at this point.

A genomics approach to understanding the role of auxin in apple (Malus x domestica) fruit size control

PubMed Central

2012-01-01

Background Auxin is an important phytohormone for fleshy fruit development, having been shown to be involved in the initial signal for fertilisation, fruit size through the control of cell division and cell expansion, and ripening related events. There is considerable knowledge of auxin-related genes, mostly from work in model species. With the apple genome now available, it is possible to carry out genomics studies on auxin-related genes to identify genes that may play roles in specific stages of apple fruit development. Results High amounts of auxin in the seed compared with the fruit cortex were observed in 'Royal Gala' apples, with amounts increasing through fruit development. Injection of exogenous auxin into developing apples at the start of cell expansion caused an increase in cell size. An expression analysis screen of auxin-related genes involved in auxin reception, homeostasis, and transcriptional regulation showed complex patterns of expression in each class of gene. Two mapping populations were phenotyped for fruit size over multiple seasons, and multiple quantitative trait loci (QTLs) were observed. One QTL mapped to a region containing an Auxin Response Factor (ARF106). This gene is expressed during cell division and cell expansion stages, consistent with a potential role in the control of fruit size. Conclusions The application of exogenous auxin to apples increased cell expansion, suggesting that endogenous auxin concentrations are at least one of the limiting factors controlling fruit size. The expression analysis of ARF106 linked to a strong QTL for fruit weight suggests that the auxin signal regulating fruit size could partially be modulated through the function of this gene. One class of gene (GH3) removes free auxin by conjugation to amino acids. The lower expression of these GH3 genes during rapid fruit expansion is consistent with the apple maximising auxin concentrations at this point. PMID:22243694

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

PubMed

Chung, Eun Young; Shin, Soon Young; Lee, Young Han

2007-07-05

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

Mapping of the Lassa virus LAMP1 binding site reveals unique determinants not shared by other old world arenaviruses.

PubMed

Israeli, Hadar; Cohen-Dvashi, Hadas; Shulman, Anastasiya; Shimon, Amir; Diskin, Ron

2017-04-01

Cell entry of many enveloped viruses occurs by engagement with cellular receptors, followed by internalization into endocytic compartments and pH-induced membrane fusion. A previously unnoticed step of receptor switching was found to be critical during cell entry of two devastating human pathogens: Ebola and Lassa viruses. Our recent studies revealed the functional role of receptor switching to LAMP1 for triggering membrane fusion by Lassa virus and showed the involvement of conserved histidines in this switching, suggesting that other viruses from this family may also switch to LAMP1. However, when we investigated viruses that are genetically close to Lassa virus, we discovered that they cannot bind LAMP1. A crystal structure of the receptor-binding module from Morogoro virus revealed structural differences that allowed mapping of the LAMP1 binding site to a unique set of Lassa residues not shared by other viruses in its family, illustrating a key difference in the cell-entry mechanism of Lassa virus that may contribute to its pathogenicity.

Single-unit labeling of medial olivocochlear neurons: the cochlear frequency map for efferent axons.

PubMed

Brown, M Christian

2014-06-01

Medial olivocochlear (MOC) neurons are efferent neurons that project axons from the brain to the cochlea. Their action on outer hair cells reduces the gain of the "cochlear amplifier," which shifts the dynamic range of hearing and reduces the effects of noise masking. The MOC effects in one ear can be elicited by sound in that ipsilateral ear or by sound in the contralateral ear. To study how MOC neurons project onto the cochlea to mediate these effects, single-unit labeling in guinea pigs was used to study the mapping of MOC neurons for neurons responsive to ipsilateral sound vs. those responsive to contralateral sound. MOC neurons were sharply tuned to sound frequency with a well-defined characteristic frequency (CF). However, their labeled termination spans in the organ of Corti ranged from narrow to broad, innervating between 14 and 69 outer hair cells per axon in a "patchy" pattern. For units responsive to ipsilateral sound, the midpoint of innervation was mapped according to CF in a relationship generally similar to, but with more variability than, that of auditory-nerve fibers. Thus, based on CF mappings, most of the MOC terminations miss outer hair cells involved in the cochlear amplifier for their CF, which are located more basally. Compared with ipsilaterally responsive neurons, contralaterally responsive neurons had an apical offset in termination and a larger span of innervation (an average of 10.41% cochlear distance), suggesting that when contralateral sound activates the MOC reflex, the actions are different than those for ipsilateral sound. Copyright © 2014 the American Physiological Society.

Single-unit labeling of medial olivocochlear neurons: the cochlear frequency map for efferent axons

PubMed Central

2014-01-01

Medial olivocochlear (MOC) neurons are efferent neurons that project axons from the brain to the cochlea. Their action on outer hair cells reduces the gain of the “cochlear amplifier,” which shifts the dynamic range of hearing and reduces the effects of noise masking. The MOC effects in one ear can be elicited by sound in that ipsilateral ear or by sound in the contralateral ear. To study how MOC neurons project onto the cochlea to mediate these effects, single-unit labeling in guinea pigs was used to study the mapping of MOC neurons for neurons responsive to ipsilateral sound vs. those responsive to contralateral sound. MOC neurons were sharply tuned to sound frequency with a well-defined characteristic frequency (CF). However, their labeled termination spans in the organ of Corti ranged from narrow to broad, innervating between 14 and 69 outer hair cells per axon in a “patchy” pattern. For units responsive to ipsilateral sound, the midpoint of innervation was mapped according to CF in a relationship generally similar to, but with more variability than, that of auditory-nerve fibers. Thus, based on CF mappings, most of the MOC terminations miss outer hair cells involved in the cochlear amplifier for their CF, which are located more basally. Compared with ipsilaterally responsive neurons, contralaterally responsive neurons had an apical offset in termination and a larger span of innervation (an average of 10.41% cochlear distance), suggesting that when contralateral sound activates the MOC reflex, the actions are different than those for ipsilateral sound. PMID:24598524

Impact of cell size on inventory and mapping errors in a cellular geographic information system

NASA Technical Reports Server (NTRS)

Wehde, M. E. (Principal Investigator)

1979-01-01

The author has identified the following significant results. The effect of grid position was found insignificant for maps but highly significant for isolated mapping units. A modelable relationship between mapping error and cell size was observed for the map segment analyzed. Map data structure was also analyzed with an interboundary distance distribution approach. Map data structure and the impact of cell size on that structure were observed. The existence of a model allowing prediction of mapping error based on map structure was hypothesized and two generations of models were tested under simplifying assumptions.

Pleiotrophin Exerts Its Migration and Invasion Effect through the Neuropilin-1 Pathway

PubMed Central

Elahouel, Rania; Blanc, Charly; Carpentier, Gilles; Frechault, Sophie; Cascone, Ilaria; Destouches, Damien; Delbé, Jean; Courty, José; Hamma-Kourbali, Yamina

2015-01-01

Pleiotrophin (PTN) is a pleiotropic growth factor that exhibits angiogenic properties and is involved in tumor growth and metastasis. Although it has been shown that PTN is expressed in tumor cells, few studies have investigated its receptors and their involvement in cell migration and invasion. Neuropilin-1 (NRP-1) is a receptor for multiple growth factors that mediates cell motility and plays an important role in angiogenesis and tumor progression. Here we provide evidence for the first time that NRP-1 is crucial for biological activities of PTN. We found that PTN interacted directly with NRP-1 through its thrombospondin type-I repeat domains. Importantly, binding of PTN to NRP-1 stimulated the internalization and recycling of NRP-1 at the cell surface. Invalidation of NRP-1 by RNA interference in human carcinoma cells inhibited PTN-induced intracellular signaling of the serine-threonine kinase, mitogen-activated protein MAP kinase, and focal adhesion kinase pathways. Accordingly, NRP-1 silencing or blocking by antibody inhibited PTN-induced human umbilical vein endothelial cell migration and tumor cell invasion. These results suggest that NRP-1/PTN interaction provides a novel mechanism for controlling the response of endothelial and tumoral cells to PTN and may explain, at least in part, how PTN contributes to tumor angiogenesis and cancer progression. PMID:26408254

Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

PubMed Central

Celada, Lindsay J.; Whalen, Margaret M.

2013-01-01

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

From a 2DE-gel spot to protein function: lesson learned from HS1 in chronic lymphocytic leukemia.

PubMed

Apollonio, Benedetta; Bertilaccio, Maria Teresa Sabrina; Restuccia, Umberto; Ranghetti, Pamela; Barbaglio, Federica; Ghia, Paolo; Caligaris-Cappio, Federico; Scielzo, Cristina

2014-10-19

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease's biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as "spots" on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Hyperprolactinemia following chronic alcohol administration.

PubMed

Sarkar, Dipak K

2010-01-01

There are several reports showing evidence for the existence of high levels of prolactin (PRL) in alcoholic men and women. Alcohol-induced hyperprolactinemia has also been demonstrated in nonhuman primates and laboratory animals. Therefore, the clinical data as well as animal data suggest that ethanol consumption is a positive risk factor for hyperprolactinemia. In animal studies, it was found that chronic ethanol administration not only elevates plasma levels of PRL but also increases proliferation of pituitary lactotropes. Ethanol action on lactotropes involves crosstalk with estradiol-responsive signaling cascade or estradiol-regulated cell-cell communication. Additionally, it involves suppression of dopamine D2 receptors inhibition of G proteins and intracellular cyclic adenosine monophosphate (cAMP), modulation of transforming growth factor-beta (TGF-beta) isoforms and their receptors (TbetaRII), as well as factors secondary to TGF-beta actions, including production of beta-fibroblast growth factor (bFGF) from follicular-stellate cells. The downstream signaling that governs b-FGF production and secretion involves activation of the MAP kinase p44/42-dependent pathway. A coordinated suppression of D2 receptor- and TbetaRII receptor-mediated signaling as well as enhancement of bFGF activity might be critical for ethanol action on PRL production and cell proliferation in lactotropes. Copyright (c) 2010 S. Karger AG, Basel.

Genetic mapping of the human amphiphysin gene (AMPH) at 7p14-p13 excludes its involvement in retinitis pigmentosa 9 or dominant cystoid macular dystrophy

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

Yamamoto, R.; Kilimann, M.W.; Li, X.

1995-10-01

Amphiphysin is a protein concentrated in neuronal synapses and peripherally associated with synaptic vesicles. It is expressed in many neurons of the central and peripheral nervous systems and also in the adrenal medulla, the anterior and posterior pituitary, cell lines of the endocrine pancreas, and male germ cells. Its subcellular localization and tissue distribution indicate a potential involvement in mechanisms of regulated exocytosis. A role in the dynamic organization of the membrane-associated cytoskeleton is suggested by sequence similarity to the products of two yeast genes, RVS161 and RVS167. Mutation of either of these results in an abnormal actin distribution, disturbsmore » budding morphology, and impairs cell entry into stationary phase. Amphiphysin is the dominant autoantigen in paraneoplastic Stiff-Man syndrome, a neurological autoimmune disorder characterized by progressive rigidity of the body musculature with superimposed painful spasms. 9 refs., 1 fig., 1 tab.« less

The neuronal differentiation process involves a series of antioxidant proteins.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Hengstschläger, M; Pollak, A; Lubec, G

2005-11-01

Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP. 16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively. Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.

Cocaine induces astrocytosis through ER stress-mediated activation of autophagy

PubMed Central

Periyasamy, Palsamy; Guo, Ming-Lei; Buch, Shilpa

2016-01-01

ABSTRACT Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation. PMID:27337297

Cocaine induces astrocytosis through ER stress-mediated activation of autophagy.

PubMed

Periyasamy, Palsamy; Guo, Ming-Lei; Buch, Shilpa

2016-08-02

Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation.

Epitope mapping and evaluation of specificity of T-helper sites in four major antigenic peptides of chicken riboflavin carrier protein in outbred rats.

PubMed

Subramanian, Sarada; Andal, S; Karande, Anjali A; Radhakantha Adiga, P

2003-11-07

This paper reviews our studies on synthetic peptides spanning the major antigenic determinants of the chicken riboflavin carrier protein (RCP; 219 AA). These determinants are composed of residues 4-24 (YGC), 64-83 (CED), 130-147 (GEN), and 200-219 (HAC) and function as minivaccines in terms of eliciting anti-peptide antibodies which recognize the native protein and are particularly promising contraceptive vaccine candidates. We have used 15-residue synthetic peptides to define short sequences involved in interaction with antibody and with T-cells. We have mapped the boundaries of T-cell epitopes of these peptides in outbred rats by immunizing the animals with each peptide and assaying the popliteal lymph node cell proliferation against a series of overlapping synthetic 15-mers covering the entire length of the individual peptides. The peptides YGC, GEN, and HAC harboured a single T-cell epitope each whereas the peptide CED exhibited bimodal response possessing two epitopes, one at N-terminus and the other at the C-terminus. These studies provide insight into the way in which an immunogen is viewed by the immune system. In addition, preferential T-cell helper function for B cells recognizing unique determinants on the same molecule was demonstrated. This information helps in exploiting synthetic peptides in the construction of designer immunogens which have potential as candidate vaccines.

Cell type-selective disease-association of genes under high regulatory load.

PubMed

Galhardo, Mafalda; Berninger, Philipp; Nguyen, Thanh-Phuong; Sauter, Thomas; Sinkkonen, Lasse

2015-10-15

We previously showed that disease-linked metabolic genes are often under combinatorial regulation. Using the genome-wide ChIP-Seq binding profiles for 93 transcription factors in nine different cell lines, we show that genes under high regulatory load are significantly enriched for disease-association across cell types. We find that transcription factor load correlates with the enhancer load of the genes and thereby allows the identification of genes under high regulatory load by epigenomic mapping of active enhancers. Identification of the high enhancer load genes across 139 samples from 96 different cell and tissue types reveals a consistent enrichment for disease-associated genes in a cell type-selective manner. The underlying genes are not limited to super-enhancer genes and show several types of disease-association evidence beyond genetic variation (such as biomarkers). Interestingly, the high regulatory load genes are involved in more KEGG pathways than expected by chance, exhibit increased betweenness centrality in the interaction network of liver disease genes, and carry longer 3' UTRs with more microRNA (miRNA) binding sites than genes on average, suggesting a role as hubs integrating signals within regulatory networks. In summary, epigenetic mapping of active enhancers presents a promising and unbiased approach for identification of novel disease genes in a cell type-selective manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The time course of saccadic decision making: dynamic field theory.

PubMed

Wilimzig, Claudia; Schneider, Stefan; Schöner, Gregor

2006-10-01

Making a saccadic eye movement involves two decisions, the decision to initiate the saccade and the selection of the visual target of the saccade. Here we provide a theoretical account for the time-courses of these two processes, whose instabilities are the basis of decision making. We show how the cross-over from spatial averaging for fast saccades to selection for slow saccades arises from the balance between excitatory and inhibitory processes. Initiating a saccade involves overcoming fixation, as can be observed in the countermanding paradigm, which we model accounting both for the temporal evolution of the suppression probability and its dependence on fixation activity. The interaction between the two forms of decision making is demonstrated by predicting how the cross-over from averaging to selection depends on the fixation stimulus in gap-step-overlap paradigms. We discuss how the activation dynamics of our model may be mapped onto neuronal structures including the motor map and the fixation cells in superior colliculus.

The Global Regulatory Architecture of Transcription during the Caulobacter Cell Cycle

PubMed Central

Zhou, Bo; Schrader, Jared M.; Kalogeraki, Virginia S.; Abeliuk, Eduardo; Dinh, Cong B.; Pham, James Q.; Cui, Zhongying Z.; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5′ RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle. PMID:25569173

The global regulatory architecture of transcription during the Caulobacter cell cycle.

PubMed

Zhou, Bo; Schrader, Jared M; Kalogeraki, Virginia S; Abeliuk, Eduardo; Dinh, Cong B; Pham, James Q; Cui, Zhongying Z; Dill, David L; McAdams, Harley H; Shapiro, Lucy

2015-01-01

Each Caulobacter cell cycle involves differentiation and an asymmetric cell division driven by a cyclical regulatory circuit comprised of four transcription factors (TFs) and a DNA methyltransferase. Using a modified global 5' RACE protocol, we globally mapped transcription start sites (TSSs) at base-pair resolution, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Out of 2726 TSSs, 586 were shown to be cell cycle-regulated and we identified 529 binding sites for the cell cycle master regulators. Twenty-three percent of the cell cycle-regulated promoters were found to be under the combinatorial control of two or more of the global regulators. Previously unknown features of the core cell cycle circuit were identified, including 107 antisense TSSs which exhibit cell cycle-control, and 241 genes with multiple TSSs whose transcription levels often exhibited different cell cycle timing. Cumulatively, this study uncovered novel new layers of transcriptional regulation mediating the bacterial cell cycle.

N-acetylcysteine attenuates TNF-α-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells

PubMed Central

Hashimoto, Shu; Gon, Yasuhiro; Matsumoto, Ken; Takeshita, Ikuko; Horie, Takashi

2001-01-01

We have previously shown that tumour necrosis factor-α (TNF-α) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF-α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-α-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC-treated cells. NAC attenuated TNF-α-induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-α-stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury. PMID:11156586

N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells.

PubMed

Hashimoto, S; Gon, Y; Matsumoto, K; Takeshita, I; Horie, T

2001-01-01

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

DOE PAGES

Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; ...

2013-04-22

The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize themore » cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.« less

Direct Ca2+-dependent Heterophilic Interaction between Desmosomal Cadherins, Desmoglein and Desmocollin, Contributes to Cell–Cell Adhesion

PubMed Central

Chitaev, Nikolai A.; Troyanovsky, Sergey M.

1997-01-01

Human fibrosarcoma cells, HT-1080, feature extensive adherens junctions, lack mature desmosomes, and express a single known desmosomal protein, Desmoglein 2 (Dsg2). Transfection of these cells with bovine Desmocollin 1a (Dsc1a) caused dramatic changes in the subcellular distribution of endogenous Dsg2. Both cadherins clustered in the areas of the adherens junctions, whereas only a minor portion of Dsg2 was seen in these areas in the parental cells. Deletion mapping showed that intact extracellular cadherin-like repeats of Dsc1a (Arg1-Thr170) are required for the translocation of Dsg2. Deletion of the intracellular C-domain that mediates the interaction of Dsc1a with plakoglobin, or the CSI region that is involved in the binding to desmoplakin, had no effect. Coimmunoprecipitation experiments of cell lysates stably expressing Dsc1a with anti-Dsc or -Dsg antibodies demonstrate that the desmosomal cadherins, Dsg2 and Dsc1a, are involved in a direct Ca2+-dependent interaction. This conclusion was further supported by the results of solid phase binding experiments. These showed that the Dsc1a fragment containing cadherin-like repeats 1 and 2 binds directly to the extracellular portion of Dsg in a Ca2+-dependent manner. The contribution of the Dsg/ Dsc interaction to cell–cell adhesion was tested by coculturing HT-1080 cells expressing Dsc1a with HT-1080 cells lacking Dsc but expressing myc-tagged plakoglobin (MPg). In the latter cells, MPg and the endogenous Dsg form stable complexes. The observed specific coimmunoprecipitation of MPg by anti-Dsc antibodies in coculture indicates that an intercellular interaction between Dsc1 and Dsg is involved in cell–cell adhesion. PMID:9214392

A novel isoform of MAP4 organises the paraxial microtubule array required for muscle cell differentiation

PubMed Central

Mogessie, Binyam; Roth, Daniel; Rahil, Zainab; Straube, Anne

2015-01-01

The microtubule cytoskeleton is critical for muscle cell differentiation and undergoes reorganisation into an array of paraxial microtubules, which serves as template for contractile sarcomere formation. In this study, we identify a previously uncharacterised isoform of microtubule-associated protein MAP4, oMAP4, as a microtubule organising factor that is crucial for myogenesis. We show that oMAP4 is expressed upon muscle cell differentiation and is the only MAP4 isoform essential for normal progression of the myogenic differentiation programme. Depletion of oMAP4 impairs cell elongation and cell–cell fusion. Most notably, oMAP4 is required for paraxial microtubule organisation in muscle cells and prevents dynein- and kinesin-driven microtubule–microtubule sliding. Purified oMAP4 aligns dynamic microtubules into antiparallel bundles that withstand motor forces in vitro. We propose a model in which the cooperation of dynein-mediated microtubule transport and oMAP4-mediated zippering of microtubules drives formation of a paraxial microtubule array that provides critical support for the polarisation and elongation of myotubes. DOI: http://dx.doi.org/10.7554/eLife.05697.001 PMID:25898002

The Genetics of Chemoreception in the Labella and Tarsi of Aedes aegypti

DTIC Science & Technology

2014-01-01

molecular pathway also involved in DEET perception in the dipteran relative Drosophila melanogaster (Ditzen et al., 2008; DeGennaro et al., 2013). Transgenic...Downloads/). Output Fastq Illumina files were map- ped to the reference genome with TopHat (Trapnell et al., 2009). The unambiguous sequence alignment...Q., Pikielny, C.W., 2002. Novel genes expressing in subsets of chemosensory sensilla on the front legs of male Drosophila melanogaster . Cell. Tissue

Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

PubMed

Savopoulos, John W; Dowd, Stephen; Armour, Carolyn; Carter, Paul S; Greenwood, Catherine J; Mills, David; Powell, David; Pettman, Gary R; Jenkins, Owen; Walsh, Frank S; Philpott, Karen L

2002-02-01

The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays. Copyright 2002 Elsevier Science (USA).

Genome-wide chromatin state transitions associated with developmental and environmental cues.

PubMed

Zhu, Jiang; Adli, Mazhar; Zou, James Y; Verstappen, Griet; Coyne, Michael; Zhang, Xiaolan; Durham, Timothy; Miri, Mohammad; Deshpande, Vikram; De Jager, Philip L; Bennett, David A; Houmard, Joseph A; Muoio, Deborah M; Onder, Tamer T; Camahort, Ray; Cowan, Chad A; Meissner, Alexander; Epstein, Charles B; Shoresh, Noam; Bernstein, Bradley E

2013-01-31

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming. Copyright © 2013 Elsevier Inc. All rights reserved.

Development and validation of a whole-cell inhibition assay for bacterial methionine aminopeptidase by surface-enhanced laser desorption ionization-time of flight mass spectrometry.

PubMed

Greis, Kenneth D; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-08-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP.

Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

PubMed

Huang, Shuaishuai; He, Zhangjiang; Zhang, Shiwei; Keyhani, Nemat O; Song, Yulin; Yang, Zhi; Jiang, Yahui; Zhang, Wenli; Pei, Yan; Zhang, Yongjun

2015-10-01

The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFβ1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFβ1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

Neurotensin-induced proinflammatory signaling in human colonocytes is regulated by β-arrestins and endothelin-converting enzyme-1-dependent endocytosis and resensitization of neurotensin receptor 1.

PubMed

Law, Ivy Ka Man; Murphy, Jane E; Bakirtzi, Kyriaki; Bunnett, Nigel W; Pothoulakis, Charalabos

2012-04-27

The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of βARR1 or βARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves βARRs and ECE-1, respectively. Our results also indicate that βARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT.

Pneumolysin-induced CXCL8 production by nasopharyngeal epithelial cells is dependent on calcium flux and MAPK activation via Toll-like receptor 4.

PubMed

Dogan, Semih; Zhang, Qibo; Pridmore, Alison C; Mitchell, Timothy J; Finn, Adam; Murdoch, Craig

2011-01-01

The natural niche of Streptococcus pneumoniae is the nasopharyngeal mucosa and nasopharyngeal carriage of pneumococci is widely prevalent. Pneumolysin (Ply), a pore-forming protein produced by S. pneumonia, may be important in driving the innate immune response of the nasopharynx. We studied the Ply-induced production of CXCL8 by nasopharyngeal cells and further analysed the mechanism of this induction. Detroit nasopharyngeal cells were stimulated with supernatants derived from bacterial cultures of Ply-deficient, wild-type pneumococci and recombinant Ply, and CXCL8 measured by ELISA. The role of MAP kinase family members in Ply-induced CXCL8 production was analysed using specific inhibitors, NF-κB activity was measured by immunoblot and Ply-mediated TLR4 activation analysed by a CXCL8 promotor luciferase assay. Ply significantly increased production of CXCL8 in Detroit and primary nasal cells. Flow cytometric analysis showed that Detroit cells express cell surface TLR4. CXCL8 production was dependent on changes in the intracellular Ca(2+) levels and also by NF-κB via activation of TLR4, and MAP kinase signalling. Ply induces production of CXCL8 by nasopharyngeal cells using signalling mechanisms involving Ca(2+) mobilisation and activation of MAPK and NF-κB via TLR4. This may be important in regulating nasopharyngeal immunity against pneumococcal colonization. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy.

PubMed

Lan, Xiaoyang; Jörg, David J; Cavalli, Florence M G; Richards, Laura M; Nguyen, Long V; Vanner, Robert J; Guilhamon, Paul; Lee, Lilian; Kushida, Michelle M; Pellacani, Davide; Park, Nicole I; Coutinho, Fiona J; Whetstone, Heather; Selvadurai, Hayden J; Che, Clare; Luu, Betty; Carles, Annaick; Moksa, Michelle; Rastegar, Naghmeh; Head, Renee; Dolma, Sonam; Prinos, Panagiotis; Cusimano, Michael D; Das, Sunit; Bernstein, Mark; Arrowsmith, Cheryl H; Mungall, Andrew J; Moore, Richard A; Ma, Yussanne; Gallo, Marco; Lupien, Mathieu; Pugh, Trevor J; Taylor, Michael D; Hirst, Martin; Eaves, Connie J; Simons, Benjamin D; Dirks, Peter B

2017-09-14

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Comprehensive QTL mapping survey dissects the complex fruit texture physiology in apple (Malus x domestica Borkh.).

PubMed

Longhi, Sara; Moretto, Marco; Viola, Roberto; Velasco, Riccardo; Costa, Fabrizio

2012-02-01

Fruit ripening is a complex physiological process in plants whereby cell wall programmed changes occur mainly to promote seed dispersal. Cell wall modification also directly regulates the textural properties, a fundamental aspect of fruit quality. In this study, two full-sib populations of apple, with 'Fuji' as the common maternal parent, crossed with 'Delearly' and 'Pink Lady', were used to understand the control of fruit texture by QTL mapping and in silico gene mining. Texture was dissected with a novel high resolution phenomics strategy, simultaneously profiling both mechanical and acoustic fruit texture components. In 'Fuji × Delearly' nine linkage groups were associated with QTLs accounting from 15.6% to 49% of the total variance, and a highly significant QTL cluster for both textural components was mapped on chromosome 10 and co-located with Md-PG1, a polygalacturonase gene that, in apple, is known to be involved in cell wall metabolism processes. In addition, other candidate genes related to Md-NOR and Md-RIN transcription factors, Md-Pel (pectate lyase), and Md-ACS1 were mapped within statistical intervals. In 'Fuji × Pink Lady', a smaller set of linkage groups associated with the QTLs identified for fruit texture (15.9-34.6% variance) was observed. The analysis of the phenotypic variance over a two-dimensional PCA plot highlighted a transgressive segregation for this progeny, revealing two QTL sets distinctively related to both mechanical and acoustic texture components. The mining of the apple genome allowed the discovery of the gene inventory underlying each QTL, and functional profile assessment unravelled specific gene expression patterns of these candidate genes.

The emergence of optical elastography in biomedicine

NASA Astrophysics Data System (ADS)

Kennedy, Brendan F.; Wijesinghe, Philip; Sampson, David D.

2017-04-01

Optical elastography, the use of optics to characterize and map the mechanical properties of biological tissue, involves measuring the deformation of tissue in response to a load. Such measurements may be used to form an image of a mechanical property, often elastic modulus, with the resulting mechanical contrast complementary to the more familiar optical contrast. Optical elastography is experiencing new impetus in response to developments in the closely related fields of cell mechanics and medical imaging, aided by advances in photonics technology, and through probing the microscale between that of cells and whole tissues. Two techniques -- optical coherence elastography and Brillouin microscopy -- have recently shown particular promise for medical applications, such as in ophthalmology and oncology, and as new techniques in cell mechanics.

Integrating Structure to Protein-Protein Interaction Networks That Drive Metastasis to Brain and Lung in Breast Cancer

PubMed Central

Engin, H. Billur; Guney, Emre; Keskin, Ozlem; Oliva, Baldo; Gursoy, Attila

2013-01-01

Blocking specific protein interactions can lead to human diseases. Accordingly, protein interactions and the structural knowledge on interacting surfaces of proteins (interfaces) have an important role in predicting the genotype-phenotype relationship. We have built the phenotype specific sub-networks of protein-protein interactions (PPIs) involving the relevant genes responsible for lung and brain metastasis from primary tumor in breast cancer. First, we selected the PPIs most relevant to metastasis causing genes (seed genes), by using the “guilt-by-association” principle. Then, we modeled structures of the interactions whose complex forms are not available in Protein Databank (PDB). Finally, we mapped mutations to interface structures (real and modeled), in order to spot the interactions that might be manipulated by these mutations. Functional analyses performed on these sub-networks revealed the potential relationship between immune system-infectious diseases and lung metastasis progression, but this connection was not observed significantly in the brain metastasis. Besides, structural analyses showed that some PPI interfaces in both metastasis sub-networks are originating from microbial proteins, which in turn were mostly related with cell adhesion. Cell adhesion is a key mechanism in metastasis, therefore these PPIs may be involved in similar molecular pathways that are shared by infectious disease and metastasis. Finally, by mapping the mutations and amino acid variations on the interface regions of the proteins in the metastasis sub-networks we found evidence for some mutations to be involved in the mechanisms differentiating the type of the metastasis. PMID:24278371

Mouse chromosomal mapping of a murine leukemia virus integration region (Mis-1) first identified in rat thymic leukemia.

PubMed Central

Jolicoeur, P; Villeneuve, L; Rassart, E; Kozak, C

1985-01-01

We have previously identified a region of genomic DNA which constitutes the site of frequent provirus integration in rat thymomas induced by Moloney murine leukemia virus (Lemay and Jolicoeur, Proc. Natl. Acad. Sci. USA 81:38-42, 1984). This genetic locus is now designated Mis-1 (Moloney integration site). Cellular sequences homologous to Mis-1 are present in mouse DNA. Using a series of hamster-mouse somatic cell hybrids, we mapped the Mis-1 locus to mouse chromosome 15. Frequent chromosome 15 aberrations have been described in mouse thymomas. Mis-1 represents a putative new oncogene which might be involved in the initiation or maintenance or both of these neoplasms. Images PMID:4068142

Dual fiber microprobe for mapping elemental distributions in biological cells

DOEpatents

Martin, Rodger C [Powell, TN; Martin, Madhavi Z [Powell, TN

2007-07-31

Laser-induced breakdown spectroscopy (LIBS) is applied on a microscale for in situ elemental analysis and spatial mapping in biological cells. A high power laser beam is focused onto a cell surface using a dual branching optical fiber probe for optical excitation of the cell constituents. Dual spectrometers and ICCD detectors capture the emission spectra from the excited cell(s). Repeated probing or repositioning of the laser beam with respect to the cell can provide 2-D or 3-D mapping of the cell.

A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

PubMed Central

Fritsch, Emilie S.; Chabbert, Christophe D.; Klaus, Bernd; Steinmetz, Lars M.

2014-01-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance. PMID:25081569

A genome-wide map of mitochondrial DNA recombination in yeast.

PubMed

Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M

2014-10-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance. Copyright © 2014 by the Genetics Society of America.

[INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

PubMed

Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

2015-01-01

To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only phospho-ERK2 could be detected after 60 min of endocytosis. In both cases, MAP-kinase activation dynamics was significantly different from the control. Our results suggest involvement of EGF-stimulated MAP-kinase pathway in cytoskeleton regulation. At the same time, they demonstrate that the two studied and widely used inhibitors are not equivalent with respect to not only the effect on MAP-kinase activity but also to such interdependent processes such as changes in cytoskeleton organization and signaling receptor' endocytosis.

GSTM3 and GSTP1: novel players driving tumor progression in cervical cancer.

PubMed

Checa-Rojas, Alberto; Delgadillo-Silva, Luis Fernando; Velasco-Herrera, Martín Del Castillo; Andrade-Domínguez, Andrés; Gil, Jeovanis; Santillán, Orlando; Lozano, Luis; Toledo-Leyva, Alfredo; Ramírez-Torres, Alberto; Talamas-Rohana, Patricia; Encarnación-Guevara, Sergio

2018-04-24

The molecular processes and proteomic markers leading to tumor progression (TP) in cervical cancer (CC) are either unknown or only partially understood. TP affects metabolic and regulatory mechanisms that can be identified as proteomic changes. To identify which proteins are differentially expressed and to understand the mechanisms of cancer progression, we analyzed the dynamics of the tumor proteome in CC cell lines. This analysis revealed two proteins that are up-regulated during TP, GSTM3 and GSTP1. These proteins are involved in cell maintenance, cell survival and the cellular stress response via the NF-κB and MAP kinase pathways during TP. Furthermore, GSTM3 and GSTP1 knockdown showed that evasion of apoptosis was affected, and tumor proliferation was significantly reduced. Our data indicate the critical role of GST proteins in the regulation and progression of cervical cancer cells. Hence, we suggest GSTM3 and GSTP1 as novel biomarkers and potential therapeutic targets for treating cervical cancer. CC is particularly hazardous in the advanced stages, and there are few therapeutic strategies specifically targeting these stages. We performed analyses on CC tumor proteome dynamics and identified GSTM3 and GSTP1 as novel potential therapeutic targets. Knockdown of these proteins showed that they are involved in cell survival, cell proliferation and cellular evasion of apoptosis.

T cells are influenced by a long non-coding RNA in the autoimmune associated PTPN2 locus.

PubMed

Houtman, Miranda; Shchetynsky, Klementy; Chemin, Karine; Hensvold, Aase Haj; Ramsköld, Daniel; Tandre, Karolina; Eloranta, Maija-Leena; Rönnblom, Lars; Uebe, Steffen; Catrina, Anca Irinel; Malmström, Vivianne; Padyukov, Leonid

2018-06-01

Non-coding SNPs in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) locus have been linked with several autoimmune diseases, including rheumatoid arthritis, type I diabetes, and inflammatory bowel disease. However, the functional consequences of these SNPs are poorly characterized. Herein, we show in blood cells that SNPs in the PTPN2 locus are highly correlated with DNA methylation levels at four CpG sites downstream of PTPN2 and expression levels of the long non-coding RNA (lncRNA) LINC01882 downstream of these CpG sites. We observed that LINC01882 is mainly expressed in T cells and that anti-CD3/CD28 activated naïve CD4 + T cells downregulate the expression of LINC01882. RNA sequencing analysis of LINC01882 knockdown in Jurkat T cells, using a combination of antisense oligonucleotides and RNA interference, revealed the upregulation of the transcription factor ZEB1 and kinase MAP2K4, both involved in IL-2 regulation. Overall, our data suggests the involvement of LINC01882 in T cell activation and hints towards an auxiliary role of these non-coding SNPs in autoimmunity associated with the PTPN2 locus. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

2',5'-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation.

PubMed

Lee, Yi-Nan; Yeh, Hung-I; Tian, Tin-Yi; Lu, Wen-Wei; Ko, Yu-Shien; Tsai, Cheng-Ho

2002-09-30

We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.

A Public Involvement Road Map

DOT National Transportation Integrated Search

1998-09-16

In order to have effective public involvement, governments need a road map for : the decision-making process. Yet, citizens from small and medium sized cities : frequently do not have the resources to use sophisticated technology for public : involve...

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

PubMed

Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

2017-07-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Pleiotrophin exerts its migration and invasion effect through the neuropilin-1 pathway.

PubMed

Elahouel, Rania; Blanc, Charly; Carpentier, Gilles; Frechault, Sophie; Cascone, Ilaria; Destouches, Damien; Delbé, Jean; Courty, José; Hamma-Kourbali, Yamina

2015-08-01

Pleiotrophin (PTN) is a pleiotropic growth factor that exhibits angiogenic properties and is involved in tumor growth and metastasis. Although it has been shown that PTN is expressed in tumor cells, few studies have investigated its receptors and their involvement in cell migration and invasion. Neuropilin-1 (NRP-1) is a receptor for multiple growth factors that mediates cell motility and plays an important role in angiogenesis and tumor progression. Here we provide evidence for the first time that NRP-1 is crucial for biological activities of PTN. We found that PTN interacted directly with NRP-1 through its thrombospondin type-I repeat domains. Importantly, binding of PTN to NRP-1 stimulated the internalization and recycling of NRP-1 at the cell surface. Invalidation of NRP-1 by RNA interference in human carcinoma cells inhibited PTN-induced intracellular signaling of the serine-threonine kinase, mitogen-activated protein MAP kinase, and focal adhesion kinase pathways. Accordingly, NRP-1 silencing or blocking by antibody inhibited PTN-induced human umbilical vein endothelial cell migration and tumor cell invasion. These results suggest that NRP-1/PTN interaction provides a novel mechanism for controlling the response of endothelial and tumoral cells to PTN and may explain, at least in part, how PTN contributes to tumor angiogenesis and cancer progression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

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

Li, Mi; University of Chinese Academy of Sciences, Beijing 100049; Liu, Lianqing, E-mail: lqliu@sia.cn

Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measuremore » the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.« less

Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Highly Connected Viral Component

PubMed Central

Bouraï, Mehdi; Lucas-Hourani, Marianne; Gad, Hans Henrik; Drosten, Christian; Jacob, Yves; Tafforeau, Lionel; Cassonnet, Patricia; Jones, Louis M.; Judith, Delphine; Couderc, Thérèse; Lecuit, Marc; André, Patrice; Kümmerer, Beate Mareike; Lotteau, Vincent; Desprès, Philippe; Vidalain, Pierre-Olivier

2012-01-01

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus. PMID:22258240

Mobile mapping of sporting event spectators using bluetooth sensors: tour of flanders 2011.

PubMed

Versichele, Mathias; Neutens, Tijs; Goudeseune, Stephanie; van Bossche, Frederik; van de Weghe, Nico

2012-10-22

Accurate spatiotemporal information on crowds is a necessity for a better management in general and for the mitigation of potential security risks. The large numbers of individuals involved and their mobility, however, make generation of this information non-trivial. This paper proposes a novel methodology to estimate and map crowd sizes using mobile Bluetooth sensors and examines to what extent this methodology represents a valuable alternative to existing traditional crowd density estimation methods. The proposed methodology is applied in a unique case study that uses Bluetooth technology for the mobile mapping of spectators of the Tour of Flanders 2011 road cycling race. The locations of nearly 16,000 cell phones of spectators along the race course were registered and detailed views of the spatiotemporal distribution of the crowd were generated. Comparison with visual head counts from camera footage delivered a detection ratio of 13.0 ± 2.3%, making it possible to estimate the crowd size. To our knowledge, this is the first study that uses mobile Bluetooth sensors to count and map a crowd over space and time.

Mobile Mapping of Sporting Event Spectators Using Bluetooth Sensors: Tour of Flanders 2011

PubMed Central

Versichele, Mathias; Neutens, Tijs; Goudeseune, Stephanie; van Bossche, Frederik; van de Weghe, Nico

2012-01-01

Accurate spatiotemporal information on crowds is a necessity for a better management in general and for the mitigation of potential security risks. The large numbers of individuals involved and their mobility, however, make generation of this information non-trivial. This paper proposes a novel methodology to estimate and map crowd sizes using mobile Bluetooth sensors and examines to what extent this methodology represents a valuable alternative to existing traditional crowd density estimation methods. The proposed methodology is applied in a unique case study that uses Bluetooth technology for the mobile mapping of spectators of the Tour of Flanders 2011 road cycling race. The locations of nearly 16,000 cell phones of spectators along the race course were registered and detailed views of the spatiotemporal distribution of the crowd were generated. Comparison with visual head counts from camera footage delivered a detection ratio of 13.0 ± 2.3%, making it possible to estimate the crowd size. To our knowledge, this is the first study that uses mobile Bluetooth sensors to count and map a crowd over space and time. PMID:23202044

cChIP-seq: a robust small-scale method for investigation of histone modifications.

PubMed

Valensisi, Cristina; Liao, Jo Ling; Andrus, Colin; Battle, Stephanie L; Hawkins, R David

2015-12-21

ChIP-seq is highly utilized for mapping histone modifications that are informative about gene regulation and genome annotations. For example, applying ChIP-seq to histone modifications such as H3K4me1 has facilitated generating epigenomic maps of putative enhancers. This powerful technology, however, is limited in its application by the large number of cells required. ChIP-seq involves extensive manipulation of sample material and multiple reactions with limited quality control at each step, therefore, scaling down the number of cells required has proven challenging. Recently, several methods have been proposed to overcome this limit but most of these methods require extensive optimization to tailor the protocol to the specific antibody used or number of cells being profiled. Here we describe a robust, yet facile method, which we named carrier ChIP-seq (cChIP-seq), for use on limited cell amounts. cChIP-seq employs a DNA-free histone carrier in order to maintain the working ChIP reaction scale, removing the need to tailor reactions to specific amounts of cells or histone modifications to be assayed. We have applied our method to three different histone modifications, H3K4me3, H3K4me1 and H3K27me3 in the K562 cell line, and H3K4me1 in H1 hESCs. We successfully obtained epigenomic maps for these histone modifications starting with as few as 10,000 cells. We compared cChIP-seq data to data generated as part of the ENCODE project. ENCODE data are the reference standard in the field and have been generated starting from tens of million of cells. Our results show that cChIP-seq successfully recapitulates bulk data. Furthermore, we showed that the differences observed between small-scale ChIP-seq data and ENCODE data are largely to be due to lab-to-lab variability rather than operating on a reduced scale. Data generated using cChIP-seq are equivalent to reference epigenomic maps from three orders of magnitude more cells. Our method offers a robust and straightforward approach to scale down ChIP-seq to as low as 10,000 cells. The underlying principle of our strategy makes it suitable for being applied to a vast range of chromatin modifications without requiring expensive optimization. Furthermore, our strategy of a DNA-free carrier can be adapted to most ChIP-seq protocols.

STATs shape the active enhancer landscape of T cell populations.

PubMed

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-Wei; Sartorelli, Vittorio; Kanno, Yuka; O'Shea, John J

2012-11-21

Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. Although enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4(+) T cells as a model of differentiation, mapping the activity of cell-type-specific enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. Copyright © 2012 Elsevier Inc. All rights reserved.

STATs Shape the Active Enhancer Landscape of T Cell Populations

PubMed Central

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-wei; Sartorelli, Vittorio; Kanno, Yuka; O’Shea, John J.

2012-01-01

SUMMARY Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. While enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4+ T cells as a model of differentiation, mapping the acquisition of cell-type-specific enhancer elements in T-helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the acquisition of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. PMID:23178119

Synchrotron radiation induced X-ray emission studies of the antioxidant mechanism of the organoselenium drug ebselen.

PubMed

Aitken, Jade B; Lay, Peter A; Duong, T T Hong; Aran, Roshanak; Witting, Paul K; Harris, Hugh H; Lai, Barry; Vogt, Stefan; Giles, Gregory I

2012-04-01

Synchrotron radiation induced X-ray emission (SRIXE) spectroscopy was used to map the cellular uptake of the organoselenium-based antioxidant drug ebselen using differentiated ND15 cells as a neuronal model. The cellular SRIXE spectra, acquired using a hard X-ray microprobe beam (12.8-keV), showed a large enhancement of fluorescence at the K(α) line for Se (11.2-keV) following treatment with ebselen (10 μM) at time periods from 60 to 240 min. Drug uptake was quantified and ebselen was shown to induce time-dependent changes in cellular elemental content that were characteristic of oxidative stress with the efflux of K, Cl, and Ca species. The SRIXE cellular Se distribution map revealed that ebselen was predominantly localized to a discreet region of the cell which, by comparison with the K and P elemental maps, is postulated to correspond to the endoplasmic reticulum. On the basis of these findings, it is hypothesized that a major outcome of ebselen redox catalysis is the induction of cellular stress. A mechanism of action of ebselen is proposed that involves the cell responding to drug-induced stress by increasing the expression of antioxidant genes. This hypothesis is supported by the observation that ebselen also regulated the homeostasis of the transition metals Mn, Cu, Fe, and Zn, with increases in transition metal uptake paralleling known induction times for the expression of antioxidant metalloenzymes. © SBIC 2012

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells.

PubMed

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-11-07

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK -shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK -shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3'- and 5'-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein.

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

PubMed Central

Fiume, Giuseppe; Scialdone, Annarita; Rizzo, Francesca; De Filippo, Maria Rosaria; Laudanna, Carmelo; Albano, Francesco; Golino, Gaetanina; Vecchio, Eleonora; Pontoriero, Marilena; Mimmi, Selena; Ceglia, Simona; Pisano, Antonio; Iaccino, Enrico; Palmieri, Camillo; Paduano, Sergio; Viglietto, Giuseppe; Weisz, Alessandro; Scala, Giuseppe; Quinto, Ileana

2016-01-01

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein. PMID:27827994

Local delivery of molecules from a nanopipette for quantitative receptor mapping on live cells.

PubMed

Babakinejad, Babak; Jönsson, Peter; López Córdoba, Ainara; Actis, Paolo; Novak, Pavel; Takahashi, Yasufumi; Shevchuk, Andrew; Anand, Uma; Anand, Praveen; Drews, Anna; Ferrer-Montiel, Antonio; Klenerman, David; Korchev, Yuri E

2013-10-01

Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level of single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nanopipette, the magnitude of the applied pressure or voltage, which drives the delivery, and the distance to the underlying surface influences the number and spatial distribution of the delivered molecules. Analytical expressions describing the delivery are derived and compared with the results from finite element simulations and experiments on delivery from a 100 nm nanopipette in bulk solution and to the surface of sensory neurons. We then developed a setup for rapid and quantitative delivery to multiple subcellular areas, delivering the molecule capsaicin to stimulate opening of Transient Receptor Potential Vanilloid subfamily member 1 (TRPV1) channels, membrane receptors involved in pain sensation. Overall, precise and quantitative delivery of molecules from nanopipettes has been demonstrated, opening up many applications in biology such as locally stimulating and mapping receptors on the surface of live cells.

The transition between immune and disease states in a cellular automaton model of clonal immune response

NASA Astrophysics Data System (ADS)

Bezzi, Michele; Celada, Franco; Ruffo, Stefano; Seiden, Philip E.

1997-02-01

In this paper we extend the Celada-Seiden (CS) model of the humoral immune response to include infections virus and killer T cells (cellular response). The model represents molecules and cells with bitstrings. The response of the system to virus involves a competition between the ability of the virus to kill the host cells and the host's ability to eliminate the virus. We find two basins of attraction in the dynamics of this system, one is identified with disease and the other with the immune state. There is also an oscillating state that exists on the border of these two stable states. Fluctuations in the population of virus or antibody can end the oscillation and drive the system into one of the stable states. The introduction of mechanisms of cross-regulation between the two responses can bias the system towards one of them. We also study a mean field model, based on coupled maps, to investigate virus-like infections. This simple model reproduces the attractors for average populations observed in the cellular automaton. All the dynamical behavior connected to spatial extension is lost, as is the oscillating feature. Thus the mean field approximation introduced with coupled maps destroys oscillations.

Inhibition of beta-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules.

PubMed

Cheng, Guangmao; Qiao, Fei; Gallien, Thomas N; Kuppuswamy, Dhandapani; Cooper, George

2005-03-01

Decreased beta-adrenergic receptor (beta-AR) number occurs both in animal models of cardiac hypertrophy and failure and in patients. beta-AR recycling is an important mechanism for the beta-AR resensitization that maintains a normal complement of cell surface beta-ARs. We have shown that 1) in severe pressure overload cardiac hypertrophy, there is extensive microtubule-associated protein 4 (MAP4) decoration of a dense microtubule network; and 2) MAP4 microtubule decoration inhibits muscarinic acetylcholine receptor recycling in neuroblastoma cells. We asked here whether MAP4 microtubule decoration inhibits beta-AR recycling in adult cardiocytes. [(3)H]CGP-12177 was used as a beta-AR ligand, and feline cardiocytes were isolated and infected with adenovirus containing MAP4 (AdMAP4) or beta-galactosidase (Adbeta-gal) cDNA. MAP4 decorated the microtubules extensively only in AdMAP4 cardiocytes. beta-AR agonist exposure reduced cell surface beta-AR number comparably in AdMAP4 and Adbeta-gal cardiocytes; however, after agonist withdrawal, the cell surface beta-AR number recovered to 78.4 +/- 2.9% of the pretreatment value in Adbeta-gal cardiocytes but only to 56.8 +/- 1.4% in AdMAP4 cardiocytes (P < 0.01). This result was confirmed in cardiocytes isolated from transgenic mice having cardiac-restricted MAP4 overexpression. In functional terms of cAMP generation, beta-AR agonist responsiveness of AdMAP4 cells was 47% less than that of Adbeta-gal cells. We conclude that MAP4 microtubule decoration interferes with beta-AR recycling and that this may be one mechanism for beta-AR downregulation in heart failure.

Numerical prediction of algae cell mixing feature in raceway ponds using particle tracing methods.

PubMed

Ali, Haider; Cheema, Taqi A; Yoon, Ho-Sung; Do, Younghae; Park, Cheol W

2015-02-01

In the present study, a novel technique, which involves numerical computation of the mixing length of algae particles in raceway ponds, was used to evaluate the mixing process. A value of mixing length that is higher than the maximum streamwise distance (MSD) of algae cells indicates that the cells experienced an adequate turbulent mixing in the pond. A coupling methodology was adapted to map the pulsating effects of a 2D paddle wheel on a 3D raceway pond in this study. The turbulent mixing was examined based on the computations of mixing length, residence time, and algae cell distribution in the pond. The results revealed that the use of particle tracing methodology is an improved approach to define the mixing phenomenon more effectively. Moreover, the algae cell distribution aided in identifying the degree of mixing in terms of mixing length and residence time. © 2014 Wiley Periodicals, Inc.

MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/β-catenin signaling pathway, and enhancing Smac expression

PubMed Central

Jiang, Yilin; Miao, Junjie; Wang, Dongliang; Zhou, Jingru; Liu, Bo; Jiao, Feng; Liang, Jiangfeng; Wang, Yangshuo; Fan, Cungang; Zhang, Qingjun

2018-01-01

Significant antitumor activity of Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) purified from Momordica charantia has been the subject of previous research. However, the effective mechanism of MAP30 on malignant glioma cells has not yet been clarified. The aim of the present study was to investigate the effects and mechanism of MAP30 on U87 and U251 cell lines. A Cell Counting Kit-8 assay, wound healing assay and Transwell assay were used to detect the effects on U87 and U251 cells treated with different concentrations of MAP30 (0.5, 1, 2, 4, 8 and 16 µM) over different periods of time. Proliferation, migration and invasion of each cell line were markedly inhibited by MAP30 in a dose- and time-dependent manner. Flow cytometry and fluorescence staining demonstrated that apoptosis increased and the cell cycle was arrested in S-phase in the two investigated cell lines following MAP30 treatment. Western blot analysis demonstrated that leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) expression and key proteins in the Wnt/β-catenin signaling pathway were apparently decreased, whereas second mitochondria-derived activator of caspase (Smac) protein expression significantly increased with MAP30 treatment in the same manner. These results suggest that MAP30 markedly induces apoptosis in U87 and U251 cell lines by suppressing LGR5 and the Wnt/β-catenin signaling pathway, and enhancing Smac expression in a dose- and time-dependent manner. PMID:29556310

The transcription factor Rap1p is required for tolerance to cell-wall perturbing agents and for cell-wall maintenance in Saccharomyces cerevisiae.

PubMed

Azad, Gajendra Kumar; Singh, Vikash; Baranwal, Shivani; Thakare, Mayur Jankiram; Tomar, Raghuvir S

2015-01-02

Yeast repressor activator protein (Rap1p) is involved in genomic stability and transcriptional regulation. We explored the function of Rap1p in yeast physiology using Rap1p truncation mutants. Our results revealed that the N-terminal truncation of Rap1p (Rap1ΔN) leads to hypersensitivity towards elevated temperature and cell-wall perturbing agents. Cell wall analysis showed an increase in the chitin and glucan content in Rap1ΔN cells as compared with wild type cells. Accordingly, mutant cells had a twofold thicker cell wall, as observed by electron microscopy. Furthermore, Rap1ΔN cells had increased levels of phosphorylated Slt2p, a MAP kinase of the cell wall integrity pathway. Mutant cells also had elevated levels of cell wall integrity response transcripts. Taken together, our findings suggest a connection between Rap1p and cell wall homeostasis. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility

PubMed Central

Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

2014-01-01

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4+ T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis. PMID:24997565

Epigenomic analysis of primary human T cells reveals enhancers associated with TH2 memory cell differentiation and asthma susceptibility.

PubMed

Seumois, Grégory; Chavez, Lukas; Gerasimova, Anna; Lienhard, Matthias; Omran, Nada; Kalinke, Lukas; Vedanayagam, Maria; Ganesan, Asha Purnima V; Chawla, Ashu; Djukanović, Ratko; Ansel, K Mark; Peters, Bjoern; Rao, Anjana; Vijayanand, Pandurangan

2014-08-01

A characteristic feature of asthma is the aberrant accumulation, differentiation or function of memory CD4(+) T cells that produce type 2 cytokines (TH2 cells). By mapping genome-wide histone modification profiles for subsets of T cells isolated from peripheral blood of healthy and asthmatic individuals, we identified enhancers with known and potential roles in the normal differentiation of human TH1 cells and TH2 cells. We discovered disease-specific enhancers in T cells that differ between healthy and asthmatic individuals. Enhancers that gained the histone H3 Lys4 dimethyl (H3K4me2) mark during TH2 cell development showed the highest enrichment for asthma-associated single nucleotide polymorphisms (SNPs), which supported a pathogenic role for TH2 cells in asthma. In silico analysis of cell-specific enhancers revealed transcription factors, microRNAs and genes potentially linked to human TH2 cell differentiation. Our results establish the feasibility and utility of enhancer profiling in well-defined populations of specialized cell types involved in disease pathogenesis.

Nitric Oxide and Protein S-Nitrosylation Are Integral to Hydrogen Peroxide-Induced Leaf Cell Death in Rice1[W][OA

PubMed Central

Lin, Aihong; Wang, Yiqin; Tang, Jiuyou; Xue, Peng; Li, Chunlai; Liu, Linchuan; Hu, Bin; Yang, Fuquan; Loake, Gary J.; Chu, Chengcai

2012-01-01

Nitric oxide (NO) is a key redox-active, small molecule involved in various aspects of plant growth and development. Here, we report the identification of an NO accumulation mutant, nitric oxide excess1 (noe1), in rice (Oryza sativa), the isolation of the corresponding gene, and the analysis of its role in NO-mediated leaf cell death. Map-based cloning revealed that NOE1 encoded a rice catalase, OsCATC. Furthermore, noe1 resulted in an increase of hydrogen peroxide (H2O2) in the leaves, which consequently promoted NO production via the activation of nitrate reductase. The removal of excess NO reduced cell death in both leaves and suspension cultures derived from noe1 plants, implicating NO as an important endogenous mediator of H2O2-induced leaf cell death. Reduction of intracellular S-nitrosothiol (SNO) levels, generated by overexpression of rice S-nitrosoglutathione reductase gene (GSNOR1), which regulates global levels of protein S-nitrosylation, alleviated leaf cell death in noe1 plants. Thus, S-nitrosylation was also involved in light-dependent leaf cell death in noe1. Utilizing the biotin-switch assay, nanoliquid chromatography, and tandem mass spectrometry, S-nitrosylated proteins were identified in both wild-type and noe1 plants. NO targets identified only in noe1 plants included glyceraldehyde 3-phosphate dehydrogenase and thioredoxin, which have been reported to be involved in S-nitrosylation-regulated cell death in animals. Collectively, our data suggest that both NO and SNOs are important mediators in the process of H2O2-induced leaf cell death in rice. PMID:22106097

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

NASA Technical Reports Server (NTRS)

Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

2001-01-01

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

RRP1B Targets PP1 to Mammalian Cell Nucleoli and Is Associated with Pre-60S Ribosomal Subunits

PubMed Central

Chamousset, Delphine; De Wever, Veerle; Moorhead, Greg B.; Chen, Yan; Boisvert, Francois-Michel; Lamond, Angus I.

2010-01-01

A pool of protein phosphatase 1 (PP1) accumulates within nucleoli and accounts for a large fraction of the serine/threonine protein phosphatase activity in this subnuclear structure. Using a combination of fluorescence imaging with quantitative proteomics, we mapped the subnuclear localization of the three mammalian PP1 isoforms stably expressed as GFP-fusions in live cells and identified RRP1B as a novel nucleolar targeting subunit that shows a specificity for PP1β and PP1γ. RRP1B, one of two mammalian orthologues of the yeast Rrp1p protein, shows an RNAse-dependent localization to the granular component of the nucleolus and distributes in a similar manner throughout the cell cycle to proteins involved in later steps of rRNA processing. Quantitative proteomic analysis of complexes containing both RRP1B and PP1γ revealed enrichment of an overlapping subset of large (60S) ribosomal subunit proteins and pre-60S nonribosomal proteins involved in mid-late processing. Targeting of PP1 to this complex by RRP1B in mammalian cells is likely to contribute to modulation of ribosome biogenesis by mechanisms involving reversible phosphorylation events, thus playing a role in the rapid transduction of cellular signals that call for regulation of ribosome production in response to cellular stress and/or changes in growth conditions. PMID:20926688

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

Moon, Yuseok; Yang, Hyun; Kim, Yung Bu

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and inflammation and have also received considerable attention because of their preventive effects against human cancer. However, the drug application is sometimes limited by the severe gastrointestinal ulcers and mucosal complications. In the present study, NSAID sulindac sulfide was investigated for the cytotoxic injury in the intestinal epithelial cells in association with an immediate inducible factor, early growth response gene 1 (EGR-1). Previously we reported that sulindac sulfide can suppress tumor cell invasion by inducing EGR-1. Extending the previous study, EGR-1 induction by sulindac sulfide was observed both in the non-transformedmore » and transformed human intestinal epithelial cell lines. In terms of signaling pathway, ERK1/2 MAP kinases and its substrate Elk-1 transcription factor were involved in the sulindac sulfide-induced EGR-1 gene expression. Moreover, sulindac sulfide stimulated the nuclear translocation of the transcription factor EGR-1, which was also mediated by ERK1/2 signaling pathway. The roles of EGR-1 signals in the apoptotic cell death were assessed in the intestinal epithelial cells. Suppression of EGR-1 expression retarded cellular growth and colony forming activity in the intestinal epithelial cells. Moreover, induced EGR-1 ameliorated sulindac sulfide-mediated apoptotic cell death and enhanced the cellular survival. Taken all together, sulindac sulfide activated ERK1/2 MAP kinases which then mediated EGR-1 induction and nuclear translocation, all of which played important roles in the cellular survival from NSAID-mediated cytotoxicity in the human intestinal epithelial cells, implicating the protective roles of EGR-1 in the NSAID-mediated mucosal injuries.« less

Curcumin Stimulates Proliferation of Spinal Cord Neural Progenitor Cells via a Mitogen-Activated Protein Kinase Signaling Pathway

PubMed Central

Son, Sihoon; Cho, Dae-Chul; Kim, Hye-Jeong; Sung, Joo-Kyung; Bae, Jae-Sung

2014-01-01

Objective The aims of our study are to evaluate the effect of curcumin on spinal cord neural progenitor cell (SC-NPC) proliferation and to clarify the mechanisms of mitogen-activated protein (MAP) kinase signaling pathways in SC-NPCs. Methods We established cultures of SC-NPCs, extracted from the spinal cord of Sprague-Dawley rats weighing 250 g to 350 g. We measured proliferation rates of SC-NPCs after curcumin treatment at different dosage. The immuno-blotting method was used to evaluate the MAP kinase signaling protein that contains extracellular signal-regulated kinases (ERKs), p38, c-Jun NH2-terminal kinases (JNKs) and β-actin as the control group. Results Curcumin has a biphasic effect on SC-NPC proliferation. Lower dosage (0.1, 0.5, 1 µM) of curcumin increased SC-NPC proliferation. However, higher dosage decreased SC-NPC proliferation. Also, curcumin stimulates proliferation of SC-NPCs via the MAP kinase signaling pathway, especially involving the p-ERK and p-38 protein. The p-ERK protein and p38 protein levels varied depending on curcumin dosage (0.5 and 1 µM, p<0.05). Conclusion Curcumin can stimulate proliferation of SC-NPCs via ERKs and the p38 signaling pathway in low concentrations. PMID:25289117

[Physical mapping of the genes px and cld coding peroxidase and cold-regulated protein in maize (Zea mays L.)].

PubMed

Ning, S B; Wang, L; Song, Y C

2000-01-01

Peroxidase plays a key role in plant disease resistance, cold stress and some developmental processes, and cold-regulated protein functions necessarily in reaction of plants on cold or heat stress. Recent studies showed that these processes in plant cells were involved in programmed cell death (PCD). Using a biotin-labelled in situ hybridization (ISH) technique, we physically mapped the genes px and cld coding peroxidase and cold-regulated protein respectively onto maize chromosomes. Both DAB and fluorescence detection systems gave the identical results, the probe uaz235 corresponding to gene px was localized onto the long arm of chromosome 2 (2L) and 7L, and csu19 corresponding to gene cld was hybridized onto 4L and 5L. The percentage distances (from the hybridization sites to centromeres) of uaz235 in 2L and 7L were 45.4 +/- 1.3 and 67.4 +/- 3.7 respectively, and those of csu19 in 4L and 5L were 68.6 +/- 2.6 and 58.2 +/- 1.6 respectively. The physical positions of px in 2L and cld in 4L coincide with those in their genetic map pattern. The results also show that both of these genes have duplicated sites in maize genome.

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation.

PubMed

Liao, C L; Lin, Y L; Wu, B C; Tsao, C H; Wang, M C; Liu, C I; Huang, Y L; Chen, J H; Wang, J P; Chen, L K

2001-09-01

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.

A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors*

PubMed Central

Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian

2010-01-01

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. PMID:20952396

The effect of climate on the presence of Culicoides imicola in Italy.

PubMed

Conte, A; Giovannini, A; Savini, L; Goffredo, M; Calistri, P; Meiswinkel, R

2003-04-01

A model was developed to classify the Italian territories in relation to their suitability to harbour populations of Culicoides imicola and, as a consequence, also able to sustain a bluetongue (BT) epidemic. Italy was subdivided into 3507 10 x 10 km cells. In 546 cells at least one collection was made. The cell was considered the unit for all subsequent analyses. Culicoides were collected using Onderstepoort-type blacklight traps. Some traps were operated weekly at chosen sites; the remainder were moved almost daily to new sites. Only the results obtained during the peak August-November period were used, to exclude bias caused by the seasonality of C. imicola. Climate data for the period 1999-2001 were obtained from 80 weather stations. Multiple logistic regression was performed using the presence or absence of C. imicola in a specific cell as the dependent variable. Annual means of daily values for minimum temperature and minimum relative humidity, and the mean altitude above sea level, were the independent variables. The probability of occurrence of C. imicola in each grid cell was used to create a prediction map for Italy. The model was able to correctly classify 77.5% of the 546 grid cells in which at least one collection had been made. Culicoides imicola was found frequently through much of Sardinia, in parts of southern Italy, and further north along the Tyrrhenian coast, but was absent from along most of the Adriatic coast, and the internal mainland, and from most of Sicily. Six detailed maps are provided. Also mapped are areas where the probability of the occurrence of C. imicola is lower than 5%. This identification of possible mountainous C. imicola-free areas in central Italy could facilitate safer animal trade and transhumance, even if BT infections in traded animals or moving stock, were to go undetected. Needless to say this depends upon no cool-adapted species of Culicoides being involved in the transmission of BT disease.

Development and Validation of a Whole-Cell Inhibition Assay for Bacterial Methionine Aminopeptidase by Surface-Enhanced Laser Desorption Ionization-Time of Flight Mass Spectrometry

PubMed Central

Greis, Kenneth D.; Zhou, Songtao; Siehnel, Richard; Klanke, Chuck; Curnow, Alan; Howard, Jeremy; Layh-Schmitt, Gerlinde

2005-01-01

Bacterial methionine aminopeptidase (MAP) is a protease that removes methionine from the N termini of newly synthesized bacterial proteins after the peptide deformylase enzyme cleaves the formyl group from the initiator formylmethionine. MAP is an essential bacterial gene product and thus represents a potential target for therapeutic intervention. A fundamental challenge in the antibacterial drug discovery field is demonstrating conclusively that compounds with in vitro enzyme inhibition activity produce the desired antibacterial effect by interfering with the same target in whole bacterial cells. One way to address the activity of inhibitor compounds is by profiling cellular biomarkers in whole bacterial cells using compounds that are known inhibitors of a particular target. However, in the case of MAP, no specific inhibitors were available for such studies. Instead, a genetically attenuated MAP strain was generated in which MAP expression was placed under the control of an inducible arabinose promoter. Thus, MAP inhibition in whole cells could be mimicked by growth in the absence of arabinose. This genetically attenuated strain was used as a benchmark for MAP inhibition by profiling whole-cell lysates for unprocessed proteins using surface-enhanced laser desorption ionization-time of flight mass spectrometry (MS). Eight proteins between 4 and 14 kDa were confirmed as being unprocessed and containing the initiator methionine by adding back purified MAP to the preparations prior to MS analysis. Upon establishing these unprocessed proteins as biomarkers for MAP inhibition, the assay was used to screen small-molecule chemical inhibitors of purified MAP for whole-cell activity. Fifteen compound classes yielded three classes of compound with whole-cell activity for further optimization by chemical expansion. This report presents the development, validation, and implementation of a whole-cell inhibition assay for MAP. PMID:16048957

Hydrogen Production Cost Analysis Map (Text Version) | Hyrdrogen and Fuel

Science.gov Websites

Cells | Hydrogen and Fuel Cells | NREL Analysis Map (Text Version) Hydrogen Production Cost Analysis Map (Text Version) Below is a text version of the U.S. map that provides the results of NREL's

Fuel Cell Backup Power Geographical Visualization Map (Fact Sheet)

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

Not Available

2012-12-01

This NREL Hydrogen and Fuel Cell Technical Highlight describes a time-lapse geographical visualization map of early market use of fuel cells for telecommunications backup power. The map synthesizes data being analyzed by NREL's Technology Validation team for the U.S. Department of Energy (DOE) Fuel Cell Technologies Program with DOE's publicly available annual summaries of electric disturbance events.

Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

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

Chou, C.-T.; Department of Biological Sciences, National Sun Yat-sen University, 804, Taiwan; He Shiping

2007-02-01

Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38more » MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.« less

The Effectiveness of Concept Mapping and Retrieval Practice as Learning Strategies in an Undergraduate Physiology Course

ERIC Educational Resources Information Center

Burdo, Joseph; O'Dwyer, Laura

2015-01-01

Concept mapping and retrieval practice are both educational methods that have separately been reported to provide significant benefits for learning in diverse settings. Concept mapping involves diagramming a hierarchical representation of relationships between distinct pieces of information, whereas retrieval practice involves retrieving…

A nanobiosensor for dynamic single cell analysis during microvascular self-organization.

PubMed

Wang, S; Sun, J; Zhang, D D; Wong, P K

2016-10-14

The formation of microvascular networks plays essential roles in regenerative medicine and tissue engineering. Nevertheless, the self-organization mechanisms underlying the dynamic morphogenic process are poorly understood due to a paucity of effective tools for mapping the spatiotemporal dynamics of single cell behaviors. By establishing a single cell nanobiosensor along with live cell imaging, we perform dynamic single cell analysis of the morphology, displacement, and gene expression during microvascular self-organization. Dynamic single cell analysis reveals that endothelial cells self-organize into subpopulations with specialized phenotypes to form microvascular networks and identifies the involvement of Notch1-Dll4 signaling in regulating the cell subpopulations. The cell phenotype correlates with the initial Dll4 mRNA expression level and each subpopulation displays a unique dynamic Dll4 mRNA expression profile. Pharmacological perturbations and RNA interference of Notch1-Dll4 signaling modulate the cell subpopulations and modify the morphology of the microvascular network. Taken together, a nanobiosensor enables a dynamic single cell analysis approach underscoring the importance of Notch1-Dll4 signaling in microvascular self-organization.

Using in situ hybridization and PFGE Southern hybridization to detect translocation breakpoints in a BOR/TRPS patient cell line

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

Gu, J.Z.; Sapru, M.; Smith, D.

Branchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by ear malformations, cervical fistulae, hearing loss and renal abnormalities. We have integrated the Genethon YAC contig maps with additional markers in the chromosome 8q region genetically linked by a unique patient cell line. This cell line is from a patient who has both the branchio-oto-renal syndrome and tricho-rhino-phalangeal syndrome (TRPS). High resolution cytogenetics demonstrated a direct insertion of materials from 8q13.3q21.13 to 8q24.11. TRPS has been previously linked to deletions involving 8q24.11-q24.13. The rearrangement in this patient suggests that TRPS results from loss of gene function due to insertion atmore » the 8q24.11 breakpoint and the possible location for the BOR gene is at either of the two breakpoints of 8q13.3 and 8q21.13. We have constructed cosmid contigs in 8q24.11. In situ hybridization with cosmids mapped to these locations as probes has helped to narrow down the breakpoints. Combinations of cosmids on either side or overlapping the 8q24.11 breakpoint show split signals on one chromosome 8q arm due to insertion of the materials from the proximal region. Cosmids mapped to the TRPS deletion region have been used to hybridize to pulsed field gel genomic blots of DNA from the patient cell line and detected rearranged genomic fragments. Both in situ hybridization and genomic PFGE Southern blot will be used to precisely locate the breakpoints.« less

The MAPKK FgMkk1 of Fusarium graminearum regulates vegetative differentiation, multiple stress response, and virulence via the cell wall integrity and high-osmolarity glycerol signaling pathways.

PubMed

Yun, Yingzi; Liu, Zunyong; Zhang, Jingze; Shim, Won-Bo; Chen, Yun; Ma, Zhonghua

2014-07-01

Mitogen-activated protein (MAP) kinases play crucial roles in regulating fungal development, growth and pathogenicity, and in responses to the environment. In this study, we characterized a MAP kinase kinase FgMkk1 in Fusarium graminearum, the causal agent of wheat head blight. Phenotypic analyses of the FgMKK1 mutant (ΔFgMKK1) showed that FgMkk1 is involved in the regulation of hyphal growth, pigmentation, conidiation, deoxynivalenol biosynthesis and virulence of F. graminearum. ΔFgMKK1 also showed increased sensitivity to cell wall-damaging agents, and to osmotic and oxidative stresses, but exhibited decreased sensitivity to the fungicides iprodione and fludioxonil. In addition, the mutant revealed increased sensitivity to a biocontrol agent, Trichoderma atroviride. Western blot assays revealed that FgMkk1 positively regulates phosphorylation of the MAP kinases Mgv1 and FgOs-2, the key component in the cell wall integrity (CWI) and high-osmolarity glycerol (HOG) signalling pathway respectively. Yeast two-hybrid assay indicated that Mgv1 interacts with a transcription factor FgRlm1. The FgRLM1 mutant (ΔFgRLM1) showed increased sensitivity to cell wall-damaging agents and exhibited decreased virulence. Taken together, our data indicated that FgMkk1 is an upstream component of Mgv1, and regulates vegetative differentiation, multiple stress response and virulence via the CWI and HOG signalling pathways. FgRlm1 may be a downstream component of Mgv1 in the CWI pathway in F. graminearum. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Mapping cell populations in flow cytometry data for cross-sample comparison using the Friedman-Rafsky test statistic as a distance measure.

PubMed

Hsiao, Chiaowen; Liu, Mengya; Stanton, Rick; McGee, Monnie; Qian, Yu; Scheuermann, Richard H

2016-01-01

Flow cytometry (FCM) is a fluorescence-based single-cell experimental technology that is routinely applied in biomedical research for identifying cellular biomarkers of normal physiological responses and abnormal disease states. While many computational methods have been developed that focus on identifying cell populations in individual FCM samples, very few have addressed how the identified cell populations can be matched across samples for comparative analysis. This article presents FlowMap-FR, a novel method for cell population mapping across FCM samples. FlowMap-FR is based on the Friedman-Rafsky nonparametric test statistic (FR statistic), which quantifies the equivalence of multivariate distributions. As applied to FCM data by FlowMap-FR, the FR statistic objectively quantifies the similarity between cell populations based on the shapes, sizes, and positions of fluorescence data distributions in the multidimensional feature space. To test and evaluate the performance of FlowMap-FR, we simulated the kinds of biological and technical sample variations that are commonly observed in FCM data. The results show that FlowMap-FR is able to effectively identify equivalent cell populations between samples under scenarios of proportion differences and modest position shifts. As a statistical test, FlowMap-FR can be used to determine whether the expression of a cellular marker is statistically different between two cell populations, suggesting candidates for new cellular phenotypes by providing an objective statistical measure. In addition, FlowMap-FR can indicate situations in which inappropriate splitting or merging of cell populations has occurred during gating procedures. We compared the FR statistic with the symmetric version of Kullback-Leibler divergence measure used in a previous population matching method with both simulated and real data. The FR statistic outperforms the symmetric version of KL-distance in distinguishing equivalent from nonequivalent cell populations. FlowMap-FR was also employed as a distance metric to match cell populations delineated by manual gating across 30 FCM samples from a benchmark FlowCAP data set. An F-measure of 0.88 was obtained, indicating high precision and recall of the FR-based population matching results. FlowMap-FR has been implemented as a standalone R/Bioconductor package so that it can be easily incorporated into current FCM data analytical workflows. © The Authors. Published by Wiley Periodicals, Inc. on behalf of ISAC.

Single-molecule optical genome mapping of a human HapMap and a colorectal cancer cell line.

PubMed

Teo, Audrey S M; Verzotto, Davide; Yao, Fei; Nagarajan, Niranjan; Hillmer, Axel M

2015-01-01

Next-generation sequencing (NGS) technologies have changed our understanding of the variability of the human genome. However, the identification of genome structural variations based on NGS approaches with read lengths of 35-300 bases remains a challenge. Single-molecule optical mapping technologies allow the analysis of DNA molecules of up to 2 Mb and as such are suitable for the identification of large-scale genome structural variations, and for de novo genome assemblies when combined with short-read NGS data. Here we present optical mapping data for two human genomes: the HapMap cell line GM12878 and the colorectal cancer cell line HCT116. High molecular weight DNA was obtained by embedding GM12878 and HCT116 cells, respectively, in agarose plugs, followed by DNA extraction under mild conditions. Genomic DNA was digested with KpnI and 310,000 and 296,000 DNA molecules (≥ 150 kb and 10 restriction fragments), respectively, were analyzed per cell line using the Argus optical mapping system. Maps were aligned to the human reference by OPTIMA, a new glocal alignment method. Genome coverage of 6.8× and 5.7× was obtained, respectively; 2.9× and 1.7× more than the coverage obtained with previously available software. Optical mapping allows the resolution of large-scale structural variations of the genome, and the scaffold extension of NGS-based de novo assemblies. OPTIMA is an efficient new alignment method; our optical mapping data provide a resource for genome structure analyses of the human HapMap reference cell line GM12878, and the colorectal cancer cell line HCT116.

Single-Frame Terrain Mapping Software for Robotic Vehicles

NASA Technical Reports Server (NTRS)

Rankin, Arturo L.

2011-01-01

This software is a component in an unmanned ground vehicle (UGV) perception system that builds compact, single-frame terrain maps for distribution to other systems, such as a world model or an operator control unit, over a local area network (LAN). Each cell in the map encodes an elevation value, terrain classification, object classification, terrain traversability, terrain roughness, and a confidence value into four bytes of memory. The input to this software component is a range image (from a lidar or stereo vision system), and optionally a terrain classification image and an object classification image, both registered to the range image. The single-frame terrain map generates estimates of the support surface elevation, ground cover elevation, and minimum canopy elevation; generates terrain traversability cost; detects low overhangs and high-density obstacles; and can perform geometry-based terrain classification (ground, ground cover, unknown). A new origin is automatically selected for each single-frame terrain map in global coordinates such that it coincides with the corner of a world map cell. That way, single-frame terrain maps correctly line up with the world map, facilitating the merging of map data into the world map. Instead of using 32 bits to store the floating-point elevation for a map cell, the vehicle elevation is assigned to the map origin elevation and reports the change in elevation (from the origin elevation) in terms of the number of discrete steps. The single-frame terrain map elevation resolution is 2 cm. At that resolution, terrain elevation from 20.5 to 20.5 m (with respect to the vehicle's elevation) is encoded into 11 bits. For each four-byte map cell, bits are assigned to encode elevation, terrain roughness, terrain classification, object classification, terrain traversability cost, and a confidence value. The vehicle s current position and orientation, the map origin, and the map cell resolution are all included in a header for each map. The map is compressed into a vector prior to delivery to another system.

Involvement of GTA protein NC2β in Neuroblastoma pathogenesis suggests that it physiologically participates in the regulation of cell proliferation

PubMed Central

Di Pietro, Cinzia; Ragusa, Marco; Barbagallo, Davide; Duro, Laura R; Guglielmino, Maria R; Majorana, Alessandra; Giunta, Veronica; Rapisarda, Antonella; Tricarichi, Elisa; Miceli, Marco; Angelica, Rosario; Grillo, Agata; Banelli, Barbara; Defferari, Isabella; Forte, Stefano; Laganà, Alessandro; Bosco, Camillo; Giugno, Rosalba; Pulvirenti, Alfredo; Ferro, Alfredo; Grzeschik, Karl H; Di Cataldo, Andrea; Tonini, Gian P; Romani, Massimo; Purrello, Michele

2008-01-01

Background The General Transcription Apparatus (GTA) comprises more than one hundred proteins, including RNA Polymerases, GTFs, TAFs, Mediator, and cofactors such as heterodimeric NC2. This complexity contrasts with the simple mechanical role that these proteins are believed to perform and suggests a still uncharacterized participation to important biological functions, such as the control of cell proliferation. Results To verify our hypothesis, we analyzed the involvement in Neuroblastoma (NB) pathogenesis of GTA genes localized at 1p, one of NB critical regions: through RT-PCR of fifty eight NB biopsies, we demonstrated the statistically significant reduction of the mRNA for NC2β (localized at 1p22.1) in 74% of samples (p = 0.0039). Transcripts from TAF13 and TAF12 (mapping at 1p13.3 and 1p35.3, respectively) were also reduced, whereas we didn't detect any quantitative alteration of the mRNAs from GTF2B and NC2α (localized at 1p22-p21 and 11q13.3, respectively). We confirmed these data by comparing tumour and constitutional DNA: most NB samples with diminished levels of NC2β mRNA had also genomic deletions at the corresponding locus. Conclusion Our data show that NC2β is specifically involved in NB pathogenesis and may be considered a new NB biomarker: accordingly, we suggest that NC2β, and possibly other GTA members, are physiologically involved in the control of cell proliferation. Finally, our studies unearth complex selective mechanisms within NB cells. PMID:18538002

Involvement of GTA protein NC2beta in neuroblastoma pathogenesis suggests that it physiologically participates in the regulation of cell proliferation.

PubMed

Di Pietro, Cinzia; Ragusa, Marco; Barbagallo, Davide; Duro, Laura R; Guglielmino, Maria R; Majorana, Alessandra; Giunta, Veronica; Rapisarda, Antonella; Tricarichi, Elisa; Miceli, Marco; Angelica, Rosario; Grillo, Agata; Banelli, Barbara; Defferari, Isabella; Forte, Stefano; Laganà, Alessandro; Bosco, Camillo; Giugno, Rosalba; Pulvirenti, Alfredo; Ferro, Alfredo; Grzeschik, Karl H; Di Cataldo, Andrea; Tonini, Gian P; Romani, Massimo; Purrello, Michele

2008-06-06

The General Transcription Apparatus (GTA) comprises more than one hundred proteins, including RNA Polymerases, GTFs, TAFs, Mediator, and cofactors such as heterodimeric NC2. This complexity contrasts with the simple mechanical role that these proteins are believed to perform and suggests a still uncharacterized participation to important biological functions, such as the control of cell proliferation. To verify our hypothesis, we analyzed the involvement in neuroblastoma (NB) pathogenesis of GTA genes localized at 1p, one of NB critical regions: through RT-PCR of fifty eight NB biopsies, we demonstrated the statistically significant reduction of the mRNA for NC2beta (localized at 1p22.1) in 74% of samples (p = 0.0039). Transcripts from TAF13 and TAF12 (mapping at 1p13.3 and 1p35.3, respectively) were also reduced, whereas we didn't detect any quantitative alteration of the mRNAs from GTF2B and NC2alpha (localized at 1p22-p21 and 11q13.3, respectively). We confirmed these data by comparing tumour and constitutional DNA: most NB samples with diminished levels of NC2beta mRNA had also genomic deletions at the corresponding locus. Our data show that NC2beta is specifically involved in NB pathogenesis and may be considered a new NB biomarker: accordingly, we suggest that NC2beta, and possibly other GTA members, are physiologically involved in the control of cell proliferation. Finally, our studies unearth complex selective mechanisms within NB cells.

Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae.

PubMed

Morimoto, Yuji; Tani, Motohiro

2015-02-01

Complex sphingolipids play important roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened yeast mutant strains showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide (MIPC) synthesis and found that a specific group of glycosyltransferases involved in the synthesis of mannan-type N-glycans is essential for the growth of cells lacking MIPC synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of MNN2, which encodes alpha-1,2-mannosyltransferase that synthesizes mannan-type N-glycans, by a tetracycline-regulatable system. MNN2-repressed sur1Δ csh1Δ cells exhibited high sensitivity to zymolyase treatment, and caffeine and sodium dodecyl sulfate (SDS) strongly inhibited the growth of sur1Δ csh1Δ cells, suggesting impairment of cell integrity due to the loss of MIPC synthesis. The phosphorylated form of Slt2, a mitogen-activated protein (MAP) kinase activated by impaired cell integrity, increased in sur1Δ csh1Δ cells, and this increase was dramatically enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2-repressed sur1Δ csh1Δ cells was enhanced by the deletion of SLT2 or RLM1 encoding a downstream target of Slt2. These results indicated that loss of MIPC synthesis causes impairment of cell integrity, and this effect is enhanced by impaired synthesis of mannan-type N-glycans. © 2014 John Wiley & Sons Ltd.

Passive mapping and intermittent exploration for mobile robots

NASA Technical Reports Server (NTRS)

Engleson, Sean P.

1994-01-01

An adaptive state space architecture is combined with diktiometric representation to provide the framework for designing a robot mapping system with flexible navigation planning tasks. This involves indexing waypoints described as expectations, geometric indexing, and perceptual indexing. Matching and updating the robot's projected position and sensory inputs with indexing waypoints involves matchers, dynamic priorities, transients, and waypoint restructuring. The robot's map learning can be opganized around the principles of passive mapping.

Accurate and reproducible functional maps in 127 human cell types via 2D genome segmentation

PubMed Central

Hardison, Ross C.

2017-01-01

Abstract The Roadmap Epigenomics Consortium has published whole-genome functional annotation maps in 127 human cell types by integrating data from studies of multiple epigenetic marks. These maps have been widely used for studying gene regulation in cell type-specific contexts and predicting the functional impact of DNA mutations on disease. Here, we present a new map of functional elements produced by applying a method called IDEAS on the same data. The method has several unique advantages and outperforms existing methods, including that used by the Roadmap Epigenomics Consortium. Using five categories of independent experimental datasets, we compared the IDEAS and Roadmap Epigenomics maps. While the overall concordance between the two maps is high, the maps differ substantially in the prediction details and in their consistency of annotation of a given genomic position across cell types. The annotation from IDEAS is uniformly more accurate than the Roadmap Epigenomics annotation and the improvement is substantial based on several criteria. We further introduce a pipeline that improves the reproducibility of functional annotation maps. Thus, we provide a high-quality map of candidate functional regions across 127 human cell types and compare the quality of different annotation methods in order to facilitate biomedical research in epigenomics. PMID:28973456

Neurotensin-induced Proinflammatory Signaling in Human Colonocytes Is Regulated by β-Arrestins and Endothelin-converting Enzyme-1-dependent Endocytosis and Resensitization of Neurotensin Receptor 1*

PubMed Central

Law, Ivy Ka Man; Murphy, Jane E.; Bakirtzi, Kyriaki; Bunnett, Nigel W.; Pothoulakis, Charalabos

2012-01-01

The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of βARR1 or βARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves βARRs and ECE-1, respectively. Our results also indicate that βARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT. PMID:22416137

Revising the embryonic origin of thyroid C cells in mice and humans

PubMed Central

Johansson, Ellen; Andersson, Louise; Örnros, Jessica; Carlsson, Therese; Ingeson-Carlsson, Camilla; Liang, Shawn; Dahlberg, Jakob; Jansson, Svante; Parrillo, Luca; Zoppoli, Pietro; Barila, Guillermo O.; Altschuler, Daniel L.; Padula, Daniela; Lickert, Heiko; Fagman, Henrik; Nilsson, Mikael

2015-01-01

Current understanding infers a neural crest origin of thyroid C cells, the major source of calcitonin in mammals and ancestors to neuroendocrine thyroid tumors. The concept is primarily based on investigations in quail–chick chimeras involving fate mapping of neural crest cells to the ultimobranchial glands that regulate Ca2+ homeostasis in birds, reptiles, amphibians and fishes, but whether mammalian C cell development involves a homologous ontogenetic trajectory has not been experimentally verified. With lineage tracing, we now provide direct evidence that Sox17+ anterior endoderm is the only source of differentiated C cells and their progenitors in mice. Like many gut endoderm derivatives, embryonic C cells were found to coexpress pioneer factors forkhead box (Fox) a1 and Foxa2 before neuroendocrine differentiation takes place. In the ultimobranchial body epithelium emerging from pharyngeal pouch endoderm in early organogenesis, differential Foxa1/Foxa2 expression distinguished two spatially separated pools of C cell precursors with different growth properties. A similar expression pattern was recapitulated in medullary thyroid carcinoma cells in vivo, consistent with a growth-promoting role of Foxa1. In contrast to embryonic precursor cells, C cell-derived tumor cells invading the stromal compartment downregulated Foxa2, foregoing epithelial-to-mesenchymal transition designated by loss of E-cadherin; both Foxa2 and E-cadherin were re-expressed at metastatic sites. These findings revise mammalian C cell ontogeny, expand the neuroendocrine repertoire of endoderm and redefine the boundaries of neural crest diversification. The data further underpin distinct functions of Foxa1 and Foxa2 in both embryonic and tumor development. PMID:26395490

Mapping of Variable DNA Methylation Across Multiple Cell Types Defines a Dynamic Regulatory Landscape of the Human Genome.

PubMed

Gu, Junchen; Stevens, Michael; Xing, Xiaoyun; Li, Daofeng; Zhang, Bo; Payton, Jacqueline E; Oltz, Eugene M; Jarvis, James N; Jiang, Kaiyu; Cicero, Theodore; Costello, Joseph F; Wang, Ting

2016-04-07

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Many studies have mapped DNA methylation changes associated with embryogenesis, cell differentiation, and cancer at a genome-wide scale. Our understanding of genome-wide DNA methylation changes in a developmental or disease-related context has been steadily growing. However, the investigation of which CpGs are variably methylated in different normal cell or tissue types is still limited. Here, we present an in-depth analysis of 54 single-CpG-resolution DNA methylomes of normal human cell types by integrating high-throughput sequencing-based methylation data. We found that the ratio of methylated to unmethylated CpGs is relatively constant regardless of cell type. However, which CpGs made up the unmethylated complement was cell-type specific. We categorized the 26,000,000 human autosomal CpGs based on their methylation levels across multiple cell types to identify variably methylated CpGs and found that 22.6% exhibited variable DNA methylation. These variably methylated CpGs formed 660,000 variably methylated regions (VMRs), encompassing 11% of the genome. By integrating a multitude of genomic data, we found that VMRs enrich for histone modifications indicative of enhancers, suggesting their role as regulatory elements marking cell type specificity. VMRs enriched for transcription factor binding sites in a tissue-dependent manner. Importantly, they enriched for GWAS variants, suggesting that VMRs could potentially be implicated in disease and complex traits. Taken together, our results highlight the link between CpG methylation variation, genetic variation, and disease risk for many human cell types. Copyright © 2016 Gu et al.

A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells.

PubMed

Miyoshi, Yukihiro; Okada, Sanae; Uchimura, Tai; Satoh, Eiichi

2006-07-01

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

Electrical coupling in ensembles of nonexcitable cells: modeling the spatial map of single cell potentials.

PubMed

Cervera, Javier; Manzanares, Jose Antonio; Mafe, Salvador

2015-02-19

We analyze the coupling of model nonexcitable (non-neural) cells assuming that the cell membrane potential is the basic individual property. We obtain this potential on the basis of the inward and outward rectifying voltage-gated channels characteristic of cell membranes. We concentrate on the electrical coupling of a cell ensemble rather than on the biochemical and mechanical characteristics of the individual cells, obtain the map of single cell potentials using simple assumptions, and suggest procedures to collectively modify this spatial map. The response of the cell ensemble to an external perturbation and the consequences of cell isolation, heterogeneity, and ensemble size are also analyzed. The results suggest that simple coupling mechanisms can be significant for the biophysical chemistry of model biomolecular ensembles. In particular, the spatiotemporal map of single cell potentials should be relevant for the uptake and distribution of charged nanoparticles over model cell ensembles and the collective properties of droplet networks incorporating protein ion channels inserted in lipid bilayers.

House dust mite induces expression of intercellular adhesion molecule-1 in EoL-1 human eosinophilic leukemic cells.

PubMed

Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn; Shin, Myeong Heon

2007-10-01

The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-kappaB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-kappaB and JNK.

House Dust Mite Induces Expression of Intercellular Adhesion Molecule-1 in EoL-1 Human Eosinophilic Leukemic Cells

PubMed Central

Kwon, Byoung Chul; Sohn, Myung Hyun; Kim, Kyung Won; Kim, Eun Soo; Kim, Kyu-Earn

2007-01-01

The house dust mite (HDM) is considered to be the most common indoor allergen associated with bronchial asthma. In this study, we investigated whether crude extract of the HDM Dermatophagoides farinae could activate human eosinophilic leukemic cells (EoL-1) to induce upregulation of cell-surface adhesion molecules. When EoL-1 cells were incubated with D. farinae extract, expression of intercellular adhesion molecule-1 (ICAM-1) significantly increased on the cell surfaces compared to cells incubated with medium alone. In contrast, surface expression of CD11b and CD49d in EoL-1 cells was not affected by D. farinae extract. In addition, pretreatment of cells with NF-κB inhibitor (MG-132) or JNK inhibitor (SP600125) significantly inhibited ICAM-1 expression promoted by HDM extract. However, neither p38 MAP kinase inhibitor nor MEK inhibitor prevented HDM-induced ICAM-1 expression in EoL-1 cells. These results suggest that crude extract of D. farinae induces ICAM-1 expression in EoL-1 cells through signaling pathways involving both NF-κB and JNK. PMID:17982228

Map Projections and the Visual Detective: How to Tell if a Map Is Equal-Area, Conformal, or Neither

ERIC Educational Resources Information Center

Olson, Judy M.

2006-01-01

The ability to see whether a map is equal-area, conformal, or neither is useful for looking intelligently at large-area maps. For example, only if a map is equal-area can reliable judgments of relative size be made. If a map is equal-area, latitude-longitude cells are equal in size between a given pair of parallels, the cells between a given pair…

Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation

PubMed Central

Matias, Andreza Cândido; Manieri, Tânia Maria; Cerchiaro, Giselle

2016-01-01

We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation. PMID:27123155

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

PubMed Central

Lan, Xiaoyang; Jörg, David J.; Cavalli, Florence M. G.; Richards, Laura M.; Nguyen, Long V.; Vanner, Robert J.; Guilhamon, Paul; Lee, Lilian; Kushida, Michelle; Pellacani, Davide; Park, Nicole I.; Coutinho, Fiona J.; Whetstone, Heather; Selvadurai, Hayden J.; Che, Clare; Luu, Betty; Carles, Annaick; Moksa, Michelle; Rastegar, Naghmeh; Head, Renee; Dolma, Sonam; Prinos, Panagiotis; Cusimano, Michael D.; Das, Sunit; Bernstein, Mark; Arrowsmith, Cheryl H.; Mungall, Andrew J.; Moore, Richard A.; Ma, Yussanne; Gallo, Marco; Lupien, Mathieu; Pugh, Trevor J.; Taylor, Michael D.; Hirst, Martin; Eaves, Connie J.; Simons, Benjamin D.; Dirks, Peter B.

2017-01-01

Summary Human glioblastomas (GBMs) harbour a subpopulation of glioblastoma stem cells (GSCs) that drive tumourigenesis. However, the origin of intra-tumoural functional heterogeneity between GBM cells remains poorly understood. Here we study the clonal evolution of barcoded GBM cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of GBM clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in GSCs. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, that in turn generates non-proliferative cells. We also identify rare “outlier” clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant GSCs. Finally, we show that functionally distinct GSCs can be separately targeted using epigenetic compounds, suggesting new avenues for GBM targeted therapy. PMID:28854171

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

PubMed Central

Planas, Delphine; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R. M.

2017-01-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation. PMID:28727807

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

Elucidation of the mechanism of homozygous deletion of 3p12-13 in the U2020 cell line reveals the unexpected involvement of other chromosomes.

PubMed

Heppell-Parton, A C; Nacheva, E; Carter, N P; Bergh, J; Ogilvie, D; Rabbitts, P H

1999-06-01

Homozygous deletions in tumor cells have been useful in the localization and validation of tumor suppressor genes. We have described a homozygous deletion in a lung cancer cell line (U2020) which is located within the most proximal of the three regions on the short arm of chromosome 3 believed to be lost in lung cancer development. Construction of a YAC contig map indicates that the deletion spans around 8 Mb, but no large deletion was apparent on conventional cytogenetic analysis of the cell line. To investigate this paradox, whole chromosome, arm-specific, and regional paints have been used. This analysis has revealed that genetic loss has occurred by complex rearrangements of chromosomes 3, rather than simple interstitial deletion. These studies emphasize the power of molecular cytogenetics to disclose unsuspected tumor-specific translocations within the extremely complex karyotypes characteristic of solid tumors.

GSTM3 and GSTP1: novel players driving tumor progression in cervical cancer

PubMed Central

Checa-Rojas, Alberto; Delgadillo-Silva, Luis Fernando; Velasco-Herrera, Martín del Castillo; Andrade-Domínguez, Andrés; Gil, Jeovanis; Santillán, Orlando; Lozano, Luis; Toledo-Leyva, Alfredo; Ramírez-Torres, Alberto; Talamas-Rohana, Patricia; Encarnación-Guevara, Sergio

2018-01-01

The molecular processes and proteomic markers leading to tumor progression (TP) in cervical cancer (CC) are either unknown or only partially understood. TP affects metabolic and regulatory mechanisms that can be identified as proteomic changes. To identify which proteins are differentially expressed and to understand the mechanisms of cancer progression, we analyzed the dynamics of the tumor proteome in CC cell lines. This analysis revealed two proteins that are up-regulated during TP, GSTM3 and GSTP1. These proteins are involved in cell maintenance, cell survival and the cellular stress response via the NF-κB and MAP kinase pathways during TP. Furthermore, GSTM3 and GSTP1 knockdown showed that evasion of apoptosis was affected, and tumor proliferation was significantly reduced. Our data indicate the critical role of GST proteins in the regulation and progression of cervical cancer cells. Hence, we suggest GSTM3 and GSTP1 as novel biomarkers and potential therapeutic targets for treating cervical cancer. Significance CC is particularly hazardous in the advanced stages, and there are few therapeutic strategies specifically targeting these stages. We performed analyses on CC tumor proteome dynamics and identified GSTM3 and GSTP1 as novel potential therapeutic targets. Knockdown of these proteins showed that they are involved in cell survival, cell proliferation and cellular evasion of apoptosis. PMID:29774096

Far-field photostable optical nanoscopy (PHOTON) for real-time super-resolution single-molecular imaging of signaling pathways of single live cells

NASA Astrophysics Data System (ADS)

Huang, Tao; Browning, Lauren M.; Xu, Xiao-Hong Nancy

2012-04-01

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11739h

EphrinA5 protein distribution in the developing mouse brain

PubMed Central

2010-01-01

Background EphrinA5 is one of the best-studied members of the Eph-ephrin family of guidance molecules, known to be involved in brain developmental processes. Using in situ hybridization, ephrinA5 mRNA expression has been detected in the retinotectal, the thalamocortical, and the olfactory systems; however, no study focused on the distribution of the protein. Considering that this membrane-anchored molecule may act far from the neuron soma expressing the transcript, it is of a crucial interest to localize ephrinA5 protein to better understand its function. Results Using immunohistochemistry, we found that ephrinA5 protein is highly expressed in the developing mouse brain from E12.5 to E16.5. The olfactory bulb, the cortex, the striatum, the thalamus, and the colliculi showed high intensity of labelling, suggesting its implication in topographic mapping of olfactory, retinocollicular, thalamocortical, corticothalamic and mesostriatal systems. In the olfactory nerve, we found an early ephrinA5 protein expression at E12.5 suggesting its implication in the guidance of primary olfactory neurons into the olfactory bulb. In the thalamus, we detected a dynamic graduated protein expression, suggesting its role in the corticothalamic patterning, whereas ephrinA5 protein expression in the target region of mesencephalic dopaminergic neurones indicated its involvement in the mesostriatal topographic mapping. Following E16.5, the signal faded gradually and was barely detectable at P0, suggesting a main role for ephrinA5 in primary molecular events in topographic map formation. Conclusion Our work shows that ephrinA5 protein is expressed in restrictive regions of the developing mouse brain. This expression pattern points out the potential sites of action of this molecule in the olfactory, retinotectal, thalamocortical, corticothalamic and mesostriatal systems, during development. This study is essential to better understand the role of ephrinA5 during developmental topographic mapping of connections and to further characterise the mechanisms involved in pathway restoration following cell transplantation in the damaged brain. PMID:20738842

A second generation SNP and SSR integrated linkage map and QTL mapping for the Chinese mitten crab Eriocheir sinensis

PubMed Central

Qiu, Gao-Feng; Xiong, Liang-Wei; Han, Zhi-Ke; Liu, Zhi-Qiang; Feng, Jian-Bin; Wu, Xu-Gan; Yan, Yin-Long; Shen, Hong; Huang, Long; Chen, Li

2017-01-01

The Chinese mitten crab Eriocheir sinensis is the most economically important cultivated crab species in China, and its genome has a high number of chromosomes (2n = 146). To obtain sufficient markers for construction of a dense genetic map for this species, we employed the recently developed specific-locus amplified fragment sequencing (SLAF-seq) method for large-scale SNPs screening and genotyping in a F1 full-sib family of 149 individuals. SLAF-seq generated 127,677 polymorphic SNP markers, of which 20,803 valid markers were assigned into five segregation types and were used together with previous SSR markers for linkage map construction. The final integrated genetic map included 17,680 SNP and 629 SSR markers on the 73 linkage groups (LG), and spanned 14,894.9 cM with an average marker interval of 0.81 cM. QTL mapping localized three significant growth-related QTL to a 1.2 cM region in LG53 as well as 146 sex-linked markers in LG48. Genome-wide QTL-association analysis further identified four growth-related QTL genes named LNX2, PAK2, FMRFamide and octopamine receptors. These genes are involved in a variety of different signaling pathways including cell proliferation and growth. The map and SNP markers described here will be a valuable resource for the E. sinensis genome project and selective breeding programs. PMID:28045132

Phosphorus detection in vitrified bacteria by cryo-STEM annular dark-field analysis.

PubMed

Wolf, Sharon Grayer; Rez, Peter; Elbaum, Michael

2015-11-01

Bacterial cells often contain dense granules. Among these, polyphosphate bodies (PPBs) store inorganic phosphate for a variety of essential functions. Identification of PPBs has until now been accomplished by analytical methods that required drying or chemically fixing the cells. These methods entail large electron doses that are incompatible with low-dose imaging of cryogenic specimens. We show here that Scanning Transmission Electron Microscopy (STEM) of fully hydrated, intact, vitrified bacteria provides a simple means for mapping of phosphorus-containing dense granules based on quantitative sensitivity of the electron scattering to atomic number. A coarse resolution of the scattering angles distinguishes phosphorus from the abundant lighter atoms: carbon, nitrogen and oxygen. The theoretical basis is similar to Z contrast of materials science. EDX provides a positive identification of phosphorus, but importantly, the method need not involve a more severe electron dose than that required for imaging. The approach should prove useful in general for mapping of heavy elements in cryopreserved specimens when the element identity is known from the biological context. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Immunomediated Pan-cancer Regulation Networks are Dominant Fingerprints After Treatment of Cell Lines with Demethylation.

PubMed

El Baroudi, Mariama; Cinti, Caterina; Capobianco, Enrico

2016-01-01

Pan-cancer studies are particularly relevant not only for addressing the complexity of the inherently observed heterogeneity but also for identifying clinically relevant features that may be common to the cancer types. Immune system regulations usually reveal synergistic modulation with other cancer mechanisms and in combination provide insights on possible advances in cancer immunotherapies. Network inference is a powerful approach to decipher pan-cancer systems dynamics. The methodology proposed in this study elucidates the impacts of epigenetic treatment on the drivers of complex pan-cancer regulation circuits involving cell lines of five cancer types. These patterns were observed from differential gene expression measurements following demethylation with 5-azacytidine. Networks were built to establish associations of phenotypes at molecular level with cancer hallmarks through both transcriptional and post-transcriptional regulation mechanisms. The most prominent feature that emerges from our integrative network maps, linking pathway landscapes to disease and drug-target associations, refers primarily to a mosaic of immune-system crosslinked influences. Therefore, characteristics initially evidenced in single cancer maps become motifs well summarized by network cores and fingerprints.

Comparative anatomy of the electrosensory lateral line lobe of mormyrids: the mystery of the missing map in the genus Stomatorhinus (family: Mormyridae).

PubMed

McNamara, Ann Marie; Denizot, Jean-Pierre; Hopkins, Carl D

2005-01-01

Fish in the family Mormyridae produce weak electric organ discharges that are used in orientation and communication. The peripheral and central anatomy of the electrosensory system has been well studied in the species Gnathonemus petersii, but comparative studies in other species are scarce. Here we report on one genus of mormyrid that displays a remarkable change in the electrosensory lateral line lobe (ELL), the hypertrophied rhombencephalic structure that receives primary electroreceptor input. Although all other mormyrids studied have three distinct zones on each side of the ELL, fish of the genus Stomatorhinus exhibit only two. Therefore, the two-zone ELL is a unique derived characteristic shared by Stomatorhinus. We examined the cutaneous electroreceptors that project to the ELL in Stomatorhinus. All three types of electroreceptors previously described for G. petersii were present, but there was a significant change in one type, the mormyromast. Both mormyromast sensory cell types (A- and B-cells) are present, but the B-cell is not innervated in Stomatorhinus. We conclude that, although all cutaneous sensory cells are present, the missing B-cell afferents account for the loss of the dorsolateral zone of the ELL, and therefore the loss of an entire sensory map. Because mormyromasts are involved in electrolocation behavior, this anatomical difference is probably related to differences in electrolocation abilities. Stomatorhinus could prove to be an excellent system for linking evolutionary changes in behavior with modifications in their neural substrates.

Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

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

Choi, Yoon Jung; Lee, Jue Yeon; Research Center, Nano Intelligent Biomedical Engineering Corporation

Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinicallymore » used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and according to our data doxazosin might be useful for application in the field of bone metabolism.« less

OxfordGrid: a web interface for pairwise comparative map views.

PubMed

Yang, Hongyu; Gingle, Alan R

2005-12-01

OxfordGrid is a web application and database schema for storing and interactively displaying genetic map data in a comparative, dot-plot, fashion. Its display is composed of a matrix of cells, each representing a pairwise comparison of mapped probe data for two linkage groups or chromosomes. These are arranged along the axes with one forming grid columns and the other grid rows with the degree and pattern of synteny/colinearity between the two linkage groups manifested in the cell's dot density and structure. A mouse click over the selected grid cell launches an image map-based display for the selected cell. Both individual and linear groups of mapped probes can be selected and displayed. Also, configurable links can be used to access other web resources for mapped probe information. OxfordGrid is implemented in C#/ASP.NET and the package, including MySQL schema creation scripts, is available at ftp://cggc.agtec.uga.edu/OxfordGrid/.

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

Potocki, L.; Oyer, C.E.; Tantravahi, U.

Two chromosomally male infants with partial monosomy 13q were found to have Leydig cell agenesis (LCA) and persistent muellerian ducts (PMD). Post mortem examination in each case revealed cardiovascular, gastrointestinal, genitourinary, musculoskeletal, and central nervous system abnormalities, characteristic of monosomy 13q. Histologic examination confirmed the presence of muellerian derivatives within the pelvis, and the absence of Leydig cells within the testes. Sertoli cells were present. Karyotypes revealed partial monosomy 13q secondary to an unbalanced translocation, der(13)t(1;13)(q43;q21), in one infant, and to a ring chromosome 13 involving a deletion of 13q31-qter, in the other. The etiology of male pseudohermaphroditism is heterogeneousmore » and included PMD due to absence of antimuellerian hormone (AMH) and LCA. Genitourinary abnormalities such as undescended testicles, hypospadias and micropenis have been described in monosomy 13q; however, testicular pathology in these cases has not been described. The cases presented here are the first reported cases in which male pseudohermaphroditism due to LCA and PMD is associated with monosomy 13q. This suggests the genetic locus involved in Leydig cell development may be located on the long arm of chromosome 13. The gene for AMH has been mapped to 19p13.3-13.2. The presence of muellerian structures and Sertoli cells, in the absence of abnormalities of chromosome 19p. suggests there may be genes on 13q coding for an enzyme in the pathway of AMH synthesis or for the AMH receptor. Based on these two cases, the critical region could possibly involve 13q13-qter.« less

Expression of Momordica charantia MAP30 and its antitumor effect on bladder cancer cells.

PubMed

Hlin, Hao; Zhi-Guo, Zhang; Cong-Hui, Han; Yan, Zhao; Qing, Liang; Bo, Jiang; Hou-Guang, He; Jun-Jie, Zhang; Pei-Ying, Zhang

2016-06-01

Momordica charantia (MC) is an edible medicinal plant that is known for its diversified biological functions. Momordica Antiviral Protein 30kD (MAP30) is a type I single chain ribosome-inactivating protein (RIP) isolated from the mature fruit and seeds of MC. Since MAP30 content in MC is limited, the study aim was to generate the recombinant MAP30 protein using prokaryotic expression system and determine its apoptotic/growth inhibitory effects on bladder cancer 5637 cells. MAP30 gene was amplified by PCR from MC genomic DNA and identified by sequencing. The target gene was inserted into pET-28a (+) vector and transformed into E. coli BL21 (DE3) cells. Positive clones were selected by PCR. Recombinant protein was efficiently expressed under induction with 1.0 mM Isopropylthio-β-D-galactoside (IPTG) at 30° C for 4 hours. Cytotoxicity studies were performed using MTT assay by treating 5637 bladder cancer cells with 100 µg/mL, 200 µg/mL, and 400 µg/mL concentrations of MAP30 for 24 hours and 48 hours, respectively. Flow cytometry was used to measure the apoptosis of MAP30-treatedcells in time course experiments. Full-length MAP30 gene was successfully expressed in Escherichia coli (E. coli) BL21 strain and MAP30 recombinant protein inhibited the growth of bladder cancer 5637 cells at 200 µg/mL and 400 µg/mL concentrations by inducing apoptosis of target cells in a dose- and time-dependent manner. It was, therefore, concluded that the MAP30 recombinant protein displayed potent antitumor activity in vitro.

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery.

PubMed

Luo, Zhao; Cao, Xue-Wei; Li, Chen; Wu, Miao-Dan; Yang, Xu-Zhong; Zhao, Jian; Wang, Fu-Jun

2016-11-01

Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Transcriptional Changes in the Transition from Vegetative Cells to Asexual Development in the Model Fungus Aspergillus nidulans

PubMed Central

Garzia, Aitor; Etxebeste, Oier; Rodríguez-Romero, Julio; Fischer, Reinhard; Espeso, Eduardo A.

2013-01-01

Morphogenesis encompasses programmed changes in gene expression that lead to the development of specialized cell types. In the model fungus Aspergillus nidulans, asexual development involves the formation of characteristic cell types, collectively known as the conidiophore. With the aim of determining the transcriptional changes that occur upon induction of asexual development, we have applied massive mRNA sequencing to compare the expression pattern of 19-h-old submerged vegetative cells (hyphae) with that of similar hyphae after exposure to the air for 5 h. We found that the expression of 2,222 (20.3%) of the predicted 10,943 A. nidulans transcripts was significantly modified after air exposure, 2,035 being downregulated and 187 upregulated. The activation during this transition of genes that belong specifically to the asexual developmental pathway was confirmed. Another remarkable quantitative change occurred in the expression of genes involved in carbon or nitrogen primary metabolism. Genes participating in polar growth or sexual development were transcriptionally repressed, as were those belonging to the HogA/SakA stress response mitogen-activated protein (MAP) kinase pathway. We also identified significant expression changes in several genes purportedly involved in redox balance, transmembrane transport, secondary metabolite production, or transcriptional regulation, mainly binuclear-zinc cluster transcription factors. Genes coding for these four activities were usually grouped in metabolic clusters, which may bring regulatory implications for the induction of asexual development. These results provide a blueprint for further stage-specific gene expression studies during conidiophore development. PMID:23264642

The cld mutation: narrowing the critical chromosomal region and selecting candidate genes.

PubMed

Péterfy, Miklós; Mao, Hui Z; Doolittle, Mark H

2006-10-01

Combined lipase deficiency (cld) is a recessive, lethal mutation specific to the tw73 haplotype on mouse Chromosome 17. While the cld mutation results in lipase proteins that are inactive, aggregated, and retained in the endoplasmic reticulum (ER), it maps separately from the lipase structural genes. We have narrowed the gene critical region by about 50% using the tw18 haplotype for deletion mapping and a recombinant chromosome used originally to map cld with respect to the phenotypic marker tf. The region now extends from 22 to 25.6 Mbp on the wild-type chromosome, currently containing 149 genes and 50 expressed sequence tags (ESTs). To identify the affected gene, we have selected candidates based on their known role in associated biological processes, cellular components, and molecular functions that best fit with the predicted function of the cld gene. A secondary approach was based on differences in mRNA levels between mutant (cld/cld) and unaffected (+/cld) cells. Using both approaches, we have identified seven functional candidates with an ER localization and/or an involvement in protein maturation and folding that could explain the lipase deficiency, and six expression candidates that exhibit large differences in mRNA levels between mutant and unaffected cells. Significantly, two genes were found to be candidates with regard to both function and expression, thus emerging as the strongest candidates for cld. We discuss the implications of our mapping results and our selection of candidates with respect to other genes, deletions, and mutations occurring in the cld critical region.

Genomic Restructuring in the Tasmanian Devil Facial Tumour: Chromosome Painting and Gene Mapping Provide Clues to Evolution of a Transmissible Tumour

PubMed Central

Pearse, Anne-Maree; Rens, Willem; O'Brien, Patricia C. M.; Ferguson-Smith, Malcolm A.; Cheng, Yuanyuan; Morris, Katrina; Taylor, Robyn; Stuart, Andrew; Belov, Katherine; Amemiya, Chris T.; Murchison, Elizabeth P.; Papenfuss, Anthony T.; Marshall Graves, Jennifer A.

2012-01-01

Devil facial tumour disease (DFTD) is a fatal, transmissible malignancy that threatens the world's largest marsupial carnivore, the Tasmanian devil, with extinction. First recognised in 1996, DFTD has had a catastrophic effect on wild devil numbers, and intense research efforts to understand and contain the disease have since demonstrated that the tumour is a clonal cell line transmitted by allograft. We used chromosome painting and gene mapping to deconstruct the DFTD karyotype and determine the chromosome and gene rearrangements involved in carcinogenesis. Chromosome painting on three different DFTD tumour strains determined the origins of marker chromosomes and provided a general overview of the rearrangement in DFTD karyotypes. Mapping of 105 BAC clones by fluorescence in situ hybridisation provided a finer level of resolution of genome rearrangements in DFTD strains. Our findings demonstrate that only limited regions of the genome, mainly chromosomes 1 and X, are rearranged in DFTD. Regions rearranged in DFTD are also highly rearranged between different marsupials. Differences between strains are limited, reflecting the unusually stable nature of DFTD. Finally, our detailed maps of both the devil and tumour karyotypes provide a physical framework for future genomic investigations into DFTD. PMID:22359511

IDC2 and IDC3, two genes involved in cell non-autonomous signaling of fruiting body development in the model fungus Podospora anserina.

PubMed

Lalucque, Hervé; Malagnac, Fabienne; Green, Kimberly; Gautier, Valérie; Grognet, Pierre; Chan Ho Tong, Laetitia; Scott, Barry; Silar, Philippe

2017-01-15

Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus Podospora anserina. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures. Copyright © 2016 Elsevier Inc. All rights reserved.

Delineation and physical separation of novel translocation breakpoints on chromosome 1p in two genetically closely associated childhood tumors.

PubMed

Steenman, M J; Zijlstra, N; Kruitbosch, D L; Wiesmeijer, C; Larizza, L; Voûte, P A; Westerveld, A; Mannens, M M

2000-01-01

Sporadic childhood tumors associated with Beckwith-Wiedemann syndrome (BWS) all show abnormalities of the same region on chromosome 11. In addition to chromosome 11, other chromosome regions are affected in some of these tumor types. In this study we analyzed the region on chromosome 1p involved in the etiology of BWS-associated tumors, Wilms tumor, rhabdomyosarcoma, and hepatoblastoma. For this purpose we determined the location of two novel translocation breakpoints in this chromosome region in cells from a Wilms tumor and cells from a rhabdomyosarcoma. We constructed a map of the region and found that both breakpoints are separated by at least 875 kb. We identified a PAC clone which crosses the rhabdomyosarcoma breakpoint and found several exons within this clone. We established that this breakpoint is located proximal to the PAX7 gene and, therefore, identified a new region involved in the etiology of rhabdomyosarcomas. Copyright 2000 S. Karger AG, Basel

Synchrotron-based X-ray fluorescence microscopy enables multiscale spatial visualization of ions involved in fungal lignocellulose deconstruction

NASA Astrophysics Data System (ADS)

Kirker, Grant; Zelinka, Sam; Gleber, Sophie-Charlotte; Vine, David; Finney, Lydia; Chen, Si; Hong, Young Pyo; Uyarte, Omar; Vogt, Stefan; Jellison, Jody; Goodell, Barry; Jakes, Joseph E.

2017-01-01

The role of ions in the fungal decay process of lignocellulose biomaterials, and more broadly fungal metabolism, has implications for diverse research disciplines ranging from plant pathology and forest ecology, to carbon sequestration. Despite the importance of ions in fungal decay mechanisms, the spatial distribution and quantification of ions in lignocellulosic cell walls and fungal hyphae during decay is not known. Here we employ synchrotron-based X-ray fluorescence microscopy (XFM) to map and quantify physiologically relevant ions, such as K, Ca, Mn, Fe, and Zn, in wood being decayed by the model brown rot fungus Serpula lacrymans. Two-dimensional XFM maps were obtained to study the ion spatial distributions from mm to submicron length scales in wood, fungal hyphae with the dried extracellular matrix (ECM) from the fungus, and Ca oxalate crystals. Three-dimensional ion volume reconstructions were also acquired of wood cell walls and hyphae with ECM. Results show that the fungus actively transports some ions, such as Fe, into the wood and controls the distribution of ions at both the bulk wood and cell wall length scales. These measurements provide new insights into the movement of ions during decay and illustrate how synchrotron-based XFM is uniquely suited study these ions.

Chaotic and stable perturbed maps: 2-cycles and spatial models

NASA Astrophysics Data System (ADS)

Braverman, E.; Haroutunian, J.

2010-06-01

As the growth rate parameter increases in the Ricker, logistic and some other maps, the models exhibit an irreversible period doubling route to chaos. If a constant positive perturbation is introduced, then the Ricker model (but not the classical logistic map) experiences period doubling reversals; the break of chaos finally gives birth to a stable two-cycle. We outline the maps which demonstrate a similar behavior and also study relevant discrete spatial models where the value in each cell at the next step is defined only by the values at the cell and its nearest neighbors. The stable 2-cycle in a scalar map does not necessarily imply 2-cyclic-type behavior in each cell for the spatial generalization of the map.

Three-Dimensional Gene Map of Cancer Cell Types: Structural Entropy Minimisation Principle for Defining Tumour Subtypes

PubMed Central

Li, Angsheng; Yin, Xianchen; Pan, Yicheng

2016-01-01

In this study, we propose a method for constructing cell sample networks from gene expression profiles, and a structural entropy minimisation principle for detecting natural structure of networks and for identifying cancer cell subtypes. Our method establishes a three-dimensional gene map of cancer cell types and subtypes. The identified subtypes are defined by a unique gene expression pattern, and a three-dimensional gene map is established by defining the unique gene expression pattern for each identified subtype for cancers, including acute leukaemia, lymphoma, multi-tissue, lung cancer and healthy tissue. Our three-dimensional gene map demonstrates that a true tumour type may be divided into subtypes, each defined by a unique gene expression pattern. Clinical data analyses demonstrate that most cell samples of an identified subtype share similar survival times, survival indicators and International Prognostic Index (IPI) scores and indicate that distinct subtypes identified by our algorithms exhibit different overall survival times, survival ratios and IPI scores. Our three-dimensional gene map establishes a high-definition, one-to-one map between the biologically and medically meaningful tumour subtypes and the gene expression patterns, and identifies remarkable cells that form singleton submodules. PMID:26842724

A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes.

PubMed

Kim, Young-Il; Ryu, Taewoo; Lee, Judong; Heo, Young-Shin; Ahnn, Joohong; Lee, Seung-Jae; Yoo, OokJoon

2010-01-25

Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines). We screened approximately 15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy.

Rapid 3D Refractive‐Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation

PubMed Central

Habaza, Mor; Kirschbaum, Michael; Guernth‐Marschner, Christian; Dardikman, Gili; Barnea, Itay; Korenstein, Rafi; Duschl, Claus

2016-01-01

A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label‐free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive‐index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive‐index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label‐free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label‐free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids. PMID:28251046

The Arabidopsis microtubule-associated protein MAP65-3 supports infection by filamentous biotrophic pathogens by down-regulating salicylic acid-dependent defenses.

PubMed

Quentin, Michaël; Baurès, Isabelle; Hoefle, Caroline; Caillaud, Marie-Cécile; Allasia, Valérie; Panabières, Franck; Abad, Pierre; Hückelhoven, Ralph; Keller, Harald; Favery, Bruno

2016-03-01

The oomycete Hyaloperonospora arabidopsidis and the ascomycete Erysiphe cruciferarum are obligate biotrophic pathogens causing downy mildew and powdery mildew, respectively, on Arabidopsis. Upon infection, the filamentous pathogens induce the formation of intracellular bulbous structures called haustoria, which are required for the biotrophic lifestyle. We previously showed that the microtubule-associated protein AtMAP65-3 plays a critical role in organizing cytoskeleton microtubule arrays during mitosis and cytokinesis. This renders the protein essential for the development of giant cells, which are the feeding sites induced by root knot nematodes. Here, we show that AtMAP65-3 expression is also induced in leaves upon infection by the downy mildew oomycete and the powdery mildew fungus. Loss of AtMAP65-3 function in the map65-3 mutant dramatically reduced infection by both pathogens, predominantly at the stages of leaf penetration. Whole-transcriptome analysis showed an over-represented, constitutive activation of genes involved in salicylic acid (SA) biosynthesis, signaling, and defense execution in map65-3, whereas jasmonic acid (JA)-mediated signaling was down-regulated. Preventing SA synthesis and accumulation in map65-3 rescued plant susceptibility to pathogens, but not the developmental phenotype caused by cytoskeleton defaults. AtMAP65-3 thus has a dual role. It positively regulates cytokinesis, thus plant growth and development, and negatively interferes with plant defense against filamentous biotrophs. Our data suggest that downy mildew and powdery mildew stimulate AtMAP65-3 expression to down-regulate SA signaling for infection. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Tumourigenicity and radiation resistance of mesenchymal stem cells.

PubMed

D'Andrea, Filippo P; Horsman, Michael R; Kassem, Moustapha; Overgaard, Jens; Safwat, Akmal

2012-05-01

Cancer stem cells are believed to be more radiation resistant than differentiated tumour cells of the same origin. It is not known, however, whether normal nontransformed adult stem cells share the same radioresistance as their cancerous counterpart. Nontumourigenic (TERT4) and tumourigenic (TRET20) cell lines, from an immortalised mesenchymal stem cell line, were grown in culture prior to irradiation and gene expression analysis. Radiation resistance was measured using a clonogenic assay. Differences in gene expression between the two cell lines, both under nontreated and irradiated conditions, were assessed with microarrays (Affymetrix Human Exon 1.0 ST array). The cellular functions affected by the altered gene expressions were assessed through gene pathway mapping (Ingenuity Pathway Analysis). Based on the clonogenic assay the nontumourigenic cell line was found to be more sensitive to radiation than the tumourigenic cell line. Using the exon chips, 297 genes were found altered between untreated samples of the cell lines whereas only 16 genes responded to radiation treatment. Among the genes with altered expression between the untreated samples were PLAU, PLAUR, TIMP3, MMP1 and LOX. The pathway analysis based on the alteration between the untreated samples indicated cancer and connective tissue disorders. This study has shown possible common genetic events linking tumourigenicity and radiation response. The PLAU and PLAUR genes are involved in apoptosis evasion while the genes TIMP3, MMP1 and LOX are involved in regulation of the surrounding matrix. The first group may contribute to the difference in radiation resistance observed and the latter could be a major contributor to the tumourigenic capabilities by degrading the intercellular matrix. These results also indicate that cancer stem cells are more radiation resistant than stem cells of the same origin.

The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection

PubMed Central

2013-01-01

Background Chlamydia trachomatis infection results in reproductive damage in some women. The process and factors involved in this immunopathology are not well understood. This study aimed to investigate the role of primary human cellular responses to chlamydial stress response proteases and chlamydial infection to further identify the immune processes involved in serious disease sequelae. Results Laboratory cell cultures and primary human reproductive epithelial cultures produced IL-6 in response to chlamydial stress response proteases (CtHtrA and CtTsp), UV inactivated Chlamydia, and live Chlamydia. The magnitude of the IL-6 response varied considerably (up to 1000 pg ml-1) across different primary human reproductive cultures. Thus different levels of IL-6 production by reproductive epithelia may be a determinant in disease outcome. Interestingly, co-culture models with either THP-1 cells or autologous primary human PBMC generally resulted in increased levels of IL-6, except in the case of live Chlamydia where the level of IL-6 was decreased compared to the epithelial cell culture only, suggesting this pathway may be able to be modulated by live Chlamydia. PBMC responses to the stress response proteases (CtTsp and CtHtrA) did not significantly vary for the different participant cohorts. Therefore, these proteases may possess conserved innate PAMPs. MAP kinases appeared to be involved in this IL-6 induction from human cells. Finally, we also demonstrated that IL-6 was induced by these proteins and Chlamydia from mouse primary reproductive cell cultures (BALB/C mice) and mouse laboratory cell models. Conclusions We have demonstrated that IL-6 may be a key factor for the chlamydial disease outcome in humans, given that primary human reproductive epithelial cell culture showed considerable variation in IL-6 response to Chlamydia or chlamydial proteins, and that the presence of live Chlamydia (but not UV killed) during co-culture resulted in a reduced IL-6 response suggesting this response may be moderated by the presence of the organism. PMID:24238294

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

PubMed

Formosa, C; Lachaize, V; Galés, C; Rols, M P; Martin-Yken, H; François, J M; Duval, R E; Dague, E

2015-01-01

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

Human X-Linked genes regionally mapped utilizing X-autosome translocations and somatic cell hybrids.

PubMed Central

Shows, T B; Brown, J A

1975-01-01

Human genes coding for hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8; IMP:pyrophosphate phosphoribosyltransferase), glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49; D-glucose-6-phosphate:NADP+ 1-oxidoreductase), and phosphoglycerate kinase (PGK, EC 2.7.2.3; ATP:3-phospho-D-glycerate 1-phosphotransferase) have been assigned to specific regions on the long arm of the X chromosome by somatic cell gentic techniques. Gene assignment and linear order were determined by employing human somatic cells possessing an X/9 translocation or an X/22 translocation in man-mouse cell hybridization studies. The X/9 translocation involved the majority of the X long arm translocated to chromosome 9 and the X/22 translocation involved the distal half of the X long arm translocated to 22. In each case these rearrangements appeared to be reciprocal. Concordant segregation of X-linked enzymes and segments of the X chromosome generated by the translocations indicated assignment of the PGK gene to a proximal long arm region (q12-q22) and the HPRT and G6PD genes to the distal half (q22-qter) of the X long arm. Further evidence suggests a gene order on the X long arm of centromere-PGK-HPRT-G6PD. Images PMID:1056018

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

Soerensen, M.P.; Davidson, A.; Pedersen, N.F.

We use the method of cell-to-cell mapping to locate attractors, basins, and saddle nodes in the phase plane of a driven Josephson junction. The cell-mapping method is discussed in some detail, emphasizing its ability to provide a global view of the phase plane. Our computations confirm the existence of a previously reported interior crisis. In addition, we observe a boundary crisis for a small shift in one parameter. The cell-mapping method allows us to show both crises explicitly in the phase plane, at low computational cost.

Development of Maps of Simple and Complex Cells in the Primary Visual Cortex

PubMed Central

Antolík, Ján; Bednar, James A.

2011-01-01

Hubel and Wiesel (1962) classified primary visual cortex (V1) neurons as either simple, with responses modulated by the spatial phase of a sine grating, or complex, i.e., largely phase invariant. Much progress has been made in understanding how simple-cells develop, and there are now detailed computational models establishing how they can form topographic maps ordered by orientation preference. There are also models of how complex cells can develop using outputs from simple cells with different phase preferences, but no model of how a topographic orientation map of complex cells could be formed based on the actual connectivity patterns found in V1. Addressing this question is important, because the majority of existing developmental models of simple-cell maps group neurons selective to similar spatial phases together, which is contrary to experimental evidence, and makes it difficult to construct complex cells. Overcoming this limitation is not trivial, because mechanisms responsible for map development drive receptive fields (RF) of nearby neurons to be highly correlated, while co-oriented RFs of opposite phases are anti-correlated. In this work, we model V1 as two topographically organized sheets representing cortical layer 4 and 2/3. Only layer 4 receives direct thalamic input. Both sheets are connected with narrow feed-forward and feedback connectivity. Only layer 2/3 contains strong long-range lateral connectivity, in line with current anatomical findings. Initially all weights in the model are random, and each is modified via a Hebbian learning rule. The model develops smooth, matching, orientation preference maps in both sheets. Layer 4 units become simple cells, with phase preference arranged randomly, while those in layer 2/3 are primarily complex cells. To our knowledge this model is the first explaining how simple cells can develop with random phase preference, and how maps of complex cells can develop, using only realistic patterns of connectivity. PMID:21559067

18F-FLT uptake kinetics in head and neck squamous cell carcinoma: a PET imaging study.

PubMed

Liu, Dan; Chalkidou, Anastasia; Landau, David B; Marsden, Paul K; Fenwick, John D

2014-04-01

To analyze the kinetics of 3(')-deoxy-3(')-[F-18]-fluorothymidine (18F-FLT) uptake by head and neck squamous cell carcinomas and involved nodes imaged using positron emission tomography (PET). Two- and three-tissue compartment models were fitted to 12 tumor time-activity-curves (TACs) obtained for 6 structures (tumors or involved nodes) imaged in ten dynamic PET studies of 1 h duration, carried out for five patients. The ability of the models to describe the data was assessed using a runs test, the Akaike information criterion (AIC) and leave-one-out cross-validation. To generate parametric maps the models were also fitted to TACs of individual voxels. Correlations between maps of different parameters were characterized using Pearson'sr coefficient; in particular the phosphorylation rate-constants k3-2tiss and k5 of the two- and three-tissue models were studied alongside the flux parameters KFLT- 2tiss and KFLT of these models, and standardized uptake values (SUV). A methodology based on expectation-maximization clustering and the Bayesian information criterion ("EM-BIC clustering") was used to distil the information from noisy parametric images. Fits of two-tissue models 2C3K and 2C4K and three-tissue models 3C5K and 3C6K comprising three, four, five, and six rate-constants, respectively, pass the runs test for 4, 8, 10, and 11 of 12 tumor TACs. The three-tissue models have lower AIC and cross-validation scores for nine of the 12 tumors. Overall the 3C6K model has the lowest AIC and cross-validation scores and its fitted parameter values are of the same orders of magnitude as literature estimates. Maps of KFLT and KFLT- 2tiss are strongly correlated (r = 0.85) and also correlate closely with SUV maps (r = 0.72 for KFLT- 2tiss, 0.64 for KFLT). Phosphorylation rate-constant maps are moderately correlated with flux maps (r = 0.48 for k3-2tiss vs KFLT- 2tiss and r = 0.68 for k5 vs KFLT); however, neither phosphorylation rate-constant correlates significantly with SUV. EM-BIC clustering reduces the parametric maps to a small number of levels--on average 5.8, 3.5, 3.4, and 1.4 for KFLT- 2tiss, KFLT, k3-2tiss, and k5. This large simplification is potentially useful for radiotherapy dose-painting, but demonstrates the high noise in some maps. Statistical simulations show that voxel level noise degrades TACs generated from the 3C6K model sufficiently that the average AIC score, parameter bias, and total uncertainty of 2C4K model fits are similar to those of 3C6K fits, whereas at the whole tumor level the scores are lower for 3C6K fits. For the patients studied here, whole tumor FLT uptake time-courses are represented better overall by a three-tissue than by a two-tissue model. EM-BIC clustering simplifies noisy parametric maps, providing the best description of the underlying information they contain and is potentially useful for radiotherapy dose-painting. However, the clustering highlights the large degree of noise present in maps of the phosphorylation rate-constantsk5 and k3-2tiss, which are conceptually tightly linked to cellular proliferation. Methods must be found to make these maps more robust-either by constraining other model parameters or modifying dynamic imaging protocols. © 2014 American Association of Physicists in Medicine.

S-adenosyl-methionine (SAM) alters the transcriptome and methylome and specifically blocks growth and invasiveness of liver cancer cells

PubMed Central

Wang, Yan; Sun, ZhongSheng; Szyf, Moshe

2017-01-01

S-adenosyl methionine (SAM) is a ubiquitous methyl donor that was reported to have chemo- protective activity against liver cancer, however the molecular footprint of SAM is unknown. We show here that SAM selectively inhibits growth, transformation and invasiveness of hepatocellular carcinoma cell lines but not normal primary liver cells. Analysis of the transcriptome of SAM treated and untreated liver cancer cell lines HepG2 and SKhep1 and primary liver cells reveals pathways involved in cancer and metastasis that are upregulated in cancer cells and are downregulated by SAM. Analysis of the methylome using bisulfite mapping of captured promoters and enhancers reveals that SAM hyper-methylates and downregulates genes in pathways of growth and metastasis that are upregulated in liver cancer cells. Depletion of two SAM downregulated genes STMN1 and TAF15 reduces cellular transformation and invasiveness, providing evidence that SAM targets are genes important for cancer growth and invasiveness. Taken together these data provide a molecular rationale for SAM as an anticancer agent. PMID:29340097

S-adenosyl-methionine (SAM) alters the transcriptome and methylome and specifically blocks growth and invasiveness of liver cancer cells.

PubMed

Wang, Yan; Sun, ZhongSheng; Szyf, Moshe

2017-12-19

S-adenosyl methionine (SAM) is a ubiquitous methyl donor that was reported to have chemo- protective activity against liver cancer, however the molecular footprint of SAM is unknown. We show here that SAM selectively inhibits growth, transformation and invasiveness of hepatocellular carcinoma cell lines but not normal primary liver cells. Analysis of the transcriptome of SAM treated and untreated liver cancer cell lines HepG2 and SKhep1 and primary liver cells reveals pathways involved in cancer and metastasis that are upregulated in cancer cells and are downregulated by SAM. Analysis of the methylome using bisulfite mapping of captured promoters and enhancers reveals that SAM hyper-methylates and downregulates genes in pathways of growth and metastasis that are upregulated in liver cancer cells. Depletion of two SAM downregulated genes STMN1 and TAF15 reduces cellular transformation and invasiveness, providing evidence that SAM targets are genes important for cancer growth and invasiveness. Taken together these data provide a molecular rationale for SAM as an anticancer agent.

Involvement of Cot activity in the proliferation of ALCL lymphoma cells.

PubMed

Fernández, Margarita; Manso, Rebeca; Bernaldo de Quirós, Flavia; Bernáldez, Flavia; López, Pilar; Martín-Duce, Antonio; Alemany, Susana

2011-08-12

Anaplastic large-cell lymphoma (ALCL) cells overexpress CD30 on their cell surface, show increased levels of activated Erk1/2 and of JunB; participating JunB in the proliferative capacity of these lymphomas. Here, we show that ALCL lymphoma cells also present high expression levels of the proto-oncogenic Cot (MAP3K8). Using pharmacological drugs as well as the RNA interference technique we show that Cot protein is responsible for the constitutive Erk1/2 activation in the ALCL lymphoma cells, SUDHL-1. Besides, inhibition of Cot activity reduces the number of cell divisions which is achieved, at least in part, by the control that Cot exercises on the activation state of p70 S6K and on the expression levels of JunB. Since Cot represents an alternative mode, independently of RAF, to activate Erk1/2, all these data strongly suggest that molecular targeting of Cot may be a potential new specific strategy for ALCL lymphomas therapy, without the fully disturbance of the Erk1/2 function. Copyright © 2011. Published by Elsevier Inc.

Evolution of the hypoxia-sensitive cells involved in amniote respiratory reflexes

PubMed Central

Hockman, Dorit; Burns, Alan J; Schlosser, Gerhard; Gates, Keith P; Jevans, Benjamin; Mongera, Alessandro; Fisher, Shannon; Unlu, Gokhan; Knapik, Ela W; Kaufman, Charles K; Mosimann, Christian; Zon, Leonard I; Lancman, Joseph J; Dong, P Duc S; Lickert, Heiko; Tucker, Abigail S; Baker, Clare V H

2017-01-01

The evolutionary origins of the hypoxia-sensitive cells that trigger amniote respiratory reflexes – carotid body glomus cells, and ‘pulmonary neuroendocrine cells’ (PNECs) - are obscure. Homology has been proposed between glomus cells, which are neural crest-derived, and the hypoxia-sensitive ‘neuroepithelial cells’ (NECs) of fish gills, whose embryonic origin is unknown. NECs have also been likened to PNECs, which differentiate in situ within lung airway epithelia. Using genetic lineage-tracing and neural crest-deficient mutants in zebrafish, and physical fate-mapping in frog and lamprey, we find that NECs are not neural crest-derived, but endoderm-derived, like PNECs, whose endodermal origin we confirm. We discover neural crest-derived catecholaminergic cells associated with zebrafish pharyngeal arch blood vessels, and propose a new model for amniote hypoxia-sensitive cell evolution: endoderm-derived NECs were retained as PNECs, while the carotid body evolved via the aggregation of neural crest-derived catecholaminergic (chromaffin) cells already associated with blood vessels in anamniote pharyngeal arches. DOI: http://dx.doi.org/10.7554/eLife.21231.001 PMID:28387645

Absence of both Sos-1 and Sos-2 in peripheral CD4+ T cells leads to PI3K pathway activation and defects in migration

PubMed Central

Guittard, Geoffrey; Kortum, Robert L; Balagopalan, Lakshmi; Çuburu, Nicolas; Nguyen, Phan; Sommers, Connie L; Samelson, Lawrence E

2015-01-01

Sos-1 and Sos-2 are ubiquitously expressed Ras-Guanine Exchange Factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4+ T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4+ T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4+ T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration. PMID:25973715

Connexin43 synthesis, phosphorylation, and degradation in regulation of transient inhibition of gap junction intercellular communication by the phorbol ester TPA in rat liver epithelial cells.

PubMed

Rivedal, Edgar; Leithe, Edward

2005-01-15

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces transient inhibition of gap junction intercellular communication (GJIC) in several cell types. The initial block in GJIC has been attributed to protein kinase C (PKC) mediated phosphorylation of connexin gap junction proteins, including connexin43 (Cx43). Restoration of GJIC, associated with normalization of the Cx43 phosphorylation status, has been ascribed to different events, including dephosphorylation of Cx43 and de novo synthesis of Cx43 or other, non-gap junctional, proteins. The data presented suggest that restoration of GJIC during continuous TPA exposure in normal and transformed rat liver epithelial cells is dependent on synthesis of Cx43 protein, as well as the transport of already synthesized Cx43 from intracellular pools to the plasma membrane. Reactivation of inactivated Cx43 by dephosphorylation does not appear to be involved in the recovery of GJIC. Both PKC and MAP kinase is involved in TPA-induced degradation of Cx43 and inhibition of GJIC. We show that coincubation of TPA with the protein synthesis inhibitor cycloheximide or the transcription inhibitor actinomycin D results in synergistic enhancement of the level of activated ERK1/2. Together, the present data highlight Cx43 degradation and synthesis as critical determinants in TPA-induced modifications of cell-cell communication via gap junctions.

Late G1 accumulation after 2 Gy of gamma-irradiation is related to endogenous Raf-1 protein expression and intrinsic radiosensitivity in human cells.

PubMed Central

Warenius, H. M.; Jones, M.; Jones, M. D.; Browning, P. G.; Seabra, L. A.; Thompson, C. C.

1998-01-01

We have previously reported a correlation between high endogenous expression of the protein product of the RAF-1 proto-oncogene, intrinsic cellular radiosensitivity and rapid exit from a G2/M delay induced by 2 Gy of gamma-irradiation. Raf1 is a positive serine/threonine kinase signal transduction factor that relays signals from the cell membrane to the MAP kinase system further downstream and is believed to be involved in an ionizing radiation signal transduction pathway modulating the G1/S checkpoint. We therefore extended our flow cytometric studies to investigate relationships between radiosensitivity, endogenous expression of the Raf1 protein and perturbation of cell cycle checkpoints, leading to alterations in the G1, S and G2/M populations after 2 Gy of gamma-irradiation. Differences in intrinsic radiosensitivity after modulation of the G1/S checkpoint have generally been understood to involve p53 function up to the present time. A role for dominant oncogenes in control of G1/S transit in radiation-treated cells has not been identified previously. Here, we show in 12 human in vitro cancer cell lines that late G1 accumulation after 2 Gy of radiation is related to both Raf1 expression (r = 0.91, P = 0.0001) and the radiosensitivity parameter SF2 (r = -0.71, P = 0.009). PMID:9579826

Web-based network analysis and visualization using CellMaps

PubMed Central

Salavert, Francisco; García-Alonso, Luz; Sánchez, Rubén; Alonso, Roberto; Bleda, Marta; Medina, Ignacio; Dopazo, Joaquín

2016-01-01

Summary: CellMaps is an HTML5 open-source web tool that allows displaying, editing, exploring and analyzing biological networks as well as integrating metadata into them. Computations and analyses are remotely executed in high-end servers, and all the functionalities are available through RESTful web services. CellMaps can easily be integrated in any web page by using an available JavaScript API. Availability and Implementation: The application is available at: http://cellmaps.babelomics.org/ and the code can be found in: https://github.com/opencb/cell-maps. The client is implemented in JavaScript and the server in C and Java. Contact: jdopazo@cipf.es Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27296979

Web-based network analysis and visualization using CellMaps.

PubMed

Salavert, Francisco; García-Alonso, Luz; Sánchez, Rubén; Alonso, Roberto; Bleda, Marta; Medina, Ignacio; Dopazo, Joaquín

2016-10-01

: CellMaps is an HTML5 open-source web tool that allows displaying, editing, exploring and analyzing biological networks as well as integrating metadata into them. Computations and analyses are remotely executed in high-end servers, and all the functionalities are available through RESTful web services. CellMaps can easily be integrated in any web page by using an available JavaScript API. The application is available at: http://cellmaps.babelomics.org/ and the code can be found in: https://github.com/opencb/cell-maps The client is implemented in JavaScript and the server in C and Java. jdopazo@cipf.es Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

CellMap visualizes protein-protein interactions and subcellular localization

PubMed Central

Dallago, Christian; Goldberg, Tatyana; Andrade-Navarro, Miguel Angel; Alanis-Lobato, Gregorio; Rost, Burkhard

2018-01-01

Many tools visualize protein-protein interaction (PPI) networks. The tool introduced here, CellMap, adds one crucial novelty by visualizing PPI networks in the context of subcellular localization, i.e. the location in the cell or cellular component in which a PPI happens. Users can upload images of cells and define areas of interest against which PPIs for selected proteins are displayed (by default on a cartoon of a cell). Annotations of localization are provided by the user or through our in-house database. The visualizer and server are written in JavaScript, making CellMap easy to customize and to extend by researchers and developers. PMID:29497493

Labeling single cell for in-vivo study of cell fate mapping and lineage tracing

NASA Astrophysics Data System (ADS)

He, Sicong; Xu, Jin; Wu, Yi; Tian, Ye; Sun, Qiqi; Wen, Zilong; Qu, Jianan Y.

2018-02-01

Cell fate mapping and lineage tracing are significant ways to understanding the developmental origins of biological tissues. It requires labeling individual cells and tracing the development of their progeny. We develop an infrared laser-evoked gene operator heat-shock microscope system to achieve single-cell labeling in zebrafish. With a fluorescent thermometry technique, we measure the temperature increase in zebrafish tissues induced by infrared laser and identify the optimal heat shock conditions for single-cell gene induction in different types of zebrafish cells. We use this technique to study the fate mapping of T lymphocytes and discover the distinct waves of lymphopoiesis during the zebrafish development.

Assessment of yeast Saccharomyces cerevisiae component binding to Mycobacterium avium subspecies paratuberculosis using bovine epithelial cells.

PubMed

Li, Ziwei; You, Qiumei; Ossa, Faisury; Mead, Philip; Quinton, Margaret; Karrow, Niel A

2016-03-01

Since yeast Saccharomyces cerevisiae and its components are being used for the prevention and treatment of enteric diseases in different species, they may also be useful for preventing Johne's disease, a chronic inflammatory bowel disease of ruminants caused by Mycobacterium avium spp. paratuberculosis (MAP). This study aimed to identify potential yeast derivatives that may be used to help prevent MAP infection. The adherence of mCherry-labeled MAP to bovine mammary epithelial cell line (MAC-T cells) and bovine primary epithelial cells (BECs) co-cultured with yeast cell wall components (CWCs) from four different yeast strains (A, B, C and D) and two forms of dead yeast from strain A was investigated. The CWCs from all four yeast strains and the other two forms of dead yeast from strain A reduced MAP adhesion to MAC-T cells and BECs in a concentration-dependent manner after 6-h of exposure, with the dead yeast having the greatest effect. The following in vitro binding studies suggest that dead yeast and its' CWCs may be useful for reducing risk of MAP infection.

Role of extracellular matrix in regulation of staurosporine-induced apoptosis in breast cancer cells.

PubMed

Vasaturo, F; Malacrino, C; Sallusti, E; Coppotelli, G; Birarelli, P; Giuffrida, A; Albonici, L; Simonelli, L; Modesti, A; Modesti, M; Scarpa, S

2005-04-01

Autocrine and paracrine mechanisms modulate the synthesis and secretion of extracellular matrix (ECM); moreover, each component of the ECM is capable of modulating the synthesis and release of other ECM molecules. Therefore, the synthesis of ECM glycoprotein fibronectin and laminin was studied in the human breast cancer cell lines MCF7 and MDA MB 23, plated on different ECM. Our results showed that the cells plated on a fibronectin substrate increased laminin synthesis: this event correlated with an increase in alpha2 and alpha3 integrin subunits. Staurosporine-induced apoptosis was then analyzed in the cell lines plated on different ECM. Staurosporine treatment determined the apoptosis of 35 and 33% respectively of MDA MB 231 and MCF7; these values increased to 60 and 64% in cells plated on laminin, to 48 and 63% in cells plated on fibronectin and to 64 and 69% in cells plated on matrigel. Moreover, staurosporine treatment decreased bcl-2 expression in the cells plated on fibronectin and laminin. Yet, staurosporine treatment determined PARP cleavage and PARP partial disappearance when the cells were plated on matrigel. Finally, a partial loss of function mutant Ras protein that activated only Raf pathway, was expressed in MCF7, in order to identify whether the increase of apoptosis induced by extracellular matrix involved the Raf/MAP kinase pathway. The increase of apoptosis of the cells plated on matrigel suggested that the activation of the Raf pathway is probably involved in the decrease of survival on matrigel. These data demonstrate that the modification of ECM modulates the apoptotic process of breast cancer cells and suggest that it is worthwhile to dissect the role of ECM in the control of apoptotic process.

Acute monocytic leukemia and multiple abnormalities in a child with duplication of 1q detected by GTG-banding and SKY.

PubMed

Scrideli, Carlos A; Baruffi, Marcelo R; Squire, Jeremy A; Ramos, Ester S; Karaskova, Jana; Heck, Benjamin; Tone, Luiz G

2005-12-01

Patients with 1q duplication have demonstrated a wide range of multiple congenital abnormalities. Alterations involving this chromosomal region have being described in hematopoietic malignancies and a series of candidate genes that may be associated with neoplasias have been mapped in this region. We describe a case of partial trisomy 1q "syndrome" and acute monocytic leukemia. Cytogenetic study of the bone marrow cells by GTG-banding and spectral karyotyping (SKY) showed dup(1)(q23q44) in all cells analyzed. The dismorphological features with the dup(1q) suggest a constitutional chromosome alteration and the first, in our knowledge, association of a trisomy 1q "syndrome" with AML.

Solving a four-destination traveling salesman problem using Escherichia coli cells as biocomputers.

PubMed

Esau, Michael; Rozema, Mark; Zhang, Tuo Huang; Zeng, Dawson; Chiu, Stephanie; Kwan, Rachel; Moorhouse, Cadence; Murray, Cameron; Tseng, Nien-Tsu; Ridgway, Doug; Sauvageau, Dominic; Ellison, Michael

2014-12-19

The Traveling Salesman Problem involves finding the shortest possible route visiting all destinations on a map only once before returning to the point of origin. The present study demonstrates a strategy for solving Traveling Salesman Problems using modified E. coli cells as processors for massively parallel computing. Sequential, combinatorial DNA assembly was used to generate routes, in the form of plasmids made up of marker genes, each representing a path between destinations, and short connecting linkers, each representing a given destination. Upon growth of the population of modified E. coli, phenotypic selection was used to eliminate invalid routes, and statistical analysis was performed to successfully identify the optimal solution. The strategy was successfully employed to solve a four-destination test problem.

The p110α and p110β Isoforms of Class I Phosphatidylinositol 3-Kinase Are Involved in Toll-Like Receptor 5 Signaling in Epithelial Cells

PubMed Central

Ivison, Sabine M.; Khan, Mohammed A. S.; Graham, Nicholas R.; Shobab, Leila A.; Yao, Yu; Kifayet, Arnawaz; Sly, Laura M.; Steiner, Theodore S.

2010-01-01

Background. Bacterial flagellin triggers inflammation in mammalian cells via Toll-like receptor (TLR) 5. Release of the chemokine IL-8 in response to flagellin involves NF-κB, p38 MAP kinase, and phosphatidylinositol 3-kinase (PI3K). However, PI3K has been reported to be either pro- or anti-inflammatory in different model systems. We hypothesized that this could be due to different activities of the p110α and β isoforms of PI3K. Results. PI3K and Akt were rapidly activated in Caco-2 colon carcinoma cells by flagellin. Using a plasmid-based shRNA delivery system and novel p110 isoform-specific inhibitors, we found that flagellin-induced IL-8 production was dependent on both p110α and p110β. However in the mouse, inhibition of p110β but not p110α reduced the increase of serum IL-6 levels induced by intraperitoneal injection of flagellin. Conclusions. These data demonstrate that the p110α and β isoforms of class IA PI3K are both required for the proinflammatory response to flagellin. PMID:20953381

Nucleosome Positioning and NDR Structure at RNA Polymerase III Promoters

NASA Astrophysics Data System (ADS)

Helbo, Alexandra Søgaard; Lay, Fides D.; Jones, Peter A.; Liang, Gangning; Grønbæk, Kirsten

2017-02-01

Chromatin is structurally involved in the transcriptional regulation of all genes. While the nucleosome positioning at RNA polymerase II (pol II) promoters has been extensively studied, less is known about the chromatin structure at pol III promoters in human cells. We use a high-resolution analysis to show substantial differences in chromatin structure of pol II and pol III promoters, and between subtypes of pol III genes. Notably, the nucleosome depleted region at the transcription start site of pol III genes extends past the termination sequences, resulting in nucleosome free gene bodies. The +1 nucleosome is located further downstream than at pol II genes and furthermore displays weak positioning. The variable position of the +1 location is seen not only within individual cell populations and between cell types, but also between different pol III promoter subtypes, suggesting that the +1 nucleosome may be involved in the transcriptional regulation of pol III genes. We find that expression and DNA methylation patterns correlate with distinct accessibility patterns, where DNA methylation associates with the silencing and inaccessibility at promoters. Taken together, this study provides the first high-resolution map of nucleosome positioning and occupancy at human pol III promoters at specific loci and genome wide.

7. ENGINE TEST CELL BUILDING INTERIOR. WALL MAP IN CENTRAL ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

7. ENGINE TEST CELL BUILDING INTERIOR. WALL MAP IN CENTRAL BASEMENT OFFICE AREA. LOOKING SOUTHWEST. - Fairchild Air Force Base, Engine Test Cell Building, Near intersection of Arnold Street & George Avenue, Spokane, Spokane County, WA

Semi-automated quantification and neuroanatomical mapping of heterogeneous cell populations.

PubMed

Mendez, Oscar A; Potter, Colin J; Valdez, Michael; Bello, Thomas; Trouard, Theodore P; Koshy, Anita A

2018-07-15

Our group studies the interactions between cells of the brain and the neurotropic parasite Toxoplasma gondii. Using an in vivo system that allows us to permanently mark and identify brain cells injected with Toxoplasma protein, we have identified that Toxoplasma-injected neurons (TINs) are heterogeneously distributed throughout the brain. Unfortunately, standard methods to quantify and map heterogeneous cell populations onto a reference brain atlas are time consuming and prone to user bias. We developed a novel MATLAB-based semi-automated quantification and mapping program to allow the rapid and consistent mapping of heterogeneously distributed cells on to the Allen Institute Mouse Brain Atlas. The system uses two-threshold background subtraction to identify and quantify cells of interest. We demonstrate that we reliably quantify and neuroanatomically localize TINs with low intra- or inter-observer variability. In a follow up experiment, we show that specific regions of the mouse brain are enriched with TINs. The procedure we use takes advantage of simple immunohistochemistry labeling techniques, use of a standard microscope with a motorized stage, and low cost computing that can be readily obtained at a research institute. To our knowledge there is no other program that uses such readily available techniques and equipment for mapping heterogeneous populations of cells across the whole mouse brain. The quantification method described here allows reliable visualization, quantification, and mapping of heterogeneous cell populations in immunolabeled sections across whole mouse brains. Copyright © 2018 Elsevier B.V. All rights reserved.

NEW DEVELOPMENTS ON INVERSE POLYGON MAPPING TO CALCULATE GRAVITATIONAL LENSING MAGNIFICATION MAPS: OPTIMIZED COMPUTATIONS

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

Mediavilla, E.; Lopez, P.; Mediavilla, T.

2011-11-01

We derive an exact solution (in the form of a series expansion) to compute gravitational lensing magnification maps. It is based on the backward gravitational lens mapping of a partition of the image plane in polygonal cells (inverse polygon mapping, IPM), not including critical points (except perhaps at the cell boundaries). The zeroth-order term of the series expansion leads to the method described by Mediavilla et al. The first-order term is used to study the error induced by the truncation of the series at zeroth order, explaining the high accuracy of the IPM even at this low order of approximation.more » Interpreting the Inverse Ray Shooting (IRS) method in terms of IPM, we explain the previously reported N {sup -3/4} dependence of the IRS error with the number of collected rays per pixel. Cells intersected by critical curves (critical cells) transform to non-simply connected regions with topological pathologies like auto-overlapping or non-preservation of the boundary under the transformation. To define a non-critical partition, we use a linear approximation of the critical curve to divide each critical cell into two non-critical subcells. The optimal choice of the cell size depends basically on the curvature of the critical curves. For typical applications in which the pixel of the magnification map is a small fraction of the Einstein radius, a one-to-one relationship between the cell and pixel sizes in the absence of lensing guarantees both the consistence of the method and a very high accuracy. This prescription is simple but very conservative. We show that substantially larger cells can be used to obtain magnification maps with huge savings in computation time.« less

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

PubMed

Deng, Youping; Xu, Hu; Riedel, Heimo

2007-02-15

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

Atomic Force Microscopy Measurements of the Mechanical Properties of Cell Walls on Living Bacterial Cells

NASA Astrophysics Data System (ADS)

Bailey, Richard; Mullin, Nic; Turner, Robert; Foster, Simon; Hobbs, Jamie

2014-03-01

Staphylococcus aureus is a major cause of infection in humans, including the Methicillin resistant strain, MRSA. However, very little is known about the mechanical properties of these cells. Our investigations use AFM to examine live S. aureus cells to quantify mechanical properties. These were explored using force spectroscopy with different trigger forces, allowing the properties to be extracted at different indentation depths. A value for the cell wall stiffness has been extracted, along with a second, higher value which is found upon indenting at higher forces. This higher value drops as the cells are exposed to high salt, sugar and detergent concentrations, implying that this measurement contains a contribution from the internal turgor pressure. We have monitored these properties as the cells progress through the cell cycle. Force maps were taken over the cells at different stages of the growth process to identify changes in the mechanics throughout the progression of growth and division. The effect of Oxacillin has also been studied, to better understand its mechanism of action. Finally mutant strains of S. aureus and a second species Bacillus subtilis have been used to link the mechanical properties of the cell walls with the chain lengths and substructures involved.

Mechanisms and dynamics of nuclear lamina-genome interactions.

PubMed

Amendola, Mario; van Steensel, Bas

2014-06-01

The nuclear lamina (NL) interacts with the genomic DNA and is thought to influence chromosome organization and gene expression. Both DNA sequences and histone modifications are important for NL tethering of the genomic DNA. These interactions are dynamic in individual cells and can change during differentiation and development. Evidence is accumulating that the NL contributes to the repression of transcription. Advances in mapping, genome-editing and microscopy techniques are increasing our understanding of the molecular mechanisms involved in NL-genome interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular mechanisms of ulcer healing.

PubMed

Tarnawski, A

2000-04-01

An ulcer in the gastrointestinal tract is a deep necrotic lesion penetrating the entire mucosal thickness and muscularis mucosae. Ulcer healing is an active process of filling the mucosal defect with proliferating and migrating epithelial and connective tissue cells. At the ulcer margin, epithelial cells proliferate and migrate onto the granulation tissue to cover (reepithelialize) the ulcer and also invade granulation tissue to reconstruct glandular structures within the ulcer scar. The reepithelialization and reconstruction of glandular structures is controlled by growth factors: trefoil peptides, EGF, HGF, bFGF and PDGF; and locally produced cytokines by regenerating cells in an orderly fashion and integrated manner to ensure the quality of mucosal restoration. These growth factors, most notably EGF, trigger cell proliferation via signal transduction pathways involving EGF-R, adapter proteins (Grb2, Shc and Sos), Ras, Raf1 and MAP (Erk1/Erk2) kinases, which, after translocation to nuclei, activate transcription factors and cell proliferation. Cell migration requires cytoskeletal rearrangements and is controlled by growth factors via Rho/Rac and signaling pathways involving PLC-gamma, PI-3 K and phosphorylation of focal adhesion proteins. Granulation tissue develops at the ulcer base. It consists of connective tissue cells: fibroblasts, macrophages and proliferating endothelial cells forming microvessels under the control of angiogenic growth factors: bFGF, VEGF and angiopoietins, which all promote angiogenesiscapillary vessel formation, essential for the restoration of microvascular network in the mucosa and thus crucial for oxygen and nutrient supply. The major mechanism of activation of angiogenic growth factors and their receptor expression appears to be hypoxia, which activates hypoxia-inducible factor, which binds to VEGF promoter.

Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

PubMed

Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

2013-01-01

Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and may therefore be important in acrolein-induced alterations in airway epithelial function, as a contributing mechanism in tobacco-related respiratory disease.

A new map of global ecological land units—An ecophysiographic stratification approach

USGS Publications Warehouse

Sayre, Roger; Dangermond, Jack; Frye, Charlie; Vaughan, Randy; Aniello, Peter; Breyer, Sean P.; Cribbs, Douglas; Hopkins, Dabney; Nauman, Richard; Derrenbacher, William; Wright, Dawn J.; Brown, Clint; Convis, Charles; Smith, Jonathan H.; Benson, Laurence; Van Sistine, Darren; Warner, Harumi; Cress, Jill Janene; Danielson, Jeffrey J.; Hamann, Sharon L.; Cecere, Thomas; Reddy, Ashwan D.; Burton, Devon; Grosse, Andrea; True, Diane; Metzger, Marc; Hartmann, Jens; Moosdorf, Nils; Durr, Hans; Paganini, Marc; Defourny, Pierre; Arino, Olivier; Maynard, Simone; Anderson, Mark; Comer, Patrick

2014-01-01

In response to the need and an intergovernmental commission for a high resolution and data-derived global ecosystem map, land surface elements of global ecological pattern were characterized in an ecophysiographic stratification of the planet. The stratification produced 3,923 terrestrial ecological land units (ELUs) at a base resolution of 250 meters. The ELUs were derived from data on land surface features in a three step approach. The first step involved acquiring or developing four global raster datalayers representing the primary components of ecosystem structure: bioclimate, landform, lithology, and land cover. These datasets generally represent the most accurate, current, globally comprehensive, and finest spatial and thematic resolution data available for each of the four inputs. The second step involved a spatial combination of the four inputs into a single, new integrated raster dataset where every cell represents a combination of values from the bioclimate, landforms, lithology, and land cover datalayers. This foundational global raster datalayer, called ecological facets (EFs), contains 47,650 unique combinations of the four inputs. The third step involved an aggregation of the EFs into the 3,923 ELUs. This subdivision of the Earth’s surface into relatively fine, ecological land areas is designed to be useful for various types of ecosystem research and management applications, including assessments of climate change impacts to ecosystems, economic and non-economic valuation of ecosystem services, and conservation planning.

PPF Dependent Fixed Point Results for Triangular α c-Admissible Mappings

PubMed Central

Ćirić, Ljubomir; Alsulami, Saud M.; Salimi, Peyman

2014-01-01

We introduce the concept of triangular α c-admissible mappings (pair of mappings) with respect to η c nonself-mappings and establish the existence of PPF dependent fixed (coincidence) point theorems for contraction mappings involving triangular α c-admissible mappings (pair of mappings) with respect to η c nonself-mappings in Razumikhin class. Several interesting consequences of our theorems are also given. PMID:24672352

Features of the Retinotopic Representation in the Visual Wulst of a Laterally Eyed Bird, the Zebra Finch (Taeniopygia guttata)

PubMed Central

Michael, Neethu; Löwel, Siegrid; Bischof, Hans-Joachim

2015-01-01

The visual wulst of the zebra finch comprises at least two retinotopic maps of the contralateral eye. As yet, it is not known how much of the visual field is represented in the wulst neuronal maps, how the organization of the maps is related to the retinal architecture, and how information from the ipsilateral eye is involved in the activation of the wulst. Here, we have used autofluorescent flavoprotein imaging and classical anatomical methods to investigate such characteristics of the most posterior map of the multiple retinotopic representations. We found that the visual wulst can be activated by visual stimuli from a large part of the visual field of the contralateral eye. Horizontally, the visual field representation extended from -5° beyond the beak tip up to +125° laterally. Vertically, a small strip from -10° below to about +25° above the horizon activated the visual wulst. Although retinal ganglion cells had a much higher density around the fovea and along a strip extending from the fovea towards the beak tip, these areas were not overrepresented in the wulst map. The wulst area activated from the foveal region of the ipsilateral eye, overlapped substantially with the middle of the three contralaterally activated regions in the visual wulst, and partially with the other two. Visual wulst activity evoked by stimulation of the frontal visual field was stronger with contralateral than with binocular stimulation. This confirms earlier electrophysiological studies indicating an inhibitory influence of the activation of the ipsilateral eye on wulst activity elicited by stimulating the contralateral eye. The lack of a foveal overrepresentation suggests that identification of objects may not be the primary task of the zebra finch visual wulst. Instead, this brain area may be involved in the processing of visual information necessary for spatial orientation. PMID:25853253

Mapping Cellular Polarity Networks Using Mass Spectrometry-based Strategies.

PubMed

Daulat, Avais M; Puvirajesinghe, Tania M; Camoin, Luc; Borg, Jean-Paul

2018-05-18

Cell polarity is a vital biological process involved in the building, maintenance and normal functioning of tissues in invertebrates and vertebrates. Unsurprisingly, molecular defects affecting polarity organization and functions have a strong impact on tissue homeostasis, embryonic development and adult life, and may directly or indirectly lead to diseases. Genetic studies have demonstrated the causative effect of several polarity genes in diseases; however, much remains to be clarified before a comprehensive view of the molecular organization and regulation of the protein networks associated with polarity proteins is obtained. This challenge can be approached head-on using proteomics to identify protein complexes involved in cell polarity and their modifications in a spatio-temporal manner. We review the fundamental basics of mass spectrometry techniques and provide an in-depth analysis of how mass spectrometry has been instrumental in understanding the complex and dynamic nature of some cell polarity networks at the tissue (apico-basal and planar cell polarities) and cellular (cell migration, ciliogenesis) levels, with the fine dissection of the interconnections between prototypic cell polarity proteins and signal transduction cascades in normal and pathological situations. This review primarily focuses on epithelial structures which are the fundamental building blocks for most metazoan tissues, used as the archetypal model to study cellular polarity. This field offers broad perspectives thanks to the ever-increasing sensitivity of mass spectrometry and its use in combination with recently developed molecular strategies able to probe in situ proteomic networks. Copyright © 2018 Elsevier Ltd. All rights reserved.

Fine mapping of the NRC-1 tumor suppressor locus within chromosome 3p12.

PubMed

Zhang, Kun; Lott, Steven T; Jin, Li; Killary, Ann McNeill

2007-08-31

Identification of tumor suppressor genes based on physical mapping exercises has proven to be a challenging endeavor, due to the difficulty of narrowing regions of loss of heterozygosity (LOH), infrequency of homozygous deletions, and the labor-intensive characterization process for screening candidates in a given genomic interval. We previously defined a chromosome 3p12 tumor suppressor locus NRC-1 (Nonpapillary Renal Carcinoma-1) by functional complementation experiments in which renal cell carcinoma microcell hybrids containing introduced normal chromosome 3p fragments were either suppressed or unsuppressed for tumorigenicity following injection into athymic nude mice. We now present the fine-scale physical mapping of NRC-1 using a QPCR-based approach for measuring copy number at sequence tagged sites (STS) which allowed a sub-exon mapping resolution. Using STS-QPCR and a novel statistical algorithm, the NRC-1 locus was narrowed to 4.615-Mb with the distal boundary mapping within a 38-Kb interval between exon 3 and exon 4 of the DUTT1/Robo1 gene, currently the only candidate tumor suppressor gene in the interval. Further mutational screening and gene expression analyses indicate that DUTT1/ROBO1 is not involved in the tumor suppressor activity of NRC-1, suggesting that there are at least two important tumor suppressor genes within the chromosome 3p12 interval.

Global, quantitative and dynamic mapping of protein subcellular localization.

PubMed

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg Hh

2016-06-09

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology.

IDC1, a pezizomycotina-specific gene that belongs to the PaMpk1 MAP kinase transduction cascade of the filamentous fungus Podospora anserina.

PubMed

Jamet-Vierny, Corinne; Debuchy, Robert; Prigent, Magali; Silar, Philippe

2007-12-01

Components involved in the activation of the MAPK cascades in filamentous fungi are not well known. Here, we provide evidence that IDC1, a pezizomycotina-specific gene is involved along with the PaNox1 NADPH oxidase in the nuclear localization of the PaMpk1 MAP kinase, a prerequisite for MAPK activity. Mutants of IDC1 display the same phenotypes as mutants in PaNox1 and PaMpk1, i.e., lack of pigment and of aerial hyphae, female sterility, impairment in hyphal interference and inability to develop Crippled Growth cell degeneration. As observed for the PaNox1 mutant, IDC1 mutants are hypostatic to PaMpk1 mutants. IDC1 seems to play a key role in sexual reproduction. Indeed, fertility is diminished in strains with lower level of IDC1. In strains over-expressing IDC1, protoperithecia reach a later stage of development towards perithecia without fertilization; however, upon fertilization maturation of fertile perithecia is diminished and delayed. In addition, heterokaryon construction shows that IDC1 is necessary together with PaNox1 in the perithecial envelope but not in the dikaryon resulting from fertilization.

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/myeloid leukemia factor 2 (MLF2).

PubMed

Kuefer, M U; Look, A T; Williams, D C; Valentine, V; Naeve, C W; Behm, F G; Mullersman, J E; Yoneda-Kato, N; Montgomery, K; Kucherlapati, R; Morris, S W

1996-07-15

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, the MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal region frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements.

Mapping the Human Toxome by Systems Toxicology

PubMed Central

Bouhifd, Mounir; Hogberg, Helena T.; Kleensang, Andre; Maertens, Alexandra; Zhao, Liang; Hartung, Thomas

2014-01-01

Toxicity testing typically involves studying adverse health outcomes in animals subjected to high doses of toxicants with subsequent extrapolation to expected human responses at lower doses. The low-throughput of current toxicity testing approaches (which are largely the same for industrial chemicals, pesticides and drugs) has led to a backlog of more than 80,000 chemicals to which human beings are potentially exposed whose potential toxicity remains largely unknown. Employing new testing strategies that employ the use of predictive, high-throughput cell-based assays (of human origin) to evaluate perturbations in key pathways, referred as pathways of toxicity, and to conduct targeted testing against those pathways, we can begin to greatly accelerate our ability to test the vast “storehouses” of chemical compounds using a rational, risk-based approach to chemical prioritization, and provide test results that are more predictive of human toxicity than current methods. The NIH Transformative Research Grant project Mapping the Human Toxome by Systems Toxicology aims at developing the tools for pathway mapping, annotation and validation as well as the respective knowledge base to share this information. PMID:24443875

Cell force mapping using a double-sided micropillar array based on the moiré fringe method

NASA Astrophysics Data System (ADS)

Zhang, F.; Anderson, S.; Zheng, X.; Roberts, E.; Qiu, Y.; Liao, R.; Zhang, X.

2014-07-01

The mapping of traction forces is crucial to understanding the means by which cells regulate their behavior and physiological function to adapt to and communicate with their local microenvironment. To this end, polymeric micropillar arrays have been used for measuring cell traction force. However, the small scale of the micropillar deflections induced by cell traction forces results in highly inefficient force analyses using conventional optical approaches; in many cases, cell forces may be below the limits of detection achieved using conventional microscopy. To address these limitations, the moiré phenomenon has been leveraged as a visualization tool for cell force mapping due to its inherent magnification effect and capacity for whole-field force measurements. This Letter reports an optomechanical cell force sensor, namely, a double-sided micropillar array (DMPA) made of poly(dimethylsiloxane), on which one side is employed to support cultured living cells while the opposing side serves as a reference pattern for generating moiré patterns. The distance between the two sides, which is a crucial parameter influencing moiré pattern contrast, is predetermined during fabrication using theoretical calculations based on the Talbot effect that aim to optimize contrast. Herein, double-sided micropillar arrays were validated by mapping mouse embryo fibroblast contraction forces and the resulting force maps compared to conventional microscopy image analyses as the reference standard. The DMPA-based approach precludes the requirement for aligning two independent periodic substrates, improves moiré contrast, and enables efficient moiré pattern generation. Furthermore, the double-sided structure readily allows for the integration of moiré-based cell force mapping into microfabricated cell culture environments or lab-on-a-chip devices.

Hydrological Simulation of Flood Events At Large Basins Using Distributed Modelling

NASA Astrophysics Data System (ADS)

Vélez, J.; Vélez, I.; Puricelli, M.; Francés, F.

Recent advances in technology allows to the scientist community advance in new pro- cedures in order to reduce the risk associated to flood events. A conceptual distributed model has been implemented to simulate the hydrological processes involved during floods. The model has been named TETIS. The basin is divided into rectangular cells, all of them connected according to the network drainage. The rainfall-runoff process is modelled using four linked tanks at each cell with different outflow relationships at each tank, which represent the ET, direct runoff, interflow and base flow, respectively. The routing along the channel network has been proposed using basin geomorpho- logic characteristics coupled to the cinematic wave procedure. The vertical movement along the cell is proposed using simple relationships based on soil properties as field capacity and the saturated hydraulic conductivities, which were previously obtained using land use, litology, edaphology and basin properties maps. The different vertical proccesses along the cell included are: capillar storage, infiltration, percolation and underground losses. Finally, snowmelting and reservoir routing has been included. TETIS has been implemented in the flood warning system of the Tagus River, with a basin of 59 200 km2. The time discretization of the input data is 15 minutes, and the cell size is 500x500 m. The basic parameter maps were estimated for the entire basin, and a calibration and validation processes were performed using some recorded events in the upper part of the basin. Calibration confirmed the initial parameter estimation. Additionally, the validation in time and space showed the robustness of these types of models

Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

PubMed

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

2009-11-26

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

PubMed Central

Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

2010-01-01

Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

The secretome of endothelial progenitor cells promotes brain endothelial cell activity through PI3-kinase and MAP-kinase.

PubMed

Di Santo, Stefano; Seiler, Stefanie; Fuchs, Anna-Lena; Staudigl, Jennifer; Widmer, Hans Rudolf

2014-01-01

Angiogenesis and vascular remodelling are crucial events in tissue repair mechanisms promoted by cell transplantation. Current evidence underscores the importance of the soluble factors secreted by stem cells in tissue regeneration. In the present study we investigated the effects of paracrine factors derived from cultured endothelial progenitor cells (EPC) on rat brain endothelial cell properties and addressed the signaling pathways involved. Endothelial cells derived from rat brain (rBCEC4) were incubated with EPC-derived conditioned medium (EPC-CM). The angiogenic response of rBCEC4 to EPC-CM was assessed as effect on cell number, migration and tubular network formation. In addition, we have compared the outcome of the in vitro experiments with the effects on capillary sprouting from rat aortic rings. The specific PI3K/AKT inhibitor LY294002 and the MEK/ERK inhibitor PD98059 were used to study the involvement of these two signaling pathways in the transduction of the angiogenic effects of EPC-CM. Viable cell number, migration and tubule network formation were significantly augmented upon incubation with EPC-CM. Similar findings were observed for aortic ring outgrowth with significantly longer sprouts. The EPC-CM-induced activities were significantly reduced by the blockage of the PI3K/AKT and MEK/ERK signaling pathways. Similarly to the outcome of the rBCEC4 experiments, inhibition of the PI3K/AKT and MEK/ERK pathways significantly interfered with capillary sprouting induced by EPC-CM. The present study demonstrates that EPC-derived paracrine factors substantially promote the angiogenic response of brain microvascular endothelial cells. In addition, our findings identified the PI3K/AKT and MEK/ERK pathways to play a central role in mediating these effects.

Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis

PubMed Central

McIntosh, Avery L.; Senthivinayagam, Subramanian; Moon, Kenneth C.; Gupta, Shipra; Lwande, Joel S.; Murphy, Cameron C.; Storey, Stephen M.

2012-01-01

Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies (E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (>60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44–57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (>100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function. PMID:22744009

Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells

PubMed Central

Skapski, Agnès; Hygonenq, Marie-Claude; Sagné, Eveline; Guiral, Sébastien; Citti, Christine; Baranowski, Eric

2011-01-01

Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions. PMID:21966487

Mapping owl's eye cells of patients with cytomegalovirus corneal endotheliitis using in vivo laser confocal microscopy.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Sugiyama, Kazuhisa

2013-01-01

To produce a two-dimensional reconstruction map of owl's eye cells using in vivo laser confocal microscopy in patients with cytomegalovirus (CMV) corneal endotheliitis, and to demonstrate any association between owl's eye cells and coin-shaped lesions observed with slit-lamp biomicroscopy. Two patients (75- and 77-year-old men) with polymerase chain reaction-proven CMV corneal endotheliitis were evaluated in this study. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Images of owl's eye cells in the endothelial cell layer were arranged and mapped into subconfluent montages. Montage images of owl's eye cells were then superimposed on a slit-lamp photo of the corresponding coin-shaped lesion. Degree of concordance between the confocal microscopic images and slit-lamp photos was evaluated. In both eyes, a two-dimensional reconstruction map of the owl's eye cells was created by computer software using acquired confocal images; the maps showed circular patterns. Superimposing montage images of owl's eye cells onto the photos of a coin-shaped lesion showed good concordance in the two eyes. This study suggests that there is an association between owl's eye cells observed by confocal microscopy and coin-shaped lesions observed by slit-lamp biomicroscopy in patients with CMV corneal endotheliitis. The use of in vivo laser confocal microscopy may provide clues as to the underlying causes of CMV corneal endotheliitis.

Spatial organization of xylem cell walls by ROP GTPases and microtubule-associated proteins.

PubMed

Oda, Yoshihisa; Fukuda, Hiroo

2013-12-01

Proper patterning of cellulosic cell walls is critical for cell shaping and differentiation of plant cells. Cortical microtubule arrays regulate the deposition patterns of cellulose microfibrils by controlling the targeting and trajectory of cellulose synthase complexes. Although some microtubule-associated proteins (MAPs) regulate the arrangement of cortical microtubules, knowledge about the overall mechanism governing the spacing of cortical microtubules is still limited. Recent studies reveal that ROP GTPases and MAPs spatially regulate the assembly and disassembly of cortical microtubules in developing xylem cells, in which localized secondary cell walls are deposited. Here, we review recent insights into the regulation of xylem cell wall patterning by cortical microtubules, ROP GTPases, and MAPs. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Novel Function of the Fe65 Neuronal Adaptor in Estrogen Receptor Action in Breast Cancer Cells*

PubMed Central

Sun, Yuefeng; Kasiappan, Ravi; Tang, Jinfu; Webb, Panida L.; Quarni, Waise; Zhang, Xiaohong; Bai, Wenlong

2014-01-01

Fe65 is a multidomain adaptor with established functions in neuronal cells and neurodegeneration diseases. It binds to the C terminus of the Aβ amyloid precursor protein and is involved in regulating gene transcription. The present studies show that Fe65 is expressed in breast cancer (BCa) cells and acts as an ERα transcriptional coregulator that is recruited by 17β-estradiol to the promoters of estrogen target genes. Deletion analyses mapped the ERα binding domain to the phosphotyrosine binding domain 2 (PTB2). Ectopic Fe65 increased the transcriptional activity of the ERα in a PTB2-dependent manner in reporter assays. Fe65 knockdown decreased, whereas its stable expression increased the transcriptional activity of endogenous ERα in BCa cells and the ability of estrogens to stimulate target gene expression, ERα, and coactivator recruitment to target gene promoters and cell growth. Furthermore, Fe65 expression decreased the antagonistic activity of tamoxifen (TAM), suggesting a role for Fe65 in TAM resistance. Overall, the studies define a novel role for the neuronal adaptor in estrogen actions in BCa cells. PMID:24619425

The stimulus selectivity and connectivity of layer six principal cells reveals cortical microcircuits underlying visual processing.

PubMed

Vélez-Fort, Mateo; Rousseau, Charly V; Niedworok, Christian J; Wickersham, Ian R; Rancz, Ede A; Brown, Alexander P Y; Strom, Molly; Margrie, Troy W

2014-09-17

Sensory computations performed in the neocortex involve layer six (L6) cortico-cortical (CC) and cortico-thalamic (CT) signaling pathways. Developing an understanding of the physiological role of these circuits requires dissection of the functional specificity and connectivity of the underlying individual projection neurons. By combining whole-cell recording from identified L6 principal cells in the mouse primary visual cortex (V1) with modified rabies virus-based input mapping, we have determined the sensory response properties and upstream monosynaptic connectivity of cells mediating the CC or CT pathway. We show that CC-projecting cells encompass a broad spectrum of selectivity to stimulus orientation and are predominantly innervated by deep layer V1 neurons. In contrast, CT-projecting cells are ultrasparse firing, exquisitely tuned to orientation and direction information, and receive long-range input from higher cortical areas. This segregation in function and connectivity indicates that L6 microcircuits route specific contextual and stimulus-related information within and outside the cortical network. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Familial skewed X inactivation: a molecular trait associated with high spontaneous-abortion rate maps to Xq28.

PubMed Central

Pegoraro, E; Whitaker, J; Mowery-Rushton, P; Surti, U; Lanasa, M; Hoffman, E P

1997-01-01

We report a family ascertained for molecular diagnosis of muscular dystrophy in a young girl, in which preferential activation (> or = 95% of cells) of the paternal X chromosome was seen in both the proband and her mother. To determine the molecular basis for skewed X inactivation, we studied X-inactivation patterns in peripheral blood and/or oral mucosal cells from 50 members of this family and from a cohort of normal females. We found excellent concordance between X-inactivation patterns in blood and oral mucosal cell nuclei in all females. Of the 50 female pedigree members studied, 16 showed preferential use (> or = 95% cells) of the paternal X chromosome; none of 62 randomly selected females showed similarly skewed X inactivation was maternally inherited in this family. A linkage study using the molecular trait of skewed X inactivation as the scored phenotype localized this trait to Xq28 (DXS1108; maximum LOD score [Zmax] = 4.34, recombination fraction [theta] = 0). Both genotyping of additional markers and FISH of a YAC probe in Xq28 showed a deletion spanning from intron 22 of the factor VIII gene to DXS115-3. This deletion completely cosegregated with the trait (Zmax = 6.92, theta = 0). Comparison of clinical findings between affected and unaffected females in the 50-member pedigree showed a statistically significant increase in spontaneous-abortion rate in the females carrying the trait (P < .02). To our knowledge, this is the first gene-mapping study of abnormalities of X-inactivation patterns and is the first association of a specific locus for recurrent spontaneous abortion in a cytogenetically normal family. The involvement of this locus in cell lethality, cell-growth disadvantage, developmental abnormalities, or the X-inactivation process is discussed. Images Figure 4 Figure 7 PMID:9245997

Effects of interleukins 2 and 12 on TBT-induced alterations of MAP kinases p38 and p44/42 in human natural killer cells.

PubMed

Aluoch, Aloice O; Whalen, Margaret M

2006-01-01

NK cells are lymphocytes in the non-adaptive immune system that protect the body against intracellular pathogens and eliminate tumor cells. Tributyltin (TBT) is a toxic chemical that has been detected in human foods as well as in human blood. The role of TBT in immunosuppression has been described, including inhibition of the human NK-cell cytotoxic function. Previous studies indicated that exposure of NK cells to TBT for 1 h induced progressive and irreversible inhibition of cytotoxic function. However, it was found that if NK cells were incubated in TBT-free media with either IL-2 or IL-12, loss of cytotoxic function was prevented/reversed within 24 h. Molecular studies established that loss of cytotoxic function is accompanied by alteration of MAP kinases (MAPKs) p38 and p44/42 phosphorylation. This study examined whether interleukin-mediated recovery of cytotoxicity involved reversal of tributyltin-altered p38 and p44/42 phosphorylation. The results indicated that there was no substantial IL-2 prevention/reversal of the TBT-induced alteration of phosphorylation of either p38 or p44/42 after either a 24 or 48 h recovery period. Additionally, IL-12 caused no substantial prevention/reversal of the TBT-induced alteration of phosphorylation of the MAPKs seen after either 24 or 48 h. These data suggest that IL-2 and/or IL-12-mediated recovery of NK cytotoxic function is not a result of prevention/reversal of TBT-induced phosphorylation of p38 and p44/42 MAPKs at the 24 or 48 h time points. Copyright 2005 John Wiley & Sons, Ltd.

Probabilistic drug connectivity mapping

PubMed Central

2014-01-01

Background The aim of connectivity mapping is to match drugs using drug-treatment gene expression profiles from multiple cell lines. This can be viewed as an information retrieval task, with the goal of finding the most relevant profiles for a given query drug. We infer the relevance for retrieval by data-driven probabilistic modeling of the drug responses, resulting in probabilistic connectivity mapping, and further consider the available cell lines as different data sources. We use a special type of probabilistic model to separate what is shared and specific between the sources, in contrast to earlier connectivity mapping methods that have intentionally aggregated all available data, neglecting information about the differences between the cell lines. Results We show that the probabilistic multi-source connectivity mapping method is superior to alternatives in finding functionally and chemically similar drugs from the Connectivity Map data set. We also demonstrate that an extension of the method is capable of retrieving combinations of drugs that match different relevant parts of the query drug response profile. Conclusions The probabilistic modeling-based connectivity mapping method provides a promising alternative to earlier methods. Principled integration of data from different cell lines helps to identify relevant responses for specific drug repositioning applications. PMID:24742351

Mapping the Views of Adolescent Health Stakeholders.

PubMed

Ewan, Lindsay A; McLinden, Daniel; Biro, Frank; DeJonckheere, Melissa; Vaughn, Lisa M

2016-01-01

Health research that includes youth and family stakeholders increases the contextual relevance of findings, which can benefit both the researchers and stakeholders involved. The goal of this study was to identify youth and family adolescent health priorities and to explore strategies to address these concerns. Stakeholders identified important adolescent health concerns, perceptions of which were then explored using concept mapping. Concept mapping is a mixed-method participatory research approach that invites input from various stakeholders. In response to prompts, stakeholders suggested ways to address the identified health conditions. Adolescent participants then sorted the statements into groups based on content similarity and rated the statements for importance and feasibility. Multidimensional scaling and cluster analysis were then applied to create the concept maps. Stakeholders identified sexually transmitted infections (STIs) and obesity as the health conditions they considered most important. The concept map for STIs identified 7 clusters: General sex education, support and empowerment, testing and treatment, community involvement and awareness, prevention and protection, parental involvement in sex education, and media. The obesity concept map portrayed 8 clusters: Healthy food choices, obesity education, support systems, clinical and community involvement, community support for exercise, physical activity, nutrition support, and nutrition education. Ratings were generally higher for importance than for feasibility. The concept maps demonstrate stakeholder-driven ideas about approaches to target STIs and obesity in this context. Strategies at multiple social ecological levels were emphasized. The concept maps can be used to generate discussion regarding these topics and to identify interventions. Copyright © 2016 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.

Hsp90 binds microtubules and is involved in the reorganization of the microtubular network in angiosperms.

PubMed

Krtková, Jana; Zimmermann, Aleksandra; Schwarzerová, Kateřina; Nick, Peter

2012-09-15

Microtubules (MTs) are essential for many processes in plant cells. MT-associated proteins (MAPs) influence MT polymerization dynamics and enable them to perform their functions. The molecular chaperone Hsp90 has been shown to associate with MTs in animal and plant cells. However, the role of Hsp90-MT binding in plants has not yet been investigated. Here, we show that Hsp90 associates with cortical MTs in tobacco cells and decorates MTs in the phragmoplast. Further, we show that tobacco Hsp90_MT binds directly to polymerized MTs in vitro. The inhibition of Hsp90 by geldanamycin (GDA) severely impairs MT re-assembly after cold-induced de-polymerization. Our results indicate that the plant Hsp90 interaction with MTs plays a key role in cellular events, where MT re-organization is needed. Copyright © 2012 Elsevier GmbH. All rights reserved.

PI3K/Akt-dependent functions of TFII-I transcription factors in mouse embryonic stem cells.

PubMed

Chimge, Nyam-Osor; Makeyev, Aleksandr V; Waigel, Sabine J; Enkhmandakh, Badam; Bayarsaihan, Dashzeveg

2012-04-01

Activation of PI3K/Akt signaling is sufficient to maintain the pluripotency of mouse embryonic stem cells (mESC) and results in down-regulation of Gtf2i and Gtf2ird1 encoding TFII-I family transcription factors. To investigate how these genes might be involved in the process of embryonic stem cell differentiation, we performed expression microarray profiling of mESC upon inhibition of PI3K by LY294002. This analysis revealed significant alterations in expression of genes for specific subsets of chromatin-modifying enzymes. Surprisingly, genome-wide promoter ChIP-chip mapping indicated that the majority of differently expressed genes could be direct targets of TFII-I regulation. The data support the hypothesis that upregulation of TFII-I factors leads to activation of a specific group of developmental genes during mESC differentiation. © 2011 Wiley Periodicals, Inc.

The cellular basis of the convergence and extension of the Xenopus neural plate.

PubMed

Keller, R; Shih, J; Sater, A

1992-03-01

There is great interest in the patterning and morphogenesis of the vertebrate nervous system, but the morphogenetic movements involved in early neural development and their underlying cellular mechanisms are poorly understood. This paper describes the cellular basis of the early neural morphogenesis of Xenopus laevis. The results have important implications for neural induction. Mapping the fate map of the midneurula (Eagleson and Harris: J. Neurobiol. 21:427-440, 1990) back to the early gastrula with time-lapse video recording demonstrates that the prospective hindbrain and spinal cord are initially very wide and very short, and thus at the beginning of gastrulation all their precursor cells lie within a few cell diameters of the inducing mesoderm. In the midgastrula, the prospective hindbrain and spinal cord undergo very strong convergence and extension movements in two phases: In the first phase they primarily undergo thinning in the radial direction and lengthening (extension) in the animal-vegetal direction, and the second phase is characterized primarily by mediolateral narrowing (convergence) and anterior-posterior lengthening (extension). These movements also occur in sandwich explants of the gastrula, thus demonstrating the local autonomy of the forces producing them. Tracing cell movements with fluorescein dextran-labeled cells in embryos or explants shows that the initial thinning and extension occurs by radial intercalation of deep cells to form fewer layers of greater area, all of which is expressed as increased length. The subsequent convergence and extension occurs by mediolateral intercalation of deep cells to form a longer, narrower array. These results establish that a similar if not identical sequence of radial and mediolateral cell intercalations underlie convergence and extension of the neural and the mesoderm tissues (Wilson and Keller: Development, 112:289-300, 1991). Moreover, these results establish that radial and mediolateral intercalation are the principal neural cell behaviors induced by the planar signals emanating from the dorsal involuting marginal zone (the Spemann organizer) in the early gastrula (Keller et al: Develop. Dynamics, 193: 218-234, 1992). Radial and mediolateral intercalation are induced among the 5 to 7 rows of cells comprising the prospective hindbrain and spinal cord, thus producing the massive convergence and extension movements that narrow and elongate these regions of the nervous system in the late gastrula. A more general significance of these results is that neural induction is best analyzed and understood in terms of the dynamics of the morphogenetic processes involved.

Digital floodplain mapping and an analysis of errors involved

USGS Publications Warehouse

Hamblen, C.S.; Soong, D.T.; Cai, X.

2007-01-01

Mapping floodplain boundaries using geographical information system (GIS) and digital elevation models (DEMs) was completed in a recent study. However convenient this method may appear at first, the resulting maps potentially can have unaccounted errors. Mapping the floodplain using GIS is faster than mapping manually, and digital mapping is expected to be more common in the future. When mapping is done manually, the experience and judgment of the engineer or geographer completing the mapping and the contour resolution of the surface topography are critical in determining the flood-plain and floodway boundaries between cross sections. When mapping is done digitally, discrepancies can result from the use of the computing algorithm and digital topographic datasets. Understanding the possible sources of error and how the error accumulates through these processes is necessary for the validation of automated digital mapping. This study will evaluate the procedure of floodplain mapping using GIS and a 3 m by 3 m resolution DEM with a focus on the accumulated errors involved in the process. Within the GIS environment of this mapping method, the procedural steps of most interest, initially, include: (1) the accurate spatial representation of the stream centerline and cross sections, (2) properly using a triangulated irregular network (TIN) model for the flood elevations of the studied cross sections, the interpolated elevations between them and the extrapolated flood elevations beyond the cross sections, and (3) the comparison of the flood elevation TIN with the ground elevation DEM, from which the appropriate inundation boundaries are delineated. The study area involved is of relatively low topographic relief; thereby, making it representative of common suburban development and a prime setting for the need of accurately mapped floodplains. This paper emphasizes the impacts of integrating supplemental digital terrain data between cross sections on floodplain delineation. ?? 2007 ASCE.

Regional gene mapping using mixed radiation hybrids and reverse chromosome painting.

PubMed

Lin, J Y; Bedford, J S

1997-11-01

We describe a new approach for low-resolution physical mapping using pooled DNA probe from mixed (non-clonal) populations of human-CHO cell hybrids and reverse chromosome painting. This mapping method is based on a process in which the human chromosome fragments bearing a complementing gene were selectively retained in a large non-clonal population of CHO-human hybrid cells during a series of 12- to 15-Gy gamma irradiations each followed by continuous growth selection. The location of the gene could then be identified by reverse chromosome painting on normal human metaphase spreads using biotinylated DNA from this population of "enriched" hybrid cells. We tested the validity of this method by correctly mapping the complementing human HPRT gene, whose location is well established. We then demonstrated the method's usefulness by mapping the chromosome location of a human gene which complemented the defect responsible for the hypersensitivity to ionizing radiation in CHO irs-20 cells. This method represents an efficient alternative to conventional concordance analysis in somatic cell hybrids where detailed chromosome analysis of numerous hybrid clones is necessary. Using this approach, it is possible to localize a gene for which there is no prior sequence or linkage information to a subchromosomal region, thus facilitating association with known mapping landmarks (e.g. RFLP, YAC or STS contigs) for higher-resolution mapping.

Cloning a balanced t(9;11)(p24;q23.1) chromosomal translocation breakpoint segregating with bipolar affective disorder in a small pedigree

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

Duggan, D.J.; Baysal, B.E.; Gollin, S.M.

A small multigenerational pedigree was previously identified in which a balanced 9;11 chromosomal translocation was cosegregating with bipolar affective disorder. We hypothesize that genes or gene regulatory sequences disrupted by the translocation are contributing to bipolar affective disorder in a dominant fashion. The general strategy involves (1) using somatic cell hybrids containing the derivative 9 or 11 chromosomes to identify the closest chromosome 9 and 11 flanking markers, (2) using the nearest markers as PCR and hybridization probes to isolate both normal DNA (YAC) and patient DNA (cosmid) adjacent to and incorporating the translocation breakpoint, and (3) identifying expressed sequencesmore » in the genomic DNA that may be disrupted by the translocation. From a fusion of the translocation patient cell line and a recipient hamster cell line, somatic cell hybrids were isolated which contain either the human derivative 9 or derivative 11 chromosome. Using PCR-based STS assays with these hybrids, the location of the translocation breakpoint was localized to an estimated 500 kb region at chromosome 11 band q23.1 and a 1 cM region in 9 band p24 (more telomeric than originally reported). From a large set of CEPH and Roswell Park yeast artificial chromosomes (YACs), six chromosome 11 YACs spanning the 11q23.1 breakpoint have now been identified. A combination of pulsed field gel eletrophoresis and YAC mapping has narrowed the chromosome 11 region to less than 430 kb. Current efforts are focused on generating new chromosome 11 probes within the flanking markers, mapping these probes back to the der(9) and der(11) containing hybrids and the chromosome 11 YAC mapping panel. As the region is physically narrowed, we will identify candidate genes whose expression may be altered by this t(9:11) translocation.« less

Mechanisms underlying modulation of monocarboxylate transporter 1 (MCT1) by somatostatin in human intestinal epithelial cells.

PubMed

Saksena, Seema; Theegala, Saritha; Bansal, Nikhil; Gill, Ravinder K; Tyagi, Sangeeta; Alrefai, Waddah A; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

2009-11-01

Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine. The present studies were undertaken to examine the effects of SST on butyrate uptake utilizing postconfluent human intestinal epithelial Caco2 cells. Apical SST treatment of Caco-2 cells for 30-60 min significantly increased butyrate uptake in a dose-dependent manner with maximal increase at 50 nM ( approximately 60%, P < 0.05). SST receptor 2 agonist, seglitide, mimicked the effects of SST on butyrate uptake. SST-mediated stimulation of butyrate uptake involved the p38 MAP kinase-dependent pathway. Kinetic studies demonstrated that SST increased the maximal velocity (V(max)) of the transporter by approximately twofold without any change in apparent Michaelis-Menten constant (K(m)). The higher butyrate uptake in response to SST was associated with an increase in the apical membrane levels of MCT1 protein parallel to a decrease in the intracellular MCT1 pool. MCT1 has been shown to interact specifically with CD147 glycoprotein/chaperone to facilitate proper expression and function of MCT1 at the cell surface. SST significantly enhanced the membrane levels of CD147 as well as its association with MCT1. This association was completely abolished by the specific p38 MAP kinase inhibitor, SB203580. Our findings demonstrate that increased MCT1 association with CD147 at the apical membrane in response to SST is p38 MAP kinase dependent and underlies the stimulatory effects of SST on butyrate uptake.

Evaluation of two novel methods for assessing intracellular oxygen

NASA Astrophysics Data System (ADS)

Williams, Catrin F.; Kombrabail, M.; Vijayalakshmi, K.; White, Nick; Krishnamoorthy, G.; Lloyd, David

2012-08-01

The ability to resolve the spatio-temporal complexity of intracellular O2 distribution is the ‘Holy Grail’ of cellular physiology. In an effort to obtain a non-invasive approach of mapping intracellular O2 tensions, two methods of phosphorescent lifetime imaging microscopy were examined in the current study. These were picosecond time-resolved epiphosphorescence microscopy (single 0.5 µm focused spot) and two-photon confocal laser scanning microscopy with pinhole shifting. Both methods utilized nanoparticle-embedded Ru complex (45 nm diameter) as the phosphorescent probe, excited using pulsed outputs of Ti-sapphire Tsunami lasers (710-1050 nm). The former method used a 1 ps pulse width excitation beam with vertical polarization via a dichroic mirror (610 nm, XF43) and a 20× objective (NA 0.55, Nikon). Transmitted luminescence (1-2 × 104 counts s-1) was collected and time-correlated single photon counted decay times measured. Alternatively, an unmodified Zeiss LSM510 Confocal NLO microscope with 40× objective (NA 1.3) used successively shifted pinhole positions to collect image data from the lagging trail of the raster scan. Images obtained from two-photon excitation of a yeast (Schizosaccharomyces pombe) and a flagellate fish parasite (Spironucleus vortens), electroporated with Ru complex, indicated the intracellular location and magnitude of O2 gradients, thus confirming the feasibility of optical mapping under different external O2 concentrations. Both methods gave similar lifetimes for Ru complex phosphorescence under aerobic and anaerobic gas phases. Estimation of O2 tensions within individual fibroblasts (human dermal fibroblast (HDF)) and mammary adenocarcinoma (MCF-7) cells was possible using epiphosphorescence microscopy. MCF-7 cells showed lower intracellular O2 concentrations than HDF cells, possibly due to higher metabolic rates in the former. Future work should involve construction of higher resolution 3D maps of Ru coordinate complex lifetime distribution in cultured cell lines.

Mapping-by-sequencing of Ligon-lintless-1 (Li 1 ) reveals a cluster of neighboring genes with correlated expression in developing fibers of Upland cotton (Gossypium hirsutum L.).

PubMed

Thyssen, Gregory N; Fang, David D; Turley, Rickie B; Florane, Christopher; Li, Ping; Naoumkina, Marina

2015-09-01

Mapping-by-sequencing and SNP marker analysis were used to fine map the Ligon-lintless-1 ( Li 1 ) short fiber mutation in tetraploid cotton to a 255-kb region that contains 16 annotated proteins. The Ligon-lintless-1 (Li 1 ) mutant of cotton (Gossypium hirsutum L.) has been studied as a model for cotton fiber development since its identification in 1929; however, the causative mutation has not been identified yet. Here we report the fine genetic mapping of the mutation to a 255-kb region that contains only 16 annotated genes in the reference Gossypium raimondii genome. We took advantage of the incompletely dominant dwarf vegetative phenotype to identify 100 mutants (Li 1 /Li 1 ) and 100 wild-type (li 1 /li 1 ) homozygotes from a mapping population of 2567 F2 plants, which we bulked and deep sequenced. Since only homozygotes were sequenced, we were able to use a high stringency in SNP calling to rapidly narrow down the region harboring the Li 1 locus, and designed subgenome-specific SNP markers to test the population. We characterized the expression of all sixteen genes in the region by RNA sequencing of elongating fibers and by RT-qPCR at seven time points spanning fiber development. One of the most highly expressed genes found in this interval in wild-type fiber cells is 40-fold under-expressed at the day of anthesis (DOA) in the mutant fiber cells.  This gene is a major facilitator superfamily protein, part of the large family of proteins that includes auxin and sugar transporters. Interestingly, nearly all genes in this region were most highly expressed at DOA and showed a high degree of co-expression. Further characterization is required to determine if transport of hormones or carbohydrates is involved in both the dwarf and lintless phenotypes of Li 1 plants.

[MAP kinases--molecular transistors in animals and plants].

PubMed

Petersen, Morten; Brodersen, Peter; Mundy, John

2002-06-10

The survival of multicellular organisms depends on the ability of their cells to communicate with each other and to respond to environmental changes. A goal of modern biology is to uncover the processes by which these cellular signals are transduced. Recent studies have shown that MAP-kinases (MAPKs) are important constituents of such signal transduction pathways. MAPKs function as modules in phosphorelay cascades to activate or repress the activity of downstream target proteins. For example, recent research with knockout mice has shown that mammalian MAPKs are involved in the control of neuronal apoptosis and the activation of immune responses. These mammalian MAPKs exert their control by both promoting and inhibiting specific processes. Surprisingly, plants also use MAPKs to control their immune responses, and plant MAPKs also seem to play dual roles as positive and negative regulators. Such mechanistic similarities provide the basis for fruitful conceptual exchange between molecular research on animals and plants.

Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

PubMed

Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

2016-09-01

Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

KSC-99pp1417

NASA Image and Video Library

1999-12-13

KENNEDY SPACE CENTER, Fla. -- Space Shuttle Endeavour is viewed atop the mobile launcher platform on its way to Launch Pad 39A for launch of mission STS-99. Named the Shuttle Radar Topography Mission (SRTM), STS-99 involves an international project spearheaded by the National Imagery and Mapping Agency and NASA, with participation of the German Aerospace Center DLR. SRTM will chart a new course, using two antennae and a 200-foot-long section of space station-derived mast protruding from its payload bay, to produce unrivaled 3-D images of the Earth's surface. The result of the Shuttle Radar Topography Mission could be close to 1 trillion measurements of the Earth's topography. Besides contributing to the production of better maps, these measurements could lead to improved water drainage modeling, more realistic flight simulators, better locations for cell phone towers, and enhanced navigation safety. STS-99 is scheduled for launch in January 2000

KSC-99pp1418

NASA Image and Video Library

1999-12-13

KENNEDY SPACE CENTER, Fla. -- Under breaking clouds, Space Shuttle Endeavour, atop the mobile launcher platform and crawler-transporter, crawls its way to Launch Pad 39A for mission STS-99. Named the Shuttle Radar Topography Mission (SRTM), STS-99 involves an international project spearheaded by the National Imagery and Mapping Agency and NASA, with participation of the German Aerospace Center DLR. SRTM will chart a new course, using two antennae and a 200-foot-long section of space station-derived mast protruding from its payload bay, to produce unrivaled 3-D images of the Earth's surface. The result of the Shuttle Radar Topography Mission could be close to 1 trillion measurements of the Earth's topography. Besides contributing to the production of better maps, these measurements could lead to improved water drainage modeling, more realistic flight simulators, better locations for cell phone towers, and enhanced navigation safety. STS-99 is scheduled for launch in January 2000

Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

PubMed Central

Zaitsevskaya-Carter, T; Cooper, J A

1997-01-01

A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

Cell Based Associations: A procedure for considering scarce and mixed mineral occurrences in predictive mapping

NASA Astrophysics Data System (ADS)

Tourlière, Bruno; Pakyuz-Charrier, Evren; Cassard, Daniel; Barbanson, Luc; Gumiaux, Charles

2015-05-01

Cell Based Association is an innovative mineral favorability procedure designed to answer special needs of the mining industry in data wise critical situations where usual favorability methods may not yield satisfactory results. Those situations relate to input data quality (e.g. clustered points, mixed and scarce data, approximate location) or some assumptions that are considered unreasonable (e.g. map areas relevance, conditional independence). The principle of CBA consists in replacing polygons of geological units with a square cell grid (hence the 'cell-based'). Each cell contains a range of units ('association') that are binary coded in terms of their presence (1) or absence (0) within study area. The loss of resolution inherent to this procedure is compensated by the enriched information contained in each cell owing to the notion of (lithological) association. Lithological associations are considered as binary spectra and as such are classified using Ascendant Hierarchical Clustering (AHC) thus obtaining a synthetic map of lithological associations. The prospectivity map shows as favourable the cells of the same AHC classes that the ones including mineral occurrences. It was observed that CBA can distinguish between different ore deposit varieties from a blended mineral occurrences data set. CBA can theoretically include any spatialized data (e.g. geophysics, structural data) as an extra variable to specify classification and narrow favourable areas. Doing so would make it an independent favorability mapping procedure and is still under development. Cell size in a grid is a critical parameter of the procedure; it must be compatible with the looked-for phenomena and should have a sufficient lithological variability. In addition to its use for producing favorability maps, a CBA-derived map could help in understanding the background information contained in geological maps. CBA can also be applied to other fields, such as agriculture and urban planning. Modelling of mineral prospectivity is a regional- or district scale mapping activity, whether field based or GIS based, which aims to delineate prospective areas for further exploration at the next higher scales of mapping. Notwithstanding the scale of mapping, mineral prospectivity is related to the degrees of presence and degrees of importance of individual pieces of spatial evidence of occurrence of mineral deposits of the type sought. That is, in a region or district under investigation, if there are more important pieces of spatial evidence in an area than in another area, then the former is considered to have higher prospectivity than the latter".

Anti-proliferative effect of rhein, an anthraquinone isolated from Cassia species, on Caco-2 human adenocarcinoma cells

PubMed Central

Aviello, Gabriella; Rowland, Ian; Gill, Christopher I; Acquaviva, Angela Maria; Capasso, Francesco; McCann, Mark; Capasso, Raffaele; Izzo, Angelo A; Borrelli, Francesca

2010-01-01

Abstract In recent years, the use of anthraquinone laxatives, in particular senna, has been associated with damage to the intestinal epithelial layer and an increased risk of developing colorectal cancer. In this study, we evaluated the cytotoxicity of rhein, the active metabolite of senna, on human colon adenocarcinoma cells (Caco-2) and its effect on cell proliferation. Cytotoxicity studies were performed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and trans-epithelial electrical resistance (TEER) assays whereas 3H-thymidine incorporation and Western blot analysis were used to evaluate the effect of rhein on cell proliferation. Moreover, for genoprotection studies Comet assay and oxidative biomarkers measurement (malondialdehyde and reactive oxygen species) were used. Rhein (0.1–10 μg/ml) had no significant cytotoxic effect on proliferating and differentiated Caco-2 cells. Rhein (0.1 and 1 μg/ml) significantly reduced cell proliferation as well as mitogen-activated protein (MAP) kinase activation; by contrast, at high concentration (10 μg/ml) rhein significantly increased cell proliferation and extracellular-signal-related kinase (ERK) phosphorylation. Moreover, rhein (0.1–10 μg/ml): (i) did not adversely affect the integrity of tight junctions and hence epithelial barrier function; (ii) did not induce DNA damage, rather it was able to reduce H2O2-induced DNA damage and (iii) significantly inhibited the increase in malondialdehyde and reactive oxygen species (ROS) levels induced by H2O2/Fe2+. Rhein was devoid of cytotoxic and genotoxic effects in colon adenocarcinoma cells. Moreover, at concentrations present in the colon after a human therapeutic dosage of senna, rhein inhibited cell proliferation via a mechanism that seems to involve directly the MAP kinase pathway. Finally, rhein prevents the DNA damage probably via an anti-oxidant mechanism. PMID:19538468

Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

PubMed

Lucas, Thaís F G; Royer, Carine; Siu, Erica R; Lazari, Maria Fatima M; Porto, Catarina S

2010-08-01

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

Gene transfer and gene mapping in mammalian cells in culture.

PubMed

Shows, T B; Sakaguchi, A Y

1980-01-01

The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being empolyed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probles. Recently, single genes have been cloned into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed.

Injury-activated glial cells promote wound healing of the adult skin in mice.

PubMed

Parfejevs, Vadims; Debbache, Julien; Shakhova, Olga; Schaefer, Simon M; Glausch, Mareen; Wegner, Michael; Suter, Ueli; Riekstina, Una; Werner, Sabine; Sommer, Lukas

2018-01-16

Cutaneous wound healing is a complex process that aims to re-establish the original structure of the skin and its functions. Among other disorders, peripheral neuropathies are known to severely impair wound healing capabilities of the skin, revealing the importance of skin innervation for proper repair. Here, we report that peripheral glia are crucially involved in this process. Using a mouse model of wound healing, combined with in vivo fate mapping, we show that injury activates peripheral glia by promoting de-differentiation, cell-cycle re-entry and dissemination of the cells into the wound bed. Moreover, injury-activated glia upregulate the expression of many secreted factors previously associated with wound healing and promote myofibroblast differentiation by paracrine modulation of TGF-β signalling. Accordingly, depletion of these cells impairs epithelial proliferation and wound closure through contraction, while their expansion promotes myofibroblast formation. Thus, injury-activated glia and/or their secretome might have therapeutic potential in human wound healing disorders.

Signalling maps in cancer research: construction and data analysis

PubMed Central

Kondratova, Maria; Sompairac, Nicolas; Barillot, Emmanuel; Zinovyev, Andrei

2018-01-01

Abstract Generation and usage of high-quality molecular signalling network maps can be augmented by standardizing notations, establishing curation workflows and application of computational biology methods to exploit the knowledge contained in the maps. In this manuscript, we summarize the major aims and challenges of assembling information in the form of comprehensive maps of molecular interactions. Mainly, we share our experience gained while creating the Atlas of Cancer Signalling Network. In the step-by-step procedure, we describe the map construction process and suggest solutions for map complexity management by introducing a hierarchical modular map structure. In addition, we describe the NaviCell platform, a computational technology using Google Maps API to explore comprehensive molecular maps similar to geographical maps and explain the advantages of semantic zooming principles for map navigation. We also provide the outline to prepare signalling network maps for navigation using the NaviCell platform. Finally, several examples of cancer high-throughput data analysis and visualization in the context of comprehensive signalling maps are presented. PMID:29688383

Spatial reconstruction of single-cell gene expression data.

PubMed

Satija, Rahul; Farrell, Jeffrey A; Gennert, David; Schier, Alexander F; Regev, Aviv

2015-05-01

Spatial localization is a key determinant of cellular fate and behavior, but methods for spatially resolved, transcriptome-wide gene expression profiling across complex tissues are lacking. RNA staining methods assay only a small number of transcripts, whereas single-cell RNA-seq, which measures global gene expression, separates cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos and generated a transcriptome-wide map of spatial patterning. We confirmed Seurat's accuracy using several experimental approaches, then used the strategy to identify a set of archetypal expression patterns and spatial markers. Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems.

Spatial reconstruction of single-cell gene expression

PubMed Central

Satija, Rahul; Farrell, Jeffrey A.; Gennert, David; Schier, Alexander F.; Regev, Aviv

2015-01-01

Spatial localization is a key determinant of cellular fate and behavior, but spatial RNA assays traditionally rely on staining for a limited number of RNA species. In contrast, single-cell RNA-seq allows for deep profiling of cellular gene expression, but established methods separate cells from their native spatial context. Here we present Seurat, a computational strategy to infer cellular localization by integrating single-cell RNA-seq data with in situ RNA patterns. We applied Seurat to spatially map 851 single cells from dissociated zebrafish (Danio rerio) embryos, inferring a transcriptome-wide map of spatial patterning. We confirmed Seurat’s accuracy using several experimental approaches, and used it to identify a set of archetypal expression patterns and spatial markers. Additionally, Seurat correctly localizes rare subpopulations, accurately mapping both spatially restricted and scattered groups. Seurat will be applicable to mapping cellular localization within complex patterned tissues in diverse systems. PMID:25867923

Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis.

PubMed

Eckelt, Elke; Jarek, Michael; Frömke, Cornelia; Meens, Jochen; Goethe, Ralph

2014-12-06

Maintenance of metal homeostasis is crucial in bacterial pathogenicity as metal starvation is the most important mechanism in the nutritional immunity strategy of host cells. Thus, pathogenic bacteria have evolved sensitive metal scavenging systems to overcome this particular host defence mechanism. The ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) displays a unique gut tropism and causes a chronic progressive intestinal inflammation. MAP possesses eight conserved lineage specific large sequence polymorphisms (LSP), which distinguish MAP from its ancestral M. avium ssp. hominissuis or other M. avium subspecies. LSP14 and LSP15 harbour many genes proposed to be involved in metal homeostasis and have been suggested to substitute for a MAP specific, impaired mycobactin synthesis. In the present study, we found that a LSP14 located putative IrtAB-like iron transporter encoded by mptABC was induced by zinc but not by iron starvation. Heterologous reporter gene assays with the lacZ gene under control of the mptABC promoter in M. smegmatis (MSMEG) and in a MSMEG∆furB deletion mutant revealed a zinc dependent, metalloregulator FurB mediated expression of mptABC via a conserved mycobacterial FurB recognition site. Deep sequencing of RNA from MAP cultures treated with the zinc chelator TPEN revealed that 70 genes responded to zinc limitation. Remarkably, 45 of these genes were located on a large genomic island of approximately 90 kb which harboured LSP14 and LSP15. Thirty-five of these genes were predicted to be controlled by FurB, due to the presence of putative binding sites. This clustering of zinc responsive genes was exclusively found in MAP and not in other mycobacteria. Our data revealed a particular genomic signature for MAP given by a unique zinc specific locus, thereby suggesting an exceptional relevance of zinc for the metabolism of MAP. MAP seems to be well adapted to maintain zinc homeostasis which might contribute to the peculiarity of MAP pathogenicity.

MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos

PubMed Central

Kisielewska, J.; Philipova, R.; Huang, J.-Y.; Whitaker, M.

2009-01-01

Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca2+ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27kip1 and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27kip1 had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation. This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells. PMID:19665013

Hematopoietic transcriptional mechanisms: from locus-specific to genome-wide vantage points.

PubMed

DeVilbiss, Andrew W; Sanalkumar, Rajendran; Johnson, Kirby D; Keles, Sunduz; Bresnick, Emery H

2014-08-01

Hematopoiesis is an exquisitely regulated process in which stem cells in the developing embryo and the adult generate progenitor cells that give rise to all blood lineages. Master regulatory transcription factors control hematopoiesis by integrating signals from the microenvironment and dynamically establishing and maintaining genetic networks. One of the most rudimentary aspects of cell type-specific transcription factor function, how they occupy a highly restricted cohort of cis-elements in chromatin, remains poorly understood. Transformative technologic advances involving the coupling of next-generation DNA sequencing technology with the chromatin immunoprecipitation assay (ChIP-seq) have enabled genome-wide mapping of factor occupancy patterns. However, formidable problems remain; notably, ChIP-seq analysis yields hundreds to thousands of chromatin sites occupied by a given transcription factor, and only a fraction of the sites appear to be endowed with critical, non-redundant function. It has become en vogue to map transcription factor occupancy patterns genome-wide, while using powerful statistical tools to establish correlations to inform biology and mechanisms. With the advent of revolutionary genome editing technologies, one can now reach beyond correlations to conduct definitive hypothesis testing. This review focuses on key discoveries that have emerged during the path from single loci to genome-wide analyses, specifically in the context of hematopoietic transcriptional mechanisms. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

PubMed

Yang, Wang-Yong; Wilson, Henry D; Velagapudi, Sai Pradeep; Disney, Matthew D

2015-04-29

One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.

Functional Toxicogenomic Assessment of Triclosan in Human HepG2 Cells Using Genome-Wide CRISPR-Cas9 Screening.

PubMed

Xia, Pu; Zhang, Xiaowei; Xie, Yuwei; Guan, Miao; Villeneuve, Daniel L; Yu, Hongxia

2016-10-04

There are thousands of chemicals used by humans and detected in the environment for which limited or no toxicological data are available. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify the potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes at IC50 (the concentration causing a 50% reduction in cell viability) were significantly enriched in the adherens junction pathway, MAPK signaling pathway, and PPAR signaling pathway, suggesting a potential role in the molecular mechanism of TCS-induced cytotoxicity. Evaluation of the top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with transcriptomic profiling of TCS at concentrations of

Functional mapping of cell surface proteins with localized stimulation of single cells

NASA Astrophysics Data System (ADS)

Sun, Bingyun; Chiu, Daniel T.

2003-11-01

This paper describes the development of using individual micro and nano meter-sized vesicles as delivery vessels to functionally map the distribution of cell surface proteins at the level of single cells. The formation of different sizes of vesicles from tens of nanometers to a few micrometers in diameter that contain the desired molecules is addressed. An optical trap is used to manipulate the loaded vesicle to specific cell morphology of interest, and a pulsed UV laser is used to photo-release the stimuli onto the cell membrane. Carbachol activated cellular calcium flux, upon binding to muscarinic acetylcholine receptors, is studied by this method, and the potential of using this method for the functional mapping of localized proteins on the cell surface membrane is discussed.

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

PubMed Central

2009-01-01

Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity. Conclusion Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression. PMID:19545448

An Inquiry-Oriented Curriculum in Map Making and Map Interpretation for the Intermediate Grades.

ERIC Educational Resources Information Center

Janeway, W. Whitney

This publication contains class activities and provocative inquiry questions for intermediate-grade teachers to use to involve students in map making and map interpretation. The author believes that the only things that are needed to develop an inquiry-oriented unit on mapping are a good map, a small group of students, and a perceptive teacher who…

WONOEP appraisal: new genetic approaches to study epilepsy

PubMed Central

Rossignol, Elsa; Kobow, Katja; Simonato, Michele; Loeb, Jeffrey A.; Grisar, Thierry; Gilby, Krista L.; Vinet, Jonathan; Kadam, Shilpa D.; Becker, Albert J.

2014-01-01

Objective New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming and optogenetic manipulations within epileptic networks are progressively unravelling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiological impacts of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. Methods This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy in Quebec, Canada. Results Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and have revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell-types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knockdown approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. Genetically-encoded cell-type labeling is also providing new means to assess the role of the non-neuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and non-coding RNAs involved in modifying gene expression following seizures. In addition, genetically-based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient iPS cells and genetically-modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Significance Genetics has considerably changed the field of epilepsy research and is paving the way for better diagnosis and therapies for patients with epilepsy. PMID:24965021

Superior cognitive mapping through single landmark-related learning than through boundary-related learning.

PubMed

Zhou, Ruojing; Mou, Weimin

2016-08-01

Cognitive mapping is assumed to be through hippocampus-dependent place learning rather than striatum-dependent response learning. However, we proposed that either type of spatial learning, as long as it involves encoding metric relations between locations and reference points, could lead to a cognitive map. Furthermore, the fewer reference points to specify individual locations, the more accurate a cognitive map of these locations will be. We demonstrated that participants have more accurate representations of vectors between 2 locations and of configurations among 3 locations when locations are individually encoded in terms of a single landmark than when locations are encoded in terms of a boundary. Previous findings have shown that learning locations relative to a boundary involve stronger place learning and higher hippocampal activation whereas learning relative to a single landmark involves stronger response learning and higher striatal activation. Recognizing this, we have provided evidence challenging the cognitive map theory but favoring our proposal. (PsycINFO Database Record (c) 2016 APA, all rights reserved).

Expression profiles of long non-coding RNAs located in autoimmune disease-associated regions reveal immune cell-type specificity.

PubMed

Hrdlickova, Barbara; Kumar, Vinod; Kanduri, Kartiek; Zhernakova, Daria V; Tripathi, Subhash; Karjalainen, Juha; Lund, Riikka J; Li, Yang; Ullah, Ubaid; Modderman, Rutger; Abdulahad, Wayel; Lähdesmäki, Harri; Franke, Lude; Lahesmaa, Riitta; Wijmenga, Cisca; Withoff, Sebo

2014-01-01

Although genome-wide association studies (GWAS) have identified hundreds of variants associated with a risk for autoimmune and immune-related disorders (AID), our understanding of the disease mechanisms is still limited. In particular, more than 90% of the risk variants lie in non-coding regions, and almost 10% of these map to long non-coding RNA transcripts (lncRNAs). lncRNAs are known to show more cell-type specificity than protein-coding genes. We aimed to characterize lncRNAs and protein-coding genes located in loci associated with nine AIDs which have been well-defined by Immunochip analysis and by transcriptome analysis across seven populations of peripheral blood leukocytes (granulocytes, monocytes, natural killer (NK) cells, B cells, memory T cells, naive CD4(+) and naive CD8(+) T cells) and four populations of cord blood-derived T-helper cells (precursor, primary, and polarized (Th1, Th2) T-helper cells). We show that lncRNAs mapping to loci shared between AID are significantly enriched in immune cell types compared to lncRNAs from the whole genome (α <0.005). We were not able to prioritize single cell types relevant for specific diseases, but we observed five different cell types enriched (α <0.005) in five AID (NK cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, and psoriasis; memory T and CD8(+) T cells in juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis; Th0 and Th2 cells for inflammatory bowel disease, juvenile idiopathic arthritis, primary biliary cirrhosis, psoriasis, and rheumatoid arthritis). Furthermore, we show that co-expression analyses of lncRNAs and protein-coding genes can predict the signaling pathways in which these AID-associated lncRNAs are involved. The observed enrichment of lncRNA transcripts in AID loci implies lncRNAs play an important role in AID etiology and suggests that lncRNA genes should be studied in more detail to interpret GWAS findings correctly. The co-expression results strongly support a model in which the lncRNA and protein-coding genes function together in the same pathways.

Physical mapping of chromosome 12q breakpoints in lipoma, pleomorphic salivary gland adenoma, uterine leiomyoma, and myxoid liposarcoma

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

Schoenmakers, E.F.P.M.; Kools, P.F.J.; Mols, R.

1994-03-15

The authors report here the physical mapping of recurrent chromosome 12q13-q15 breakpoints in cell lines derived from primary myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands. In fluorescence in situ hybridization (FISH) experiments, they first mapped the position of the chromosome 12 translocation breakpoint in uterine leiomyoma cell line LM-30.1/SV40 relative to loci COL2A1, D12S4, D12S17, D12S6, D12S19, D12S8, and D12S7. It mapped between linkage probes CRI-C86 (D12S19) and p7G11 (D12S8). They then isolated YAC clones using CRI-C86- and p7G11-derived sequence-tagged sites, constructed corresponding YAC contigs of 310 and 800 kb, respectively, and a mixture ofmore » them was used to routinely study the various tumor cell lines by FISH analysis. The chromosome 12 breakpoints of all tumor cell lines tested mapped between cosmids LLNL12NCO1-98C10 and LLNL12NCO1-113D12. None of the breakpoints appeared to map within any of the isolated YAC clones. Furthermore, FISH analysis using cosmid LLNL12-NCO1-144G3, which maps at the CHOP locus, revealed that the chromosome 12 breakpoints in all cell lines of the three benign solid tumors that were tested were located distal to the chromosome 12 translocation breakpoint with the CHOP gene in myxoid liposarcoma cells with t(12;16). In conclusion, the studies seem to indicate that the chromosome 12 breakpoints of myxoid liposarcoma, lipoma, uterine leiomyoma, and pleomorphic adenoma of the salivary glands are all clustered within the 7-cM interval between D12S19 and D12S8, with those of the benign solid tumors distal to CHOP. Finally, the MYF5 gene mapped telomeric to LLNL12NCO1-113D12, and the MIP gene mapped centromeric to the chromosome 12 translocation breakpoint in myxoid liposarcoma cells. 56 refs., 5 figs., 3 tabs.« less

MiroRNA-188 Acts as Tumor Suppressor in Non-Small-Cell Lung Cancer by Targeting MAP3K3.

PubMed

Zhao, Lili; Ni, Xin; Zhao, Linlin; Zhang, Yao; Jin, Dan; Yin, Wei; Wang, Dandan; Zhang, Wei

2018-04-02

Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer. MicroRNAs have been increasingly implicated in NSCLC and may serve as novel therapeutic targets to combat cancer. Here we investigated the functional implication of miR-188 in NSCLC. We first analyzed miR-188 expression in both NSCLC clinical samples and cancer cell lines. Next we investigated its role in A549 and H2126 cells with cell proliferation, migration, and apoptosis assays. To extend the in vitro study, we employed both xenograft model and LSL- K-ras G12D lung cancer model to examine the role of miR-188 in tumorigenesis. Last we tested MAP3K3 as miR-188 target in NSCLC model. MiR-188 expression was significantly downregulated at the NSCLC tumor sites and lung cancer cells. In vitro transfection of miR-188 reduced cell proliferation and migration potential and promoted cell apoptosis. In xenograft model, miR-188 inhibited tumor growth derived from cancer cells. Intranasal miR-188 administration reduced tumor formation in NSCLC animal model. MAP3K3 was validated as direct target of miR-188. Knocking down MAP3K3 in mice also inhibited tumorigenesis in LSL- K-ras G12D model. Our results demonstrate that miR-188 and its downstream target MAP3K3 could be a potential therapeutic target for NSCLC.

Transcript profiling by microarray and marker analysis of the short cotton (Gossypium hirsutum L.) fiber mutant Ligon lintless-1 (Li1).

PubMed

Gilbert, Matthew K; Turley, Rickie B; Kim, Hee Jin; Li, Ping; Thyssen, Gregory; Tang, Yuhong; Delhom, Christopher D; Naoumkina, Marina; Fang, David D

2013-06-17

Cotton fiber length is very important to the quality of textiles. Understanding the genetics and physiology of cotton fiber elongation can provide valuable tools to the cotton industry by targeting genes or other molecules responsible for fiber elongation. Ligon Lintless-1 (Li1) is a monogenic mutant in Upland cotton (Gossypium hirsutum) which exhibits an early cessation of fiber elongation resulting in very short fibers (< 6 mm) at maturity. This presents an excellent model system for studying the underlying molecular and cellular processes involved with cotton fiber elongation. Previous reports have characterized Li1 at early cell wall elongation and during later secondary cell wall synthesis, however there has been very limited analysis of the transition period between these developmental time points. Physical and morphological measurements of the Li1 mutant fibers were conducted, including measurement of the cellulose content during development. Affymetrix microarrays were used to analyze transcript profiles at the critical developmental time points of 3 days post anthesis (DPA), the late elongation stage of 12 DPA and the early secondary cell wall synthesis stage of 16 DPA. The results indicated severe disruption to key hormonal and other pathways related to fiber development, especially pertaining to the transition stage from elongation to secondary cell wall synthesis. Gene Ontology enrichment analysis identified several key pathways at the transition stage that exhibited altered regulation. Genes involved in ethylene biosynthesis and primary cell wall rearrangement were affected, and a primary cell wall-related cellulose synthase was transcriptionally repressed. Linkage mapping using a population of 2,553 F2 individuals identified SSR markers associated with the Li1 genetic locus on chromosome 22. Linkage mapping in combination with utilizing the diploid G. raimondii genome sequences permitted additional analysis of the region containing the Li1 gene. The early termination of fiber elongation in the Li1 mutant is likely controlled by an early upstream regulatory factor resulting in the altered regulation of hundreds of downstream genes. Several elongation-related genes that exhibited altered expression profiles in the Li1 mutant were identified. Molecular markers closely associated with the Li1 locus were developed. Results presented here will lay the foundation for further investigation of the genetic and molecular mechanisms of fiber elongation.

Profiling Bioactivity of the ToxCast Chemical Library Using BioMAP Primary Human Cell Systems

EPA Science Inventory

The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, ...

Automated cell-type classification in intact tissues by single-cell molecular profiling

PubMed Central

2018-01-01

A major challenge in biology is identifying distinct cell classes and mapping their interactions in vivo. Tissue-dissociative technologies enable deep single cell molecular profiling but do not provide spatial information. We developed a proximity ligation in situ hybridization technology (PLISH) with exceptional signal strength, specificity, and sensitivity in tissue. Multiplexed data sets can be acquired using barcoded probes and rapid label-image-erase cycles, with automated calculation of single cell profiles, enabling clustering and anatomical re-mapping of cells. We apply PLISH to expression profile ~2900 cells in intact mouse lung, which identifies and localizes known cell types, including rare ones. Unsupervised classification of the cells indicates differential expression of ‘housekeeping’ genes between cell types, and re-mapping of two sub-classes of Club cells highlights their segregated spatial domains in terminal airways. By enabling single cell profiling of various RNA species in situ, PLISH can impact many areas of basic and medical research. PMID:29319504

Mutations in IRX5 impair craniofacial development and germ cell migration via SDF1.

PubMed

Bonnard, Carine; Strobl, Anna C; Shboul, Mohammad; Lee, Hane; Merriman, Barry; Nelson, Stanley F; Ababneh, Osama H; Uz, Elif; Güran, Tülay; Kayserili, Hülya; Hamamy, Hanan; Reversade, Bruno

2012-05-13

Using homozygosity mapping and locus resequencing, we found that alterations in the homeodomain of the IRX5 transcription factor cause a recessive congenital disorder affecting face, brain, blood, heart, bone and gonad development. We found through in vivo modeling in Xenopus laevis embryos that Irx5 modulates the migration of progenitor cell populations in branchial arches and gonads by repressing Sdf1. We further found that transcriptional control by Irx5 is modulated by direct protein-protein interaction with two GATA zinc-finger proteins, GATA3 and TRPS1; disruptions of these proteins also cause craniofacial dysmorphisms. Our findings suggest that IRX proteins integrate combinatorial transcriptional inputs to regulate key signaling molecules involved in the ontogeny of multiple organs during embryogenesis and homeostasis.

Lighting a path: genetic studies pinpoint neurodevelopmental mechanisms in autism and related disorders.

PubMed

Pescosolido, Matthew F; Yang, Unikora; Sabbagh, Mark; Morrow, Eric M

2012-09-01

In this review, we outline critical molecular processes that have been implicated by discovery of genetic mutations in autism. These mechanisms need to be mapped onto the neurodevelopment step(s) gone awry that may be associated with cause in autism. Molecular mechanisms include: (i) regulation of gene expression; (ii) pre-mRNA splicing; (iii) protein localization, translation, and turnover; (iv) synaptic transmission; (v) cell signaling; (vi) the functions of cytoskeletal and scaffolding proteins; and (vii) the function of neuronal cell adhesion molecules. While the molecular mechanisms appear broad, they may converge on only one of a few steps during neurodevelopment that perturbs the structure, function, and/or plasticity of neuronal circuitry. While there are many genetic mutations involved, novel treatments may need to target only one of few developmental mechanisms.

The effect of surface tension on steadily translating bubbles in an unbounded Hele-Shaw cell

PubMed Central

2017-01-01

New numerical solutions to the so-called selection problem for one and two steadily translating bubbles in an unbounded Hele-Shaw cell are presented. Our approach relies on conformal mapping which, for the two-bubble problem, involves the Schottky-Klein prime function associated with an annulus. We show that a countably infinite number of solutions exist for each fixed value of dimensionless surface tension, with the bubble shapes becoming more exotic as the solution branch number increases. Our numerical results suggest that a single solution is selected in the limit that surface tension vanishes, with the scaling between the bubble velocity and surface tension being different to the well-studied problems for a bubble or a finger propagating in a channel geometry. PMID:28588410

Global, quantitative and dynamic mapping of protein subcellular localization

PubMed Central

Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

2016-01-01

Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

PubMed Central

Deschênes-Simard, Xavier; Gaumont-Leclerc, Marie-France; Bourdeau, Véronique; Lessard, Frédéric; Moiseeva, Olga; Forest, Valérie; Igelmann, Sebastian; Mallette, Frédérick A.; Saba-El-Leil, Marc K.; Meloche, Sylvain; Saad, Fred; Mes-Masson, Anne-Marie; Ferbeyre, Gerardo

2013-01-01

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. PMID:23599344

Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies.

PubMed

Jafri, Mohammad A; Ansari, Shakeel A; Alqahtani, Mohammed H; Shay, Jerry W

2016-06-20

Telomeres maintain genomic integrity in normal cells, and their progressive shortening during successive cell divisions induces chromosomal instability. In the large majority of cancer cells, telomere length is maintained by telomerase. Thus, telomere length and telomerase activity are crucial for cancer initiation and the survival of tumors. Several pathways that regulate telomere length have been identified, and genome-scale studies have helped in mapping genes that are involved in telomere length control. Additionally, genomic screening for recurrent human telomerase gene hTERT promoter mutations and mutations in genes involved in the alternative lengthening of telomeres pathway, such as ATRX and DAXX, has elucidated how these genomic changes contribute to the activation of telomere maintenance mechanisms in cancer cells. Attempts have also been made to develop telomere length- and telomerase-based diagnostic tools and anticancer therapeutics. Recent efforts have revealed key aspects of telomerase assembly, intracellular trafficking and recruitment to telomeres for completing DNA synthesis, which may provide novel targets for the development of anticancer agents. Here, we summarize telomere organization and function and its role in oncogenesis. We also highlight genomic mutations that lead to reactivation of telomerase, and mechanisms of telomerase reconstitution and trafficking that shed light on its function in cancer initiation and tumor development. Additionally, recent advances in the clinical development of telomerase inhibitors, as well as potential novel targets, will be summarized.

Spiking Neurons in a Hierarchical Self-Organizing Map Model Can Learn to Develop Spatial and Temporal Properties of Entorhinal Grid Cells and Hippocampal Place Cells

PubMed Central

Pilly, Praveen K.; Grossberg, Stephen

2013-01-01

Medial entorhinal grid cells and hippocampal place cells provide neural correlates of spatial representation in the brain. A place cell typically fires whenever an animal is present in one or more spatial regions, or places, of an environment. A grid cell typically fires in multiple spatial regions that form a regular hexagonal grid structure extending throughout the environment. Different grid and place cells prefer spatially offset regions, with their firing fields increasing in size along the dorsoventral axes of the medial entorhinal cortex and hippocampus. The spacing between neighboring fields for a grid cell also increases along the dorsoventral axis. This article presents a neural model whose spiking neurons operate in a hierarchy of self-organizing maps, each obeying the same laws. This spiking GridPlaceMap model simulates how grid cells and place cells may develop. It responds to realistic rat navigational trajectories by learning grid cells with hexagonal grid firing fields of multiple spatial scales and place cells with one or more firing fields that match neurophysiological data about these cells and their development in juvenile rats. The place cells represent much larger spaces than the grid cells, which enable them to support navigational behaviors. Both self-organizing maps amplify and learn to categorize the most frequent and energetic co-occurrences of their inputs. The current results build upon a previous rate-based model of grid and place cell learning, and thus illustrate a general method for converting rate-based adaptive neural models, without the loss of any of their analog properties, into models whose cells obey spiking dynamics. New properties of the spiking GridPlaceMap model include the appearance of theta band modulation. The spiking model also opens a path for implementation in brain-emulating nanochips comprised of networks of noisy spiking neurons with multiple-level adaptive weights for controlling autonomous adaptive robots capable of spatial navigation. PMID:23577130

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway.

PubMed

Liu, Kuang-Kai; Qiu, Wei-Ru; Naveen Raj, Emmanuel; Liu, Huei-Fang; Huang, Hou-Syun; Lin, Yu-Wei; Chang, Chien-Jen; Chen, Ting-Hua; Chen, Chinpiao; Chang, Huan-Cheng; Hwang, Jenn-Kang; Chao, Jui-I

2017-01-02

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.

Managing mapping data using commercial data base management software.

USGS Publications Warehouse

Elassal, A.A.

1985-01-01

Electronic computers are involved in almost every aspect of the map making process. This involvement has become so thorough that it is practically impossible to find a recently developed process or device in the mapping field which does not employ digital processing in some form or another. This trend, which has been evolving over two decades, is accelerated by the significant improvements in capility, reliability, and cost-effectiveness of electronic devices. Computerized mapping processes and devices share a common need for machine readable data. Integrating groups of these components into automated mapping systems requires careful planning for data flow amongst them. Exploring the utility of commercial data base management software to assist in this task is the subject of this paper. -Author

Mental and Physical (MAP) Training: a neurogenesis-inspired intervention that enhances health in humans.

PubMed

Shors, Tracey J; Olson, Ryan L; Bates, Marsha E; Selby, Edward A; Alderman, Brandon L

2014-11-01

New neurons are generated in the hippocampus each day and their survival is greatly enhanced through effortful learning (Shors, 2014). The numbers of cells produced can be increased by physical exercise (van Praag, Kempermann, & Gage, 1999). These findings inspired us to develop a clinical intervention for humans known as Mental and Physical Training, or MAP Training. Each session consists of 30min of mental training with focused attention meditation (20min sitting and 10min walking). Meditation is an effortful training practice that involves learning about the transient nature of thoughts and thought patterns, and acquiring skills to recognize them without necessarily attaching meaning and/or emotions to them. The mental training component is followed by physical training with 30min of aerobic exercise performed at moderate intensity. During this component, participants learn choreographed dance routines while engaging in aerobic exercise. In a pilot "proof-of-concept" study, we provided supervised MAP Training (2 sessions per week for 8weeks) to a group of young mothers in the local community who were recently homeless, most of them having previously suffered from physical and sexual abuse, addiction, and depression. Preliminary data suggest that MAP Training improves dependent measures of aerobic fitness (as assessed by maximal rate of oxygen consumed) while decreasing symptoms of depression and anxiety. Similar changes were not observed in a group of recently homeless women who did not participate in MAP Training. It is not currently possible to determine whether new neurons in the human brain increase in number as a result of MAP Training. Rather these preliminary results of MAP Training illustrate how neuroscientific research can be translated into novel clinical interventions that benefit human health and wellness. Copyright © 2014 Elsevier Inc. All rights reserved.

Potential role of p21 Activated Kinase 1 (PAK1) in the invasion and motility of oral cancer cells.

PubMed

Parvathy, Muraleedharan; Sreeja, Sreeharshan; Kumar, Rakesh; Pillai, Madhavan Radhakrishna

2016-05-16

Oral cancer malignancy consists of uncontrolled division of cells primarily in and around the floor of the oral cavity, gingiva, oropharynx, lower lip and base of the tongue. According to GLOBOCAN 2012 report, oral cancer is one of the most common cancers among males and females in India. Even though significant advancements have been made in the field of oral cancer treatment modalities, the overall prognosis for the patients has not improved in the past few decades and hence, this demands a new thrust for the identification of novel therapeutic targets in oral cancer. p21 Activated Kinases (PAKs) are potential therapeutic targets that are involved in numerous physiological functions. PAKs are serine-threonine kinases and they serve as important regulators of cytoskeletal dynamics and cell motility, transcription through MAP kinase cascades, death and survival signalling, and cell-cycle progression. Although PAKs are known to play crucial roles in cancer progression, the role and clinical significance of PAKs in oral cancer remains poorly understood. Our results suggest that PAK1 is over-expressed in oral cancer cell lines. Stimulation of Oral Squamous Cell Carcinoma (OSCC) cells with serum growth factors leads to PAK1 re-localization and might cause a profound cytoskeletal remodelling. PAK1 was also found to be involved in the invasion, migration and cytoskeletal remodelling of OSCC cells. Our study revealed that PAK1 may play a crucial role in the progression of OSCC. Studying the role of PAK1 and its substrates is likely to enhance our understanding of oral carcinogenesis and potential therapeutic value of PAKs in oral cancer.

CD4 T Cell Dependent Colitis Exacerbation Following Re-Exposure of Mycobacterium avium ssp. paratuberculosis.

PubMed

Suwandi, Abdulhadi; Bargen, Imke; Pils, Marina C; Krey, Martina; Zur Lage, Susanne; Singh, Anurag K; Basler, Tina; Falk, Christine S; Seidler, Ursula; Hornef, Mathias W; Goethe, Ralph; Weiss, Siegfried

2017-01-01

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic inflammatory bowel disease of cattle characterized by intermittent to chronic diarrhea. In addition, MAP has been isolated from Crohn's disease (CD) patients. The impact of MAP on severity of clinical symptoms in JD as well as its role in CD are yet unknown. We have previously shown that MAP is able to colonize inflamed enteric tissue and to exacerbate the inflammatory tissue response (Suwandi et al., 2014). In the present study, we analyzed how repeated MAP administration influences the course of dextran sulfate sodium (DSS)-induced colitis. In comparison to mice exposed to DSS or MAP only, repeated exposure of DSS-treated mice to MAP (DSS/MAP) revealed a significantly enhanced clinical score, reduction of colon length as well as severe CD4 + T cell infiltration into the colonic lamina propria . Functional analysis identified a critical role of CD4 + T cells in the MAP-induced disease exacerbation. Additionally, altered immune responses were observed when closely related mycobacteria species such as M. avium ssp. avium and M. avium ssp. hominissuis were administered. These data reveal the specific ability of MAP to aggravate intestinal inflammation and clinical symptoms. Overall, this phenotype is compatible with similar disease promoting capabilites of MAP in JD and CD.

CD4 T Cell Dependent Colitis Exacerbation Following Re-Exposure of Mycobacterium avium ssp. paratuberculosis

PubMed Central

Suwandi, Abdulhadi; Bargen, Imke; Pils, Marina C.; Krey, Martina; Zur Lage, Susanne; Singh, Anurag K.; Basler, Tina; Falk, Christine S.; Seidler, Ursula; Hornef, Mathias W.; Goethe, Ralph; Weiss, Siegfried

2017-01-01

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic inflammatory bowel disease of cattle characterized by intermittent to chronic diarrhea. In addition, MAP has been isolated from Crohn's disease (CD) patients. The impact of MAP on severity of clinical symptoms in JD as well as its role in CD are yet unknown. We have previously shown that MAP is able to colonize inflamed enteric tissue and to exacerbate the inflammatory tissue response (Suwandi et al., 2014). In the present study, we analyzed how repeated MAP administration influences the course of dextran sulfate sodium (DSS)-induced colitis. In comparison to mice exposed to DSS or MAP only, repeated exposure of DSS-treated mice to MAP (DSS/MAP) revealed a significantly enhanced clinical score, reduction of colon length as well as severe CD4+ T cell infiltration into the colonic lamina propria. Functional analysis identified a critical role of CD4+ T cells in the MAP-induced disease exacerbation. Additionally, altered immune responses were observed when closely related mycobacteria species such as M. avium ssp. avium and M. avium ssp. hominissuis were administered. These data reveal the specific ability of MAP to aggravate intestinal inflammation and clinical symptoms. Overall, this phenotype is compatible with similar disease promoting capabilites of MAP in JD and CD. PMID:28361039

Wnt1 from cochlear schwann cells enhances neuronal differentiation of transplanted neural stem cells in a rat spiral ganglion neuron degeneration model.

PubMed

He, Ya; Zhang, Peng-Zhi; Sun, Dong; Mi, Wen-Juan; Zhang, Xin-Yi; Cui, Yong; Jiang, Xing-Wang; Mao, Xiao-Bo; Qiu, Jian-Hua

2014-04-01

Although neural stem cell (NSC) transplantation is widely expected to become a therapy for nervous system degenerative diseases and injuries, the low neuronal differentiation rate of NSCs transplanted into the inner ear is a major obstacle for the successful treatment of spiral ganglion neuron (SGN) degeneration. In this study, we validated whether the local microenvironment influences the neuronal differentiation of transplanted NSCs in the inner ear. Using a rat SGN degeneration model, we demonstrated that transplanted NSCs were more likely to differentiate into microtubule-associated protein 2 (MAP2)-positive neurons in SGN-degenerated cochleae than in control cochleae. Using real-time quantitative PCR and an immunofluorescence assay, we also proved that the expression of Wnt1 (a ligand of Wnt signaling) increases significantly in Schwann cells in the SGN-degenerated cochlea. We further verified that NSC cultures express receptors and signaling components for Wnts. Based on these expression patterns, we hypothesized that Schwann cell-derived Wnt1 and Wnt signaling might be involved in the regulation of the neuronal differentiation of transplanted NSCs. We verified our hypothesis in vitro using a coculture system. We transduced a lentiviral vector expressing Wnt1 into cochlear Schwann cell cultures and cocultured them with NSC cultures. The coculture with Wnt1-expressing Schwann cells resulted in a significant increase in the percentage of NSCs that differentiated into MAP2-positive neurons, whereas this differentiation-enhancing effect was prevented by Dkk1 (an inhibitor of the Wnt signaling pathway). These results suggested that Wnt1 derived from cochlear Schwann cells enhanced the neuronal differentiation of transplanted NSCs through Wnt signaling pathway activation. Alterations of the microenvironment deserve detailed investigation because they may help us to conceive effective strategies to overcome the barrier of the low differentiation rate of transplanted NSCs.

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

PubMed

Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

2017-01-10

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation

PubMed Central

Khan, Mohammed A S; Kang, Jian; Steiner, Theodore S

2004-01-01

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhoea in a number of clinical settings. EAEC diarrhoea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, causes interleukin (IL)-8 release from several epithelial cell lines. The host response to flagellins from E. coli and several other bacteria is mediated by Toll-like receptor 5 (TLR5), which signals through nuclear factor kappa B (NF-κB) to induce transcription of pro-inflammatory cytokines. p38 mitogen-activating protein (MAP) kinase (MAPK) is a member of a family of stress-related kinases that influences a diverse range of cellular functions including host inflammatory responses to microbial products. We studied the role of p38 MAPK in FliC-EAEC-induced IL-8 secretion from Caco-2 human intestinal epithelial cells and THP-1 human monocytic cells. We found that IL-8 secretion from both cell types is dependent on p38 MAPK, which is phospho-activated in response to FliC-EAEC. The role of TLR5 in p38 MAPK-dependent IL-8 secretion was verified in HEp-2 cells transiently transfected with a TLR5 expression construct. Activation of interleukin-1 receptor-associated kinase (IRAK) was also observed in Caco-2 and TLR5-transfected HEp-2 cells after exposure to FliC-EAEC. Finally, we demonstrated that pharmacological inhibition of p38 MAPK reduced IL-8 transcription and mRNA levels, but did not affect NF-κB activation. Collectively, our results suggest that TLR5 mediates p38 MAPK-dependent IL-8 secretion from epithelial and monocytic cells incubated with FliC-EAEC, and that this effect requires IL-8 promoter activation independent of NF-κB nuclear migration. PMID:15270737

The evolution of mapping habitat for northern spotted owls (Strix occidentalis caurina): A comparison of photo-interpreted, Landsat-based, and lidar-based habitat maps

Treesearch

Steven H. Ackers; Raymond J. Davis; Keith A. Olsen; Katie M. Dugger

2015-01-01

Wildlife habitat mapping has evolved at a rapid pace over the last fewdecades. Beginning with simple, often subjective, hand-drawn maps, habitat mapping now involves complex species distribution models (SDMs) using mapped predictor variables derived from remotely sensed data. For species that inhabit large geographic areas, remote sensing technology is often...

Resource Needs and Pedagogical Value of Web Mapping for Spatial Thinking

ERIC Educational Resources Information Center

Manson, Steven; Shannon, Jerry; Eria, Sami; Kne, Len; Dyke, Kevin; Nelson, Sara; Batra, Lalit; Bonsal, Dudley; Kernik, Melinda; Immich, Jennifer; Matson, Laura

2014-01-01

Web mapping involves publishing and using maps via the Internet, and can range from presenting static maps to offering dynamic data querying and spatial analysis. Web mapping is seen as a promising way to support development of spatial thinking in the classroom but there are unanswered questions about how this promise plays out in reality. This…

Genome-Wide Association Study Reveals the Genetic Basis of Stalk Cell Wall Components in Maize

PubMed Central

Hu, Xiaojiao; Liu, Zhifang; Wu, Yujin; Huang, Changling

2016-01-01

Lignin, cellulose and hemicellulose are the three main components of the plant cell wall and can impact stalk quality by affecting cell wall structure and strength. In this study, we evaluated the lignin (LIG), cellulose (CEL) and hemicellulose (HC) contents in maize using an association mapping panel that included 368 inbred lines in seven environments. A genome-wide association study using approximately 0.56 million SNPs with a minor allele frequency of 0.05 identified 22, 18 and 24 loci significantly associated with LIG, CEL and HC at P < 1.0×10−4, respectively. The allelic variation of each significant association contributed 4 to 7% of the phenotypic variation. Candidate genes identified by GWAS mainly encode enzymes involved in cell wall metabolism, transcription factors, protein kinase and protein related to other biological processes. Among the association signals, six candidate genes had pleiotropic effects on lignin and cellulose content. These results provide valuable information for better understanding the genetic basis of stalk cell wall components in maize. PMID:27479588

Absence of both Sos-1 and Sos-2 in peripheral CD4(+) T cells leads to PI3K pathway activation and defects in migration.

PubMed

Guittard, Geoffrey; Kortum, Robert L; Balagopalan, Lakshmi; Çuburu, Nicolas; Nguyen, Phan; Sommers, Connie L; Samelson, Lawrence E

2015-08-01

Sos-1 and Sos-2 are ubiquitously expressed Ras-guanine exchange factors involved in Erk-MAP kinase pathway activation. Using mice lacking genes encoding Sos-1 and Sos-2, we evaluated the role of these proteins in peripheral T-cell signaling and function. Our results confirmed that TCR-mediated Erk activation in peripheral CD4(+) T cells does not depend on Sos-1 and Sos-2, although IL-2-mediated Erk activation does. Unexpectedly, however, we show an increase in AKT phosphorylation in Sos-1/2dKO CD4(+) T cells upon TCR and IL-2 stimulation. Activation of AKT was likely a consequence of increased recruitment of PI3K to Grb2 upon TCR and/or IL-2 stimulation in Sos-1/2dKO CD4(+) T cells. The increased activity of the PI3K/AKT pathway led to downregulation of the surface receptor CD62L in Sos-1/2dKO T cells and a subsequent impairment in T-cell migration. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway.

PubMed

Li, Xiao C; Hopfer, Ulrich; Zhuo, Jia L

2009-11-01

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1 (AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1 receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1 siRNAs inhibited AT1 expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by approximately 90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT1 expression or clathrin-dependent transferrin uptake. Both losartan and AT1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT1 (AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.

Information-dependent enrichment analysis reveals time-dependent transcriptional regulation of the estrogen pathway of toxicity.

PubMed

Pendse, Salil N; Maertens, Alexandra; Rosenberg, Michael; Roy, Dipanwita; Fasani, Rick A; Vantangoli, Marguerite M; Madnick, Samantha J; Boekelheide, Kim; Fornace, Albert J; Odwin, Shelly-Ann; Yager, James D; Hartung, Thomas; Andersen, Melvin E; McMullen, Patrick D

2017-04-01

The twenty-first century vision for toxicology involves a transition away from high-dose animal studies to in vitro and computational models (NRC in Toxicity testing in the 21st century: a vision and a strategy, The National Academies Press, Washington, DC, 2007). This transition requires mapping pathways of toxicity by understanding how in vitro systems respond to chemical perturbation. Uncovering transcription factors/signaling networks responsible for gene expression patterns is essential for defining pathways of toxicity, and ultimately, for determining the chemical modes of action through which a toxicant acts. Traditionally, transcription factor identification is achieved via chromatin immunoprecipitation studies and summarized by calculating which transcription factors are statistically associated with up- and downregulated genes. These lists are commonly determined via statistical or fold-change cutoffs, a procedure that is sensitive to statistical power and may not be as useful for determining transcription factor associations. To move away from an arbitrary statistical or fold-change-based cutoff, we developed, in the context of the Mapping the Human Toxome project, an enrichment paradigm called information-dependent enrichment analysis (IDEA) to guide identification of the transcription factor network. We used a test case of activation in MCF-7 cells by 17β estradiol (E2). Using this new approach, we established a time course for transcriptional and functional responses to E2. ERα and ERβ were associated with short-term transcriptional changes in response to E2. Sustained exposure led to recruitment of additional transcription factors and alteration of cell cycle machinery. TFAP2C and SOX2 were the transcription factors most highly correlated with dose. E2F7, E2F1, and Foxm1, which are involved in cell proliferation, were enriched only at 24 h. IDEA should be useful for identifying candidate pathways of toxicity. IDEA outperforms gene set enrichment analysis (GSEA) and provides similar results to weighted gene correlation network analysis, a platform that helps to identify genes not annotated to pathways.

A protein interaction map for cell polarity development

PubMed Central

Drees, Becky L.; Sundin, Bryan; Brazeau, Elizabeth; Caviston, Juliane P.; Chen, Guang-Chao; Guo, Wei; Kozminski, Keith G.; Lau, Michelle W.; Moskow, John J.; Tong, Amy; Schenkman, Laura R.; McKenzie, Amos; Brennwald, Patrick; Longtine, Mark; Bi, Erfei; Chan, Clarence; Novick, Peter; Boone, Charles; Pringle, John R.; Davis, Trisha N.; Fields, Stanley; Drubin, David G.

2001-01-01

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein–protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express ∼90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein–protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed. PMID:11489916

High Throughput T Epitope Mapping and Vaccine Development

PubMed Central

Li Pira, Giuseppina; Ivaldi, Federico; Moretti, Paolo; Manca, Fabrizio

2010-01-01

Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost. PMID:20617148

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

PubMed Central

Roth Flach, Rachel J.; Skoura, Athanasia; Matevossian, Anouch; Danai, Laura V.; Zheng, Wei; Cortes, Christian; Bhattacharya, Samit K.; Aouadi, Myriam; Hagan, Nana; Yawe, Joseph C.; Vangala, Pranitha; Menendez, Lorena Garcia; Cooper, Marcus P.; Fitzgibbons, Timothy P.; Buckbinder, Leonard; Czech, Michael P.

2015-01-01

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis. PMID:26688060

Diffusion maps for high-dimensional single-cell analysis of differentiation data.

PubMed

Haghverdi, Laleh; Buettner, Florian; Theis, Fabian J

2015-09-15

Single-cell technologies have recently gained popularity in cellular differentiation studies regarding their ability to resolve potential heterogeneities in cell populations. Analyzing such high-dimensional single-cell data has its own statistical and computational challenges. Popular multivariate approaches are based on data normalization, followed by dimension reduction and clustering to identify subgroups. However, in the case of cellular differentiation, we would not expect clear clusters to be present but instead expect the cells to follow continuous branching lineages. Here, we propose the use of diffusion maps to deal with the problem of defining differentiation trajectories. We adapt this method to single-cell data by adequate choice of kernel width and inclusion of uncertainties or missing measurement values, which enables the establishment of a pseudotemporal ordering of single cells in a high-dimensional gene expression space. We expect this output to reflect cell differentiation trajectories, where the data originates from intrinsic diffusion-like dynamics. Starting from a pluripotent stage, cells move smoothly within the transcriptional landscape towards more differentiated states with some stochasticity along their path. We demonstrate the robustness of our method with respect to extrinsic noise (e.g. measurement noise) and sampling density heterogeneities on simulated toy data as well as two single-cell quantitative polymerase chain reaction datasets (i.e. mouse haematopoietic stem cells and mouse embryonic stem cells) and an RNA-Seq data of human pre-implantation embryos. We show that diffusion maps perform considerably better than Principal Component Analysis and are advantageous over other techniques for non-linear dimension reduction such as t-distributed Stochastic Neighbour Embedding for preserving the global structures and pseudotemporal ordering of cells. The Matlab implementation of diffusion maps for single-cell data is available at https://www.helmholtz-muenchen.de/icb/single-cell-diffusion-map. fbuettner.phys@gmail.com, fabian.theis@helmholtz-muenchen.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Displays, instruments, and the multi-dimensional world of cartography

NASA Technical Reports Server (NTRS)

Mccleary, George F., Jr.

1989-01-01

Cartographers are creators and purveyors of maps. Maps are representations of space, geographical images of the environment. Maps organize spatial information for convenience, particularly for use in performing tasks which involve the environment. There are many different kinds of maps, and there are as many different uses of maps as there are spatial problems to be solved. Maps and the display instrument dichotomy are examined. Also examined are the categories of map use along with the characteristics of maps.

Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation.

PubMed

Woo, Seon Min; Min, Kyoung-Jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Chun, Kyung-Soo; Kim, Young Ho; Lee, Tae-Jin; Kim, Sang Hyun; Choi, Yung Hyun; Chang, Jong-Soo; Kwon, Taeg Kyu

2014-03-25

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Rapid disinhibition by adjustment of PV intrinsic excitability during whisker map plasticity in mouse S1.

PubMed

Gainey, Melanie A; Aman, Joseph W; Feldman, Daniel E

2018-04-20

Rapid plasticity of layer (L) 2/3 inhibitory circuits is an early step in sensory cortical map plasticity, but its cellular basis is unclear. We show that, in mice of either sex, 1 day whisker deprivation drives rapid loss of L4-evoked feedforward inhibition and more modest loss of feedforward excitation in L2/3 pyramidal (PYR) cells, increasing E-I conductance ratio. Rapid disinhibition was due to reduced L4-evoked spiking by L2/3 parvalbumin (PV) interneurons, caused by reduced PV intrinsic excitability. This included elevated PV spike threshold, associated with an increase in low-threshold, voltage activated delayed rectifier (presumed Kv1) and A-type potassium currents. Excitatory synaptic input and unitary inhibitory output of PV cells were unaffected. Functionally, the loss of feedforward inhibition and excitation were precisely coordinated in L2/3 PYR cells, so that peak feedforward synaptic depolarization remained stable. Thus, rapid plasticity of PV intrinsic excitability offsets early weakening of excitatory circuits to homeostatically stabilize synaptic potentials in PYR cells of sensory cortex. SIGNIFICANCE STATEMENT Inhibitory circuits in cerebral cortex are highly plastic, but the cellular mechanisms and functional importance of this plasticity are incompletely understood. We show that brief (1-day) sensory deprivation rapidly weakens parvalbumin (PV) inhibitory circuits by reducing the intrinsic excitability of PV neurons. This involved a rapid increase in voltage-gated potassium conductances that control near-threshold spiking excitability. Functionally, the loss of PV-mediated feedforward inhibition in L2/3 pyramidal cells was precisely balanced with the separate loss of feedforward excitation, resulting in a net homeostatic stabilization of synaptic potentials. Thus, rapid plasticity of PV intrinsic excitability implements network-level homeostasis to stabilize synaptic potentials in sensory cortex. Copyright © 2018 the authors.

N-Caffeoyltryptamine, a Potent Anti-Inflammatory Phenolic Amide, Suppressed MCP-1 Expression in LPS-stimulated THP-1 Cells and Rats Fed a High-Fat Diet.

PubMed

Park, Jae B

2017-05-27

Monocyte chemoattractant protein-1 (MCP-1) is a well-known chemokine critically involved in the pathophysiological progression of several inflammatory diseases including arthrosclerosis. N -caffeoyltryptamine is a phenolic amide with strong anti-inflammatory effects. Therefore, in this paper, the potential effect of N -caffeoyltryptamine on MCP-1 expression was investigated as a potential p38 mitogen-activated protein (MAP) kinase inhibitor in vitro and in vivo. At the concentration of 20 μM, N -caffeoyltryptamine significantly inhibited p38 MAP kinase α, β, γ and δ by 15-50% ( p < 0.05), particularly p38 MAP kinase α (IC 50 = 16.7 μM) and β (IC 50 = 18.3 μM). Also, the pretreatment of the lipopolysaccharide (LPS)-stimulated THP-1 cells with N -caffeoyltryptamine (10, 20 and 40 μM) led to significant suppression of MCP-1 production by 10-45% ( p < 0.05) in the cells. Additionally, N -caffeoyltryptamine was also able to significantly downregulate MCP-1 mRNA expression in the THP-1 cells ( p < 0.05). On the basis of this strong inhibition in vitro, an animal study was conducted to confirm this inhibitory effect in vivo. Rats were divided into three groups ( n = 8): a normal control diet (C), a high-fat diet (HF), or a high-fat diet supplemented with N -caffeoyltryptamine (2 mg per day) (HFS). After 16 weeks, blood samples were collected from the rats in each group, and MCP-1 levels were determined in plasma with other atherogenic markers (C-reactive protein and soluble E-selectin (sE-selectin)). As expected, the average MCP-1 levels of the HF group were found to be higher than those of the C group ( p < 0.05). However, the MCP-1 levels of the HFS group were significantly lower than those of the HF group ( p < 0.05), suggesting that N -caffeoyltryptamine could decrease MCP-1 expression in vivo. Related to other atherogenic markers such as C-reactive protein and sE-selectin, there was no significant difference in their levels between the HF and HFS groups. These data suggest that N -caffeoyltryptamine may specifically suppress MCP-1 expression in vitro and in vivo, possibly by inhibiting p38 MAP kinase.

The Functional Landscape of Hsp27 Reveals New Cellular Processes such as DNA Repair and Alternative Splicing and Proposes Novel Anticancer Targets*

PubMed Central

Katsogiannou, Maria; Andrieu, Claudia; Baylot, Virginie; Baudot, Anaïs; Dusetti, Nelson J.; Gayet, Odile; Finetti, Pascal; Garrido, Carmen; Birnbaum, Daniel; Bertucci, François; Brun, Christine; Rocchi, Palma

2014-01-01

Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells. PMID:25277244

Comparative proteomics of human endothelial cell caveolae and rafts using two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Sprenger, Richard R; Speijer, Dave; Back, Jaap Willem; De Koster, Chris G; Pannekoek, Hans; Horrevoets, Anton J G

2004-01-01

The human endothelial cell plasma membrane harbors two subdomains of similar lipid composition, caveolae and rafts, both crucially involved in various essential cellular processes like transcytosis, signal transduction and cholesterol homeostasis. Caveolin-enriched membranes, isolated by either cationic silica or buoyant density methods, were explored by comparing large series of two-dimensional (2-D) maps and subsequent identification of over 100 protein spots by matrix-assisted laser desorption/ionization (MALDI) peptide mass fingerprinting. Improved representation and identification of membrane proteins and valuable information on various post-translational modifications was achieved by the presented optimized procedures for solubilization, destaining and database searching/computing. Whereas the cationic silica purification yielded predominantly known endoplasmic reticulum residents, the cold-detergent method yielded a large number of known caveolae residents, including caveolin-1. Thus, a large part of this subproteome was established, including known (trans-)membrane, signal transduction and glycosyl phosphatidylinositol (GPI)-anchored proteins. Several predicted proteins from the human genome were isolated for the first time from biological samples, including SGRP58, SLP-2, C8ORF2, and XRP-2. These findings and various optimized procedures can serve as a reference to study the differential composition of endothelial cell caveolae and rafts, known to be involved in pathologies like cancer and cardiovascular disease.

In vivo laser confocal microscopy findings in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy.

PubMed

Kobayashi, Akira; Yokogawa, Hideaki; Sugiyama, Kazuhisa

2012-01-01

The purpose of this study was to investigate pathological changes of the corneal cell layer in patients with map-dot-fingerprint (epithelial basement membrane) dystrophy by in vivo laser corneal confocal microscopy. Two patients were evaluated using a cornea-specific in vivo laser scanning confocal microscope (Heidelberg Retina Tomograph 2 Rostock Cornea Module, HRT 2-RCM). The affected corneal areas of both patients were examined. Image analysis was performed to identify corneal epithelial and stromal deposits correlated with this dystrophy. Variously shaped (linear, multilaminar, curvilinear, ring-shape, geographic) highly reflective materials were observed in the "map" area, mainly in the basal epithelial cell layer. In "fingerprint" lesions, multiple linear and curvilinear hyporeflective lines were observed. Additionally, in the affected corneas, infiltration of possible Langerhans cells and other inflammatory cells was observed as highly reflective Langerhans cell-like or dot images. Finally, needle-shaped materials were observed in one patient. HRT 2-RCM laser confocal microscopy is capable of identifying corneal microstructural changes related to map-dot-fingerprint corneal dystrophy in vivo. The technique may be useful in elucidating the pathogenesis and natural course of map-dot-fingerprint corneal dystrophy and other similar basement membrane abnormalities.

Correlation of physical and genetic maps of human chromosome 16

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

Sutherland, G.R.

1991-01-01

This project aimed to divide chromosome 16 into approximately 50 intervals of {approximately}2Mb in size by constructing a series of mouse/human somatic cell hybrids each containing a rearranged chromosome 16. Using these hybrids, DNA probes would be regionally mapped by Southern blot or PCR analysis. Preference would be given to mapping probes which demonstrated polymorphisms for which the CEPH panel of families had been typed. This would allow a correlation of the physical and linkage maps of this chromosome. The aims have been substantially achieved. 49 somatic cell hybrids have been constructed which have allowed definition of 46, and potentiallymore » 57, different physical intervals on the chromosome. 164 loci have been fully mapped into these intervals. A correlation of the physical and genetic maps of the chromosome is in an advanced stage of preparation. The somatic cell hybrids constructed have been widely distributed to groups working on chromosome 16 and other genome projects.« less

Correlation of physical and genetic maps of human chromosome 16. Annual progress report, October 1, 1990--July 31, 1991

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

Sutherland, G.R.

1991-12-31

This project aimed to divide chromosome 16 into approximately 50 intervals of {approximately}2Mb in size by constructing a series of mouse/human somatic cell hybrids each containing a rearranged chromosome 16. Using these hybrids, DNA probes would be regionally mapped by Southern blot or PCR analysis. Preference would be given to mapping probes which demonstrated polymorphisms for which the CEPH panel of families had been typed. This would allow a correlation of the physical and linkage maps of this chromosome. The aims have been substantially achieved. 49 somatic cell hybrids have been constructed which have allowed definition of 46, and potentiallymore » 57, different physical intervals on the chromosome. 164 loci have been fully mapped into these intervals. A correlation of the physical and genetic maps of the chromosome is in an advanced stage of preparation. The somatic cell hybrids constructed have been widely distributed to groups working on chromosome 16 and other genome projects.« less

Identification of sequences in herpes simplex virus type 1 ICP22 that influence RNA polymerase II modification and viral late gene expression.

PubMed

Bastian, Thomas W; Rice, Stephen A

2009-01-01

Previous studies have shown that the herpes simplex virus type 1 (HSV-1) immediate-early protein ICP22 alters the phosphorylation of the host cell RNA polymerase II (Pol II) during viral infection. In this study, we have engineered several ICP22 plasmid and virus mutants in order to map the ICP22 sequences that are involved in this function. We identify a region in the C-terminal half of ICP22 (residues 240 to 340) that is critical for Pol II modification and further show that the N-terminal half of the protein (residues 1 to 239) is not required. However, immunofluorescence analysis indicates that the N-terminal half of ICP22 is needed for its localization to nuclear body structures. These results demonstrate that ICP22's effects on Pol II do not require that it accumulate in nuclear bodies. As ICP22 is known to enhance viral late gene expression during infection of certain cultured cells, including human embryonic lung (HEL) cells, we used our engineered viral mutants to map this function of ICP22. It was found that mutations in both the N- and C-terminal halves of ICP22 result in similar defects in viral late gene expression and growth in HEL cells, despite having distinctly different effects on Pol II. Thus, our results genetically uncouple ICP22's effects on Pol II from its effects on viral late gene expression. This suggests that these two functions of ICP22 may be due to distinct activities of the protein.

Characterization of a microdissection library from human chromosome region 3p14

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

Bardenheuer, W.; Szymanski, S.; Lux, A.

1994-01-15

Structural alterations in human chromosome region 3p14-p23 resulting in the inactivation of one or more tumor suppressor genes are thought to play a pathogenic role in small cell lung cancer, renal cell carcinoma, and other human neoplasms. To identify putative tumor suppressor genes, 428 recombinant clones from a microdissection library specific for human chromosome region 3p14 were isolated and characterized. Ninety-six of these (22.5%) were human single-copy DNA sequences, 57 of which were unique sequence clones. Forty-four of these were mapped to the microdissected region using a cell hybrid mapping panel. Within this mapping panel, four probes detected two newmore » chromosome breakpoints that were previously indistinguishable from the translocation breakpoint t(3;8) in 3p14.2 in hereditary renal cell carcinoma. One probe maps to the homozygously deleted region of the small cell lung cancer cell line U2020. In addition, microdissection clones have been shown to be suitable for isolation of yeast artificial chromosomes. 52 refs., 3 figs., 2 tabs.« less

viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia.

PubMed

Amir, El-ad David; Davis, Kara L; Tadmor, Michelle D; Simonds, Erin F; Levine, Jacob H; Bendall, Sean C; Shenfeld, Daniel K; Krishnaswamy, Smita; Nolan, Garry P; Pe'er, Dana

2013-06-01

New high-dimensional, single-cell technologies offer unprecedented resolution in the analysis of heterogeneous tissues. However, because these technologies can measure dozens of parameters simultaneously in individual cells, data interpretation can be challenging. Here we present viSNE, a tool that allows one to map high-dimensional cytometry data onto two dimensions, yet conserve the high-dimensional structure of the data. viSNE plots individual cells in a visual similar to a scatter plot, while using all pairwise distances in high dimension to determine each cell's location in the plot. We integrated mass cytometry with viSNE to map healthy and cancerous bone marrow samples. Healthy bone marrow automatically maps into a consistent shape, whereas leukemia samples map into malformed shapes that are distinct from healthy bone marrow and from each other. We also use viSNE and mass cytometry to compare leukemia diagnosis and relapse samples, and to identify a rare leukemia population reminiscent of minimal residual disease. viSNE can be applied to any multi-dimensional single-cell technology.

Involving Families and Communities in CSPAP Development Using Asset Mapping

ERIC Educational Resources Information Center

Allar, Ishonté; Elliott, Eloise; Jones, Emily; Kristjansson, Alfgeir L.; Taliaferro, Andrea; Bulger, Sean M.

2017-01-01

The purpose of this article is to provide an introduction to asset mapping as a systematic approach to facilitating increased family and community involvement in comprehensive school physical activity programs (CSPAP). It includes a brief summary of the literature related to the importance of family and community in children's physical activity…

The power and benefits of concept mapping: measuring use, usefulness, ease of use, and satisfaction

NASA Astrophysics Data System (ADS)

Freeman, Lee A.; Jessup, Leonard M.

2004-02-01

The power and benefits of concept mapping rest in four arenas: enabling shared understanding, the inclusion of affect, the balance of power, and client involvement. Concept mapping theory and research indicate concept maps (1) are appropriate tools to assist with communication, (2) are easy to use, and (3) are seen as beneficial by their users. An experiment was conducted to test these assertions and analyze the power and benefits of concept mapping using a typical business consulting scenario involving 16 groups of two individuals. The results were analyzed via empirical hypothesis testing and protocol analyses, and indicate an overall support of the theory and prior research and additional support of new measures of usefulness, ease of use, and satisfaction by both parties. A more thorough understanding of concept mapping is gained and available to future practitioners and researchers.

ITO/gold nanoparticle/RGD peptide composites to enhance electrochemical signals and proliferation of human neural stem cells.

PubMed

Kim, Tae-Hyung; El-Said, Waleed Ahmed; An, Jeung Hee; Choi, Jeong-Woo

2013-04-01

A cell chip composed of ITO, gold nanoparticles (GNP) and RGD-MAP-C peptide composites was fabricated to enhance the electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The structural characteristics of the fabricated surfaces were confirmed by both scanning electron microscopy and surface-enhanced Raman spectroscopy. HB1.F3 cells were allowed to attach to various composites electrodes in the cell chip and the material-dependent effects on electrochemical signals and cell proliferation were analyzed. The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to be the best material in regards to enhancing the voltammetric signals of HB1.F3 cells when exposed to cyclic voltammetry, as well as for increasing cell proliferation. Differential pulse voltammetry was performed to evaluate the adverse effects of doxorubicin on HB1.F3 cells. In these experiments, negative correlations between cell viability and chemical concentrations were obseved, which were more sensitive than MTT viability assay especially at low concentrations (<0.1 μg/mL). In this basic science study, a cell chip composed of ITO, gold nanoparticles and RGD-MAP-C peptide composites was fabricated to enhance electrochemical signals and proliferation of undifferentiated human neural stem cells (HB1.F3). The ITO/60 nm GNP/RGD-MAP-C composite electrode was found to best enhance the voltammetric signals of the studied cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Spontaneous hair cell regeneration in the mouse utricle following gentamicin ototoxicity.

PubMed

Kawamoto, Kohei; Izumikawa, Masahiko; Beyer, Lisa A; Atkin, Graham M; Raphael, Yehoash

2009-01-01

Whereas most epithelial tissues turn-over and regenerate after a traumatic lesion, this restorative ability is diminished in the sensory epithelia of the inner ear; it is absent in the cochlea and exists only in a limited capacity in the vestibular epithelium. The extent of regeneration in vestibular hair cells has been characterized for several mammalian species including guinea pig, rat, and chinchilla, but not yet in mouse. As the fundamental model species for investigating hereditary disease, the mouse can be studied using a wide variety of genetic and molecular tools. To design a mouse model for vestibular hair cell regeneration research, an aminoglycoside-induced method of complete hair cell elimination was developed in our lab and applied to the murine utricle. Loss of utricular hair cells was observed using scanning electron microscopy, and corroborated by a loss of fluorescent signal in utricles from transgenic mice with GFP-positive hair cells. Regenerative capability was characterized at several time points up to six months following insult. Using scanning electron microscopy, we observed that as early as two weeks after insult, a few immature hair cells, demonstrating the characteristic immature morphology indicative of regeneration, could be seen in the utricle. As time progressed, larger numbers of immature hair cells could be seen along with some mature cells resembling surface morphology of type II hair cells. By six months post-lesion, numerous regenerated hair cells were present in the utricle, however, neither their number nor their appearance was normal. A BrdU assay suggested that at least some of the regeneration of mouse vestibular hair cells involved mitosis. Our results demonstrate that the vestibular sensory epithelium in mice can spontaneously regenerate, elucidate the time course of this process, and identify involvement of mitosis in some cases. These data establish a road map of the murine vestibular regenerative process, which can be used for elucidating the molecular events that govern this process.

Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

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

Lee, Jeong Eun; Hanyang Biomedical Research Institute, Seoul; Park, Jae Hyeon

2012-09-01

Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity asmore » well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.« less

The roles of MHC class II genes and post-translational modification in celiac disease.

PubMed

Sollid, Ludvig M

2017-08-01

Our increasing understanding of the etiology of celiac disease, previously considered a simple food hypersensitivity disorder caused by an immune response to cereal gluten proteins, challenges established concepts of autoimmunity. HLA is a chief genetic determinant, and certain HLA-DQ allotypes predispose to the disease by presenting posttranslationally modified (deamidated) gluten peptides to CD4 + T cells. The deamidation of gluten peptides is mediated by transglutaminase 2. Strikingly, celiac disease patients generate highly disease-specific autoantibodies to the transglutaminase 2 enzyme. The dual role of transglutaminase 2 in celiac disease is hardly coincidental. This paper reviews the genetic mapping and involvement of MHC class II genes in disease pathogenesis, and discusses the evidence that MHC class II genes, via the involvement of transglutaminase 2, influence the generation of celiac disease-specific autoantibodies.

Systems Imaging of the Immune Synapse.

PubMed

Ambler, Rachel; Ruan, Xiangtao; Murphy, Robert F; Wülfing, Christoph

2017-01-01

Three-dimensional live cell imaging of the interaction of T cells with antigen-presenting cells (APCs) visualizes the subcellular distributions of signaling intermediates during T cell activation at thousands of resolved positions within a cell. These information-rich maps of local protein concentrations are a valuable resource in understanding T cell signaling. Here, we describe a protocol for the efficient acquisition of such imaging data and their computational processing to create four-dimensional maps of local concentrations. This protocol allows quantitative analysis of T cell signaling as it occurs inside live cells with resolution in time and space across thousands of cells.

Zinc Promotes Adipose-Derived Mesenchymal Stem Cell Proliferation and Differentiation towards a Neuronal Fate.

PubMed

Moon, Mi-Young; Kim, Hyun Jung; Choi, Bo Young; Sohn, Min; Chung, Tae Nyoung; Suh, Sang Won

2018-01-01

Zinc is an essential element required for cell division, migration, and proliferation. Under zinc-deficient conditions, proliferation and differentiation of neural progenitors are significantly impaired. Adipose-derived mesenchymal stem cells (AD-MSCs) are multipotent stem cells that can differentiate into neurons. The aim of this study was to evaluate the effect of zinc on AD-MSC proliferation and differentiation. We initially examined the effect of zinc on stem cell proliferation at the undifferentiated stage. AD-MSCs showed high proliferation rates on day 6 in 30  μ M and 100  μ M of ZnCl 2 . Zinc chelation inhibited AD-MSC proliferation via downregulation of ERK1/2 activity. We then assessed whether zinc was involved in cell migration and neurite outgrowth during differentiation. After three days of neuronal differentiation, TUJ-1-positive cells were observed, implying that AD-MSCs had differentiated into early neuron or neuron-like cells. Neurite outgrowth was increased in the zinc-treated group, while the CaEDTA-treated group showed diminished, shrunken neurites. Furthermore, we showed that zinc promoted neurite outgrowth via the inactivation of RhoA and led to the induction of neuronal gene expression (MAP2 and nestin) in differentiated stem cells. Taken together, zinc promoted AD-MSC proliferation and affected neuronal differentiation, mainly by increasing neurite outgrowth.

Population differences in the rate of proliferation of international HapMap cell lines.

PubMed

Stark, Amy L; Zhang, Wei; Zhou, Tong; O'Donnell, Peter H; Beiswanger, Christine M; Huang, R Stephanie; Cox, Nancy J; Dolan, M Eileen

2010-12-10

The International HapMap Project is a resource for researchers containing genotype, sequencing, and expression information for EBV-transformed lymphoblastoid cell lines derived from populations across the world. The expansion of the HapMap beyond the four initial populations of Phase 2, referred to as Phase 3, has increased the sample number and ethnic diversity available for investigation. However, differences in the rate of cellular proliferation between the populations can serve as confounders in phenotype-genotype studies using these cell lines. Within the Phase 2 populations, the JPT and CHB cell lines grow faster (p < 0.0001) than the CEU or YRI cell lines. Phase 3 YRI cell lines grow significantly slower than Phase 2 YRI lines (p < 0.0001), with no widespread genetic differences based on common SNPs. In addition, we found significant growth differences between the cell lines in the Phase 2 ASN populations and the Han Chinese from the Denver metropolitan area panel in Phase 3 (p < 0.0001). Therefore, studies that separate HapMap panels into discovery and replication sets must take this into consideration. Copyright © 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Grid cell hexagonal patterns formed by fast self-organized learning within entorhinal cortex.

PubMed

Mhatre, Himanshu; Gorchetchnikov, Anatoli; Grossberg, Stephen

2012-02-01

Grid cells in the dorsal segment of the medial entorhinal cortex (dMEC) show remarkable hexagonal activity patterns, at multiple spatial scales, during spatial navigation. It has previously been shown how a self-organizing map can convert firing patterns across entorhinal grid cells into hippocampal place cells that are capable of representing much larger spatial scales. Can grid cell firing fields also arise during navigation through learning within a self-organizing map? This article describes a simple and general mathematical property of the trigonometry of spatial navigation which favors hexagonal patterns. The article also develops a neural model that can learn to exploit this trigonometric relationship. This GRIDSmap self-organizing map model converts path integration signals into hexagonal grid cell patterns of multiple scales. GRIDSmap creates only grid cell firing patterns with the observed hexagonal structure, predicts how these hexagonal patterns can be learned from experience, and can process biologically plausible neural input and output signals during navigation. These results support an emerging unified computational framework based on a hierarchy of self-organizing maps for explaining how entorhinal-hippocampal interactions support spatial navigation. Copyright © 2010 Wiley Periodicals, Inc.

Nemo regulates cell dynamics and represses the expression of miple, a midkine/pleiotrophin cytokine, during ommatidial rotation

PubMed Central

Muñoz-Soriano, Verónica; Ruiz, Carlos; Pérez-Alonso, Manuel; Mlodzik, Marek; Paricio, Nuria

2013-01-01

Ommatidial rotation is one of the most important events for correct patterning of the Drosophila eye. Although several signaling pathways are involved in this process, few genes have been shown to specifically affect it. One of them is nemo (nmo), which encodes a MAP-like protein kinase that regulates the rate of rotation throughout the entire process, and serves as a link between core planar cell polarity (PCP) factors and the E-cadherin–β-catenin complex. To determine more precisely the role of nmo in ommatidial rotation, live-imaging analyses in nmo mutant and wild-type early pupal eye discs were performed. We demonstrate that ommatidial rotation is not a continuous process, and that rotating and non-rotating interommatidial cells are very dynamic. Our in vivo analyses also show that nmo regulates the speed of rotation and is required in cone cells for correct ommatidial rotation, and that these cells as well as interommatidial cells are less dynamic in nmo mutants. Furthermore, microarray analyses of nmo and wild-type larval eye discs led us to identify new genes and signaling pathways related to nmo function during this process. One of them, miple, encodes the Drosophila ortholog of the midkine/pleiotrophin secreted cytokines that are involved in cell migration processes. miple is highly up-regulated in nmo mutant discs. Indeed, phenotypic analyses reveal that miple overexpression leads to ommatidial rotation defects. Genetic interaction assays suggest that miple is signaling through Ptp99A, the Drosophila ortholog of the vertebrate midkine/pleiotrophin PTPζ receptor. Accordingly, we propose that one of the roles of Nmo during ommatial rotation is to repress miple expression, which may in turn affect the dynamics in E-cadherin–β-catenin complexes. PMID:23428616

Hyperforin activates gene transcription involving transient receptor potential C6 channels.

PubMed

Thiel, Gerald; Rössler, Oliver G

2017-04-01

Hypericum perforatum is one of the most prominent medical plants. Hyperforin, a main ingredient of H. perforatum, has been shown to activate transient receptor potential canonical C6 (TRPC6) channels. Alternatively, it has been proposed that hyperforin functions as a protonophore in a TRPC6-independent manner. Here, we show that hyperforin stimulation activates the transcription factor AP-1 in HEK293 cells expressing TRPC6 (T6.11 cells), but did not substantially change the AP-1 activity in HEK293 cells lacking TRPC6. We identified the AP-1 binding site as a hyperforin-responsive element. AP-1 is composed of the transcription factors c-Jun and c-Fos, or other members of the c-Jun and c-Fos families of proteins. Hyperforin stimulation increased c-Jun and c-Fos promoter activities in T6.11 cells and induced an upregulation of c-Jun and c-Fos biosynthesis. The analysis of the c-Fos promoter revealed that the cAMP-response element also functions as a hyperforin-responsive element. Hyperforin-induced upregulation of AP-1 in T6.11 cells was attenuated by preincubation of the cells with either pregnenolone or progesterone, indicating that gene regulation via TRPC6 is under control of hormones or hormonal precursors. The signal transduction of hyperforin-induced AP-1 gene transcription required an influx of Ca 2+ ions into the cells, the activation of MAP kinases, and the activation of the transcription factors c-Jun and ternary complex factor. We conclude that hyperforin regulates gene transcription via activation of TRPC6 channels, involving stimulus-regulated protein kinases and stimulus-responsive transcription factors. The fact that hyperforin regulates gene transcription may explain many of the intracellular alterations induced by this compound. Copyright © 2017 Elsevier Inc. All rights reserved.

Male Hypogonadism and Germ Cell Loss Caused by a Mutation in Polo-Like Kinase 4

PubMed Central

Harris, Rebecca M.; Weiss, Jeffrey

2011-01-01

The genetic etiologies of male infertility remain largely unknown. To identify genes potentially involved in spermatogenesis and male infertility, we performed genome-wide mutagenesis in mice with N-ethyl-N-nitrosourea and identified a line with dominant hypogonadism and patchy germ cell loss. Genomic mapping and DNA sequence analysis identified a novel heterozygous missense mutation in the kinase domain of Polo-like kinase 4 (Plk4), altering an isoleucine to asparagine at residue 242 (I242N). Genetic complementation studies using a gene trap line with disruption in the Plk4 locus confirmed that the putative Plk4 missense mutation was causative. Plk4 is known to be involved in centriole formation and cell cycle progression. However, a specific role in mammalian spermatogenesis has not been examined. PLK4 was highly expressed in the testes both pre- and postnatally. In the adult, PLK4 expression was first detected in stage VIII pachytene spermatocytes and was present through step 16 elongated spermatids. Because the homozygous Plk4I242N/I242N mutation was embryonic lethal, all analyses were performed using the heterozygous Plk4+/I242N mice. Testis size was reduced by 17%, and histology revealed discrete regions of germ cell loss, leaving only Sertoli cells in these defective tubules. Testis cord formation (embryonic day 13.5) was normal. Testis histology was also normal at postnatal day (P)1, but germ cell loss was detected at P10 and subsequent ages. We conclude that the I242N heterozygous mutation in PLK4 is causative for patchy germ cell loss beginning at P10, suggesting a role for PLK4 during the initiation of spermatogenesis. PMID:21791561

cDNA cloning, tissue distribution, and chromosomal localization of myelodysplasia/Myeloid Leukemia Factor 2 (MLF2)

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

Kuefer, M.U.; Valentine, V.; Behm, F.G.

A fusion gene between nucleophosmin (NPM) and myelodysplasia/myeloid leukemia factor 1 (MLF1) and myelodysplasia/myeloid leukemia factor 1 (MLF1) is formed by a recurrent t(3;5)(q25.1;q34) in myelodysplastic syndrome and acute myeloid leukemia. Here we report the identification of a novel gene, MLF2, which contains an open reading frame of 744 bp encoding a 248-amino-acid protein highly related to the previously identified MLF1 protein (63% similarity, 40% identity). In contrast to the tissue-restricted expression pattern of MLF1, and MLF2 messenger RNA is expressed ubiquitously. The MLF2 gene locus was mapped by fluorescence in situ hybridization to human chromosome 12p13, a chromosomal regionmore » frequently involved in translocations and deletions in acute leukemias of lymphoid or myeloid lineage. In a physical map of chromosome 12, MLF2 was found to reside on the yeast artificial chromosome clone 765b9. Southern blotting analysis of malignant cell DNAs prepared from a series of acute lymphoblastic leukemia cases with translocations involving chromosome arm 12p, as well as a group of acute myeloid leukemias with various cytogenetic abnormalities, failed to reveal MLF2 gene rearrangements. 19 refs., 2 figs.« less

Adaptive treatment-length optimization in spatiobiologically integrated radiotherapy

NASA Astrophysics Data System (ADS)

Ajdari, Ali; Ghate, Archis; Kim, Minsun

2018-04-01

Recent theoretical research on spatiobiologically integrated radiotherapy has focused on optimization models that adapt fluence-maps to the evolution of tumor state, for example, cell densities, as observed in quantitative functional images acquired over the treatment course. We propose an optimization model that adapts the length of the treatment course as well as the fluence-maps to such imaged tumor state. Specifically, after observing the tumor cell densities at the beginning of a session, the treatment planner solves a group of convex optimization problems to determine an optimal number of remaining treatment sessions, and a corresponding optimal fluence-map for each of these sessions. The objective is to minimize the total number of tumor cells remaining (TNTCR) at the end of this proposed treatment course, subject to upper limits on the biologically effective dose delivered to the organs-at-risk. This fluence-map is administered in future sessions until the next image is available, and then the number of sessions and the fluence-map are re-optimized based on the latest cell density information. We demonstrate via computer simulations on five head-and-neck test cases that such adaptive treatment-length and fluence-map planning reduces the TNTCR and increases the biological effect on the tumor while employing shorter treatment courses, as compared to only adapting fluence-maps and using a pre-determined treatment course length based on one-size-fits-all guidelines.

The morphological classification of normal and abnormal red blood cell using Self Organizing Map

NASA Astrophysics Data System (ADS)

Rahmat, R. F.; Wulandari, F. S.; Faza, S.; Muchtar, M. A.; Siregar, I.

2018-02-01

Blood is an essential component of living creatures in the vascular space. For possible disease identification, it can be tested through a blood test, one of which can be seen from the form of red blood cells. The normal and abnormal morphology of the red blood cells of a patient is very helpful to doctors in detecting a disease. With the advancement of digital image processing technology can be used to identify normal and abnormal blood cells of a patient. This research used self-organizing map method to classify the normal and abnormal form of red blood cells in the digital image. The use of self-organizing map neural network method can be implemented to classify the normal and abnormal form of red blood cells in the input image with 93,78% accuracy testing.

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes.

PubMed

Jin, Ya; Yuan, Qi; Zhang, Jun; Manabe, Takashi; Tan, Wen

2015-09-01

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an "overlap score," (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 "overlap factors," (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of a megabase deletion in a patient with brachio-oto-renal syndrome (BOR) and tricho-rhino-phalangeal syndrome (TRPS): Implications for mapping and cloning the BOR gene

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

Gu, J.Z.; Wells, D.E.; Wagner, M.J.

Genetic linkage analysis has previously mapped the locus for the autosomal dominant disorder branchio-oto-renal syndrome (BOR) to the pericentric region of chromosome 8q. A YAC contig spanning the putative BOR region, from D8S543 to D8S541, was constructed and confirmed by sequence-tagged site content mapping using microsatellite markers and by DNA hybridization analysis. YACs spanning the BOR interval were used as fluorescence in situ hybridization probes on a cell line from a patient with BO and tricho-rhino-phalangeal syndrome I that involves a chromosome 8q rearrangement. In addition to the cytogenetically defined direct insertion of material from 8q13.3-q21.13 into 8q24.11, a previouslymore » unidentified deletion of just under one megabase was found in 8q13.3. These data narrowed the most likely location of the BOR gene to a region corresponding to the proximal two-thirds of YAC 869E10 between D8S543 and D8S279. 23 refs., 3 figs.« less

A methodology for physically based rockfall hazard assessment

NASA Astrophysics Data System (ADS)

Crosta, G. B.; Agliardi, F.

Rockfall hazard assessment is not simple to achieve in practice and sound, physically based assessment methodologies are still missing. The mobility of rockfalls implies a more difficult hazard definition with respect to other slope instabilities with minimal runout. Rockfall hazard assessment involves complex definitions for "occurrence probability" and "intensity". This paper is an attempt to evaluate rockfall hazard using the results of 3-D numerical modelling on a topography described by a DEM. Maps portraying the maximum frequency of passages, velocity and height of blocks at each model cell, are easily combined in a GIS in order to produce physically based rockfall hazard maps. Different methods are suggested and discussed for rockfall hazard mapping at a regional and local scale both along linear features or within exposed areas. An objective approach based on three-dimensional matrixes providing both a positional "Rockfall Hazard Index" and a "Rockfall Hazard Vector" is presented. The opportunity of combining different parameters in the 3-D matrixes has been evaluated to better express the relative increase in hazard. Furthermore, the sensitivity of the hazard index with respect to the included variables and their combinations is preliminarily discussed in order to constrain as objective as possible assessment criteria.

Comparison of Node-Centered and Cell-Centered Unstructured Finite-Volume Discretizations. Part 1; Viscous Fluxes

NASA Technical Reports Server (NTRS)

Diskin, Boris; Thomas, James L.; Nielsen, Eric J.; Nishikawa, Hiroaki; White, Jeffery A.

2009-01-01

Discretization of the viscous terms in current finite-volume unstructured-grid schemes are compared using node-centered and cell-centered approaches in two dimensions. Accuracy and efficiency are studied for six nominally second-order accurate schemes: a node-centered scheme, cell-centered node-averaging schemes with and without clipping, and cell-centered schemes with unweighted, weighted, and approximately mapped least-square face gradient reconstruction. The grids considered range from structured (regular) grids to irregular grids composed of arbitrary mixtures of triangles and quadrilaterals, including random perturbations of the grid points to bring out the worst possible behavior of the solution. Two classes of tests are considered. The first class of tests involves smooth manufactured solutions on both isotropic and highly anisotropic grids with discontinuous metrics, typical of those encountered in grid adaptation. The second class concerns solutions and grids varying strongly anisotropically over a curved body, typical of those encountered in high-Reynolds number turbulent flow simulations. Results from the first class indicate the face least-square methods, the node-averaging method without clipping, and the node-centered method demonstrate second-order convergence of discretization errors with very similar accuracies per degree of freedom. The second class of tests are more discriminating. The node-centered scheme is always second order with an accuracy and complexity in linearization comparable to the best of the cell-centered schemes. In comparison, the cell-centered node-averaging schemes are less accurate, have a higher complexity in linearization, and can fail to converge to the exact solution when clipping of the node-averaged values is used. The cell-centered schemes using least-square face gradient reconstruction have more compact stencils with a complexity similar to the complexity of the node-centered scheme. For simulations on highly anisotropic curved grids, the least-square methods have to be amended either by introducing a local mapping of the surface anisotropy or modifying the scheme stencil to reflect the direction of strong coupling.

cDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells

PubMed Central

Shi, Ting; Mazumdar, Tapati; DeVecchio, Jennifer; Duan, Zhong-Hui; Agyeman, Akwasi; Aziz, Mohammad; Houghton, Janet A.

2010-01-01

Background Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited. Methodology/Principal Findings To identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21Cip1 (CDKN1A) and p15Ink4b (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling. Conclusions/Significance This study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary. PMID:20957031

Mapping the breakpoints of an individual with congenital glaucoma and a 6:13 translocation

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

Nishimura, D.Y.; Patil, S.; Alward, W.L.M.

1994-09-01

We have identified an individual with a balanced translocation involving the short arm of chromosome 6 (6p25) and the long arm of chromosome 13 (13q22). This person was found to have congenital glaucoma along with other anomalies. The identification of a second individual with both congenital glaucoma and an unbalanced translocation involving the distal short arm of chromosome 6 suggested that this region of chromosome 6 might be involved in at least some cases of congenital glaucoma. Therefore, we wished to characterize the two breakpoints with an emphasis on the 6p25 breakpoint. Somatic cell hybrids were used to isolate themore » two translocated chromosomes away from the normal 6 and 13. Two hybrids were identified which contained the distal region of chromosome 6p, while a third hybrid contained the reciprocal translocated chromosome. Characterization of the hybrids demonstrated that the breakpoint on chromosome 6p lies between the markers D6S344 and D6S477, while the chromosome 13 breakpoint lies between the markers D13S160 and D13S170. The distances between the flanking markers on both of the chromosomes are less than 5 cM. The lack of genetic markers within the 6p25 region made it difficult to accurately map the location of the breakpoint in this region. However, we were able to demonstrate that at least two markers are distal to the 6p25 breakpoint. We are working to improve the quality of both the genetic and physical maps of 6p25 and 13q22 in an attempt to further refine the localization of the breakpoints. Preliminary work on the individual with the unbalanced translocation suggests that the 6p deletion in this person includes the 6p25 breakpoints.« less

Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2013-01-01

A major challenge of the postgenomic era is to understand how human genes function together in normal and disease states. In microorganisms, high-density genetic interaction (GI) maps are a powerful tool to elucidate gene functions and pathways. We have developed an integrated methodology based on pooled shRNA screening in mammalian cells for genome-wide identification of genes with relevant phenotypes and systematic mapping of all GIs among them. We recently demonstrated the potential of this approach in an application to pathways controlling the susceptibility of human cells to the toxin ricin. Here we present the complete quantitative framework underlying our strategy, including experimental design, derivation of quantitative phenotypes from pooled screens, robust identification of hit genes using ultra-complex shRNA libraries, parallel measurement of tens of thousands of GIs from a single double-shRNA experiment, and construction of GI maps. We describe the general applicability of our strategy. Our pooled approach enables rapid screening of the same shRNA library in different cell lines and under different conditions to determine a range of different phenotypes. We illustrate this strategy here for single- and double-shRNA libraries. We compare the roles of genes for susceptibility to ricin and Shiga toxin in different human cell lines and reveal both toxin-specific and cell line-specific pathways. We also present GI maps based on growth and ricin-resistance phenotypes, and we demonstrate how such a comparative GI mapping strategy enables functional dissection of physical complexes and context-dependent pathways. PMID:23739767

Local Time-Dependent Charging in a Perovskite Solar Cell.

PubMed

Bergmann, Victor W; Guo, Yunlong; Tanaka, Hideyuki; Hermes, Ilka M; Li, Dan; Klasen, Alexander; Bretschneider, Simon A; Nakamura, Eiichi; Berger, Rüdiger; Weber, Stefan A L

2016-08-03

Efficient charge extraction within solar cells explicitly depends on the optimization of the internal interfaces. Potential barriers, unbalanced charge extraction, and interfacial trap states can prevent cells from reaching high power conversion efficiencies. In the case of perovskite solar cells, slow processes happening on time scales of seconds cause hysteresis in the current-voltage characteristics. In this work, we localized and investigated these slow processes using frequency-modulation Kelvin probe force microscopy (FM-KPFM) on cross sections of planar methylammonium lead iodide (MAPI) perovskite solar cells. FM-KPFM can map the charge density distribution and its dynamics at internal interfaces. Upon illumination, space charge layers formed at the interfaces of the selective contacts with the MAPI layer within several seconds. We observed distinct differences in the charging dynamics at the interfaces of MAPI with adjacent layers. Our results indicate that more than one process is involved in hysteresis. This finding is in agreement with recent simulation studies claiming that a combination of ion migration and interfacial trap states causes the hysteresis in perovskite solar cells. Such differences in the charging rates at different interfaces cannot be separated by conventional device measurements.

Vascular Endothelial Growth Factor (VEGF) Promotes Assembly of the p130Cas Interactome to Drive Endothelial Chemotactic Signaling and Angiogenesis.

PubMed

Evans, Ian M; Kennedy, Susan A; Paliashvili, Ketevan; Santra, Tapesh; Yamaji, Maiko; Lovering, Ruth C; Britton, Gary; Frankel, Paul; Kolch, Walter; Zachary, Ian C

2017-02-01

p130Cas is a polyvalent adapter protein essential for cardiovascular development, and with a key role in cell movement. In order to identify the pathways by which p130Cas exerts its biological functions in endothelial cells we mapped the p130Cas interactome and its dynamic changes in response to VEGF using high-resolution mass spectrometry and reconstruction of protein interaction (PPI) networks with the aid of multiple PPI databases. VEGF enriched the p130Cas interactome in proteins involved in actin cytoskeletal dynamics and cell movement, including actin-binding proteins, small GTPases and regulators or binders of GTPases. Detailed studies showed that p130Cas association of the GTPase-binding scaffold protein, IQGAP1, plays a key role in VEGF chemotactic signaling, endothelial polarization, VEGF-induced cell migration, and endothelial tube formation. These findings indicate a cardinal role for assembly of the p130Cas interactome in mediating the cell migratory response to VEGF in angiogenesis, and provide a basis for further studies of p130Cas in cell movement. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein tyrosine phosphatase 1B (PTP1B) is dispensable for IgE-mediated cutaneous reaction in vivo.

PubMed

Yang, Ting; Xie, Zhongping; Li, Hua; Yue, Lei; Pang, Zheng; MacNeil, Adam J; Tremblay, Michel L; Tang, Jin-Tian; Lin, Tong-Jun

2016-01-01

Mast cells play a critical role in allergic reactions. The cross-linking of FcεRI-bound IgE with multivalent antigen initiates a cascade of signaling events leading to mast cell activation. It has been well-recognized that cross linking of FcεRI mediates tyrosine phosphorylation. However, the mechanism involved in tyrosine dephosphorylation in mast cells is less clear. Here we demonstrated that protein tyrosine phosphatase 1B (PTP1B)-deficient mast cells showed increased IgE-mediated phosphorylation of the signal transducer and activator of transcription 5 (STAT5) and enhanced production of CCL9 (MIP-1γ) and IL-6 in IgE-mediated mast cells activation in vitro. However, IgE-mediated calcium mobilization, β-hexaosaminidase release (degranulation), and phosphorylation of IκB and MAP kinases were not affected by PTP1B deficiency. Furthermore, PTP1B deficient mice showed normal IgE-dependent passive cutaneous anaphylaxis and late phase cutaneous reactions in vivo. Thus, PTP1B specifically regulates IgE-mediated STAT5 pathway, but is redundant in influencing mast cell function in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.

Value of right ventricular mapping in patients with postinfarction ventricular tachycardia.

PubMed

Yokokawa, Miki; Good, Eric; Crawford, Thomas; Chugh, Aman; Pelosi, Frank; Latchamsetty, Rakesh; Oral, Hakan; Morady, Fred; Bogun, Frank

2012-06-01

Postinfarction ventricular tachycardia (VT) typically involves the left ventricular endocardium. Right ventricular involvement in the arrhythmogenic substrate of postinfarction VT is considered unusual. To assess the role of right ventricular mapping and ablation in patients with prior septal myocardial infarction. From among 37 consecutive patients with recurrent postinfarction VT, 18 patients with evidence of left ventricular septal involvement of myocardial infarction were identified; these patients were the subjects of this report. In these 18 patients, 166 VTs (cycle length 372 ± 117 ms) were induced. Right ventricular voltage mapping was performed in all 18 patients with left ventricular septal myocardial infarction. Right ventricular voltage mapping showed areas of low voltage in 11 patients; pace mapping from these areas revealed matching pace maps for 17 VTs, and radiofrequency ablation from the right ventricular endocardium but not the left ventricular endocardium eliminated 14 of 17 VTs. VTs with critical components in the right ventricle had a left bundle branch block morphology that had similar characteristics as left bundle branch block VTs with critical areas involving the left ventricular septum. Patients with right ventricular VT breakthrough sites had a lower ejection fraction than did patients without VT breaking out on the right ventricular septum (18% ± 5% vs 33% ± 15%; P = .01). Right ventricular mapping and ablation may be necessary in order to eliminate all inducible VTs in patients with postinfarction VT. More than half the patients with septal myocardial infarction have right ventricular septal areas that are critical for postinfarction VT and that cannot be eliminated by left ventricular ablation alone. Copyright © 2012 Heart Rhythm Society. All rights reserved.

Translating Research Reports into Educational Materials or How To Take a Neat Piece of Research and Turn It into a Curriculum.

ERIC Educational Resources Information Center

Brody, Michael J.

The rationale and procedures involved with the instructional strategies of concept mapping and Gowin's Vee Mapping are provided in this paper. Specific directives are outlined for building concept maps and vee maps. Examples of both types of these maps are given and explanations are offered on their form and use. Concepts and activities…

The National Council for Geographic Education Competency-Based Geography Test. Secondary Level. Form I. Parts I, II, and III.

ERIC Educational Resources Information Center

Kurfman, Dana G.; And Others

A 3-part test measures the geography knowledge, skills, and understanding of secondary level students. Part 1, map skills and location, contains 20 questions involving the use of three maps: an imaginary sketch map, a contour map, and a political map of the world. Part 2 consists of 20 questions covering physical geography. Students analyze…

Analysis of whitefly transcriptional responses to Beauveria bassiana infection reveals new insights into insect-fungus interactions.

PubMed

Xia, Jun; Zhang, Chang-Rong; Zhang, Shan; Li, Fang-Fang; Feng, Ming-Guang; Wang, Xiao-Wei; Liu, Shu-Sheng

2013-01-01

The fungal pathogen, Beauveria bassiana, is an efficient biocontrol agent against a variety of agricultural pests. A thorough understanding of the basic principles of insect-fungus interactions may enable the genetic modification of Beauveria bassiana to enhance its virulence. However, the molecular mechanism of insect response to Beauveria bassiana infection is poorly understood, let alone the identification of fungal virulent factors involved in pathogenesis. Here, next generation sequencing technology was applied to examine the expression of whitefly (Bemisia tabaci) genes in response to the infection of Beauveria bassiana. Results showed that, compared to control, 654 and 1,681genes were differentially expressed at 48 hours and 72 hours post-infected whiteflies, respectively. Functional and enrichment analyses indicated that the DNA damage stimulus response and drug metabolism were important anti-fungi strategies of the whitefly. Mitogen-activated protein kinase (MAPK) pathway was also likely involved in the whitefly defense responses. Furthermore, the notable suppression of general metabolism and ion transport genes observed in 72 hours post-infected B. tabaci might be manipulated by fungal secreted effectors. By mapping the sequencing tags to B. bassiana genome, we also identified a number of differentially expressed fungal genes between the early and late infection stages. These genes are generally associated with fungal cell wall synthesis and energy metabolism. The expression of fungal cell wall protein genes might play an important role in fungal pathogenesis and the dramatically up-regulated enzymes of carbon metabolism indicate the increasing usage of energy during the fungal infection. To our knowledge, this is the first report on the molecular mechanism of fungus-whitefly interactions. Our results provide a road map for future investigations on insect-pathogen interactions and genetically modifying the fungus to enhance its efficiency in whitefly control.

Exploring the cellular basis of human disease through a large-scale mapping of deleterious genes to cell types.

PubMed

Cornish, Alex J; Filippis, Ioannis; David, Alessia; Sternberg, Michael J E

2015-09-01

Each cell type found within the human body performs a diverse and unique set of functions, the disruption of which can lead to disease. However, there currently exists no systematic mapping between cell types and the diseases they can cause. In this study, we integrate protein-protein interaction data with high-quality cell-type-specific gene expression data from the FANTOM5 project to build the largest collection of cell-type-specific interactomes created to date. We develop a novel method, called gene set compactness (GSC), that contrasts the relative positions of disease-associated genes across 73 cell-type-specific interactomes to map genes associated with 196 diseases to the cell types they affect. We conduct text-mining of the PubMed database to produce an independent resource of disease-associated cell types, which we use to validate our method. The GSC method successfully identifies known disease-cell-type associations, as well as highlighting associations that warrant further study. This includes mast cells and multiple sclerosis, a cell population currently being targeted in a multiple sclerosis phase 2 clinical trial. Furthermore, we build a cell-type-based diseasome using the cell types identified as manifesting each disease, offering insight into diseases linked through etiology. The data set produced in this study represents the first large-scale mapping of diseases to the cell types in which they are manifested and will therefore be useful in the study of disease systems. Overall, we demonstrate that our approach links disease-associated genes to the phenotypes they produce, a key goal within systems medicine.

The cerebellum: a new key structure in the navigation system

PubMed Central

Rochefort, Christelle; Lefort, Julie M.; Rondi-Reig, Laure

2013-01-01

Early investigations of cerebellar function focused on motor learning, in particular on eyeblink conditioning and adaptation of the vestibulo-ocular reflex, and led to the general view that cerebellar long-term depression (LTD) at parallel fiber (PF)–Purkinje cell (PC) synapses is the neural correlate of cerebellar motor learning. Thereafter, while the full complexity of cerebellar plasticities was being unraveled, cerebellar involvement in more cognitive tasks—including spatial navigation—was further investigated. However, cerebellar implication in spatial navigation remains a matter of debate because motor deficits frequently associated with cerebellar damage often prevent the dissociation between its role in spatial cognition from its implication in motor function. Here, we review recent findings from behavioral and electrophysiological analyses of cerebellar mutant mouse models, which show that the cerebellum might participate in the construction of hippocampal spatial representation map (i.e., place cells) and thereby in goal-directed navigation. These recent advances in cerebellar research point toward a model in which computation from the cerebellum could be required for spatial representation and would involve the integration of multi-source self-motion information to: (1) transform the reference frame of vestibular signals and (2) distinguish between self- and externally-generated vestibular signals. We eventually present herein anatomical and functional connectivity data supporting a cerebello-hippocampal interaction. Whilst a direct cerebello-hippocampal projection has been suggested, recent investigations rather favor a multi-synaptic pathway involving posterior parietal and retrosplenial cortices, two regions critically involved in spatial navigation. PMID:23493515

Statistical parametric mapping of stimuli-evoked changes in quantitative blood flow using extended-focus optical coherence microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Marchand, Paul J.; Bouwens, Arno; Shamaei, Vincent; Nguyen, David; Extermann, Jerome; Bolmont, Tristan; Lasser, Theo

2016-03-01

Magnetic Resonance Imaging has revolutionised our understanding of brain function through its ability to image human cerebral structures non-invasively over the entire brain. By exploiting the different magnetic properties of oxygenated and deoxygenated blood, functional MRI can indirectly map areas undergoing neural activation. Alongside the development of fMRI, powerful statistical tools have been developed in an effort to shed light on the neural pathways involved in processing of sensory and cognitive information. In spite of the major improvements made in fMRI technology, the obtained spatial resolution of hundreds of microns prevents MRI in resolving and monitoring processes occurring at the cellular level. In this regard, Optical Coherence Microscopy is an ideal instrumentation as it can image at high spatio-temporal resolution. Moreover, by measuring the mean and the width of the Doppler spectra of light scattered by moving particles, OCM allows extracting the axial and lateral velocity components of red blood cells. The ability to assess quantitatively total blood velocity, as opposed to classical axial velocity Doppler OCM, is of paramount importance in brain imaging as a large proportion of cortical vascular is oriented perpendicularly to the optical axis. We combine here quantitative blood flow imaging with extended-focus Optical Coherence Microscopy and Statistical Parametric Mapping tools to generate maps of stimuli-evoked cortical hemodynamics at the capillary level.

Bioinformatics Knowledge Map for Analysis of Beta-Catenin Function in Cancer

PubMed Central

Arighi, Cecilia N.; Wu, Cathy H.

2015-01-01

Given the wealth of bioinformatics resources and the growing complexity of biological information, it is valuable to integrate data from disparate sources to gain insight into the role of genes/proteins in health and disease. We have developed a bioinformatics framework that combines literature mining with information from biomedical ontologies and curated databases to create knowledge “maps” of genes/proteins of interest. We applied this approach to the study of beta-catenin, a cell adhesion molecule and transcriptional regulator implicated in cancer. The knowledge map includes post-translational modifications (PTMs), protein-protein interactions, disease-associated mutations, and transcription factors co-activated by beta-catenin and their targets and captures the major processes in which beta-catenin is known to participate. Using the map, we generated testable hypotheses about beta-catenin biology in normal and cancer cells. By focusing on proteins participating in multiple relation types, we identified proteins that may participate in feedback loops regulating beta-catenin transcriptional activity. By combining multiple network relations with PTM proteoform-specific functional information, we proposed a mechanism to explain the observation that the cyclin dependent kinase CDK5 positively regulates beta-catenin co-activator activity. Finally, by overlaying cancer-associated mutation data with sequence features, we observed mutation patterns in several beta-catenin PTM sites and PTM enzyme binding sites that varied by tissue type, suggesting multiple mechanisms by which beta-catenin mutations can contribute to cancer. The approach described, which captures rich information for molecular species from genes and proteins to PTM proteoforms, is extensible to other proteins and their involvement in disease. PMID:26509276

Transcriptome profiling analysis of Vibrio vulnificus during human infection.

PubMed

Bisharat, Naiel; Bronstein, Michal; Korner, Mira; Schnitzer, Temima; Koton, Yael

2013-09-01

Vibrio vulnificus is a waterborne pathogen that was responsible for an outbreak of severe soft-tissue infections among fish farmers and fish consumers in Israel. Several factors have been shown to be associated with virulence. However, the transcriptome profile of the pathogen during human infection has not been determined yet. We compared the transcriptome profile, using RNA sequencing, of a human-pathogenic strain harvested directly from tissue of a patient suffering from severe soft-tissue infection with necrotizing fasciitis, with the same strain and three other environmental strains grown in vitro. The five sequenced libraries were aligned to the reference genomes of V. vulnificus strains CMCP6 and YJ016. Approximately 47.8 to 62.3 million paired-end raw reads were generated from the five runs. Nearly 84 % of the genome was covered by reads from at least one of the five runs, suggesting that nearly 16 % of the genome is not transcribed or is transcribed at low levels. We identified 123 genes that were differentially expressed during the acute phase of infection. Sixty-three genes were mapped to the large chromosome, 47 genes mapped to the small chromosome and 13 genes mapped to the YJ016 plasmid. The 123 genes fell into a variety of functional categories including transcription, signal transduction, cell motility, carbohydrate metabolism, intracellular trafficking and cell envelope biogenesis. Among the genes differentially expressed during human infection we identified genes encoding bacterial toxin (RtxA1) and genes involved in flagellar components, Flp-coding region, GGDEF family protein, iron acquisition system and sialic acid metabolism.

Negative regulatory loci coupling flagellin synthesis to flagellar assembly in Salmonella typhimurium.

PubMed Central

Gillen, K L; Hughes, K T

1991-01-01

The complex regulation of flagellin gene expression in Salmonella typhimurium was characterized in vivo by using lac transcriptional fusions to the two flagellin structural genes (fliC [H1] and fljB [H2]). Phase variation was measured as the rate of switching of flagellin gene expression. Switching frequencies varied from 1/500 per cell per generation to 1/10,000 per cell per generation depending on the particular insertion and the direction of switching. There is a 4- to 20-fold bias in favor of switching from the fljB(On) to the fljB(Off) orientation. Random Tn10dTc insertions were isolated which failed to express flagellin. While most of these insertions mapped to loci known to be required for flagellin expression, several new loci were identified. The presence of functional copies of all of the genes responsible for complete flagellar assembly, except the hook-associated proteins (flgK, flgL, and fliD gene products), were required for expression of the fliC or fljB flagellin genes. Two novel loci involved in negative regulation of fliC and fljB in fla mutant backgrounds were identified. One of these loci, designated the flgR locus, mapped to the flg operon at 23 min on the Salmonella linkage map. An flgR insertion mutation resulted in relief of repression of the fliC and fljB genes in all fla mutant backgrounds except for mutants in the positive regulatory loci (flhC, flhD, and fliA genes). PMID:1848842

Transport dynamics of molecular motors that switch between an active and inactive state

NASA Astrophysics Data System (ADS)

Pinkoviezky, I.; Gov, N. S.

2013-08-01

Molecular motors are involved in key transport processes in the cell. Many of these motors can switch from an active to a nonactive state, either spontaneously or depending on their interaction with other molecules. When active, the motors move processively along the filaments, while when inactive they are stationary. We treat here the simple case of spontaneously switching motors, between the active and inactive states, along an open linear track. We use our recent analogy with vehicular traffic, where we go beyond the mean-field description. We map the phase diagram of this system, and find that it clearly breaks the symmetry between the different phases, as compared to the standard total asymmetric exclusion process. We make several predictions that may be testable using molecular motors in vitro and in living cells.

Quantitative mapping of intracellular cations in the human amniotic membrane

NASA Astrophysics Data System (ADS)

Moretto, Ph.; Llabador, Y.; Simonoff, M.; Razafindrabe, L.; Bara, M.; Guiet-Bara, A.

1993-05-01

The effect of magnesium and taurine on the permeability of cell membranes to monovalent cations has been investigated using the Bordeaux nuclear microprobe. PIXE and RBS techniques have been used to provide quantitative measurements and ion distributions in the isolated amniotic membrane. This physiological model for cellular exchanges allowed us to reveal the distribution of most elements involved in cellular pathways and the modifications under different experimental conditions of incubation in physiological fluids. The PIXE microanalysis provided an original viewpoint on these mechanisms. Following this first study, the amnion compact lamina was found to play a role which was not, up to now, taken into account in the interpretation of electrophysiological experimentations. The release of some ionic species, such as K +, from the epithelial cells, during immersion in isotonic fluids, could have been hitherto underestimated.

Whole genome scan reveals the genetic signature of African Ankole cattle breed and potential for higher quality beef.

PubMed

Taye, Mengistie; Kim, Jaemin; Yoon, Sook Hee; Lee, Wonseok; Hanotte, Olivier; Dessie, Tadelle; Kemp, Stephen; Mwai, Okeyo Ally; Caetano-Anolles, Kelsey; Cho, Seoae; Oh, Sung Jong; Lee, Hak-Kyo; Kim, Heebal

2017-02-09

Africa is home to numerous cattle breeds whose diversity has been shaped by subtle combinations of human and natural selection. African Sanga cattle are an intermediate type of cattle resulting from interbreeding between Bos taurus and Bos indicus subspecies. Recently, research has asserted the potential of Sanga breeds for commercial beef production with better meat quality as compared to Bos indicus breeds. Here, we identified meat quality related gene regions that are positively selected in Ankole (Sanga) cattle breeds as compared to indicus (Boran, Ogaden, and Kenana) breeds using cross-population (XP-EHH and XP-CLR) statistical methods. We identified 238 (XP-EHH) and 213 (XP-CLR) positively selected genes, of which 97 were detected from both statistics. Among the genes obtained, we primarily reported those involved in different biological process and pathways associated with meat quality traits. Genes (CAPZB, COL9A2, PDGFRA, MAP3K5, ZNF410, and PKM2) involved in muscle structure and metabolism affect meat tenderness. Genes (PLA2G2A, PARK2, ZNF410, MAP2K3, PLCD3, PLCD1, and ROCK1) related to intramuscular fat (IMF) are involved in adipose metabolism and adipogenesis. MB and SLC48A1 affect meat color. In addition, we identified genes (TIMP2, PKM2, PRKG1, MAP3K5, and ATP8A1) related to feeding efficiency. Among the enriched Gene Ontology Biological Process (GO BP) terms, actin cytoskeleton organization, actin filament-based process, and protein ubiquitination are associated with meat tenderness whereas cellular component organization, negative regulation of actin filament depolymerization and negative regulation of protein complex disassembly are involved in adipocyte regulation. The MAPK pathway is responsible for cell proliferation and plays an important role in hyperplastic growth, which has a positive effect on meat tenderness. Results revealed several candidate genes positively selected in Ankole cattle in relation to meat quality characteristics. The genes identified are involved in muscle structure and metabolism, and adipose metabolism and adipogenesis. These genes help in the understanding of the biological mechanisms controlling beef quality characteristics in African Ankole cattle. These results provide a basis for further research on the genomic characteristics of Ankole and other Sanga cattle breeds for quality beef.

Mapping of Outdoor Classrooms.

ERIC Educational Resources Information Center

Horvath, Victor G.

Mapping symbols adopted by the Michigan Department of Natural Resources are presented with their explanations. In an effort to provide standardization and familiarity teachers and other school people involved in an outdoor education program are encouraged to utilize the same symbols in constructing maps. (DK)

Human Induced Pluripotent Stem Cell-Derived Cardiac Cell Sheets Expressing Genetically Encoded Voltage Indicator for Pharmacological and Arrhythmia Studies.

PubMed

Shaheen, Naim; Shiti, Assad; Huber, Irit; Shinnawi, Rami; Arbel, Gil; Gepstein, Amira; Setter, Noga; Goldfracht, Idit; Gruber, Amit; Chorna, Snizhanna V; Gepstein, Lior

2018-06-05

Fulfilling the potential of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes for studying conduction and arrhythmogenesis requires development of multicellular models and methods for long-term repeated tissue phenotyping. We generated confluent hiPSC-derived cardiac cell sheets (hiPSC-CCSs), expressing the genetically encoded voltage indicator ArcLight. ArcLight-based optical mapping allowed generation of activation and action-potential duration (APD) maps, which were validated by mapping the same hiPSC-CCSs with the voltage-sensitive dye, Di-4-ANBDQBS. ArcLight mapping allowed long-term assessment of electrical remodeling in the hiPSC-CCSs and evaluation of drug-induced conduction slowing (carbenoxolone, lidocaine, and quinidine) and APD prolongation (quinidine and dofetilide). The latter studies also enabled step-by-step depiction of drug-induced arrhythmogenesis ("torsades de pointes in the culture dish") and its prevention by MgSO 4 and rapid pacing. Phase-mapping analysis allowed biophysical characterization of spiral waves induced in the hiPSC-CCSs and their termination by electrical cardioversion and overdrive pacing. In conclusion, ArcLight mapping of hiPSC-CCSs provides a powerful tool for drug testing and arrhythmia investigation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers.

PubMed

Taraslia, Vasiliki; Lymperi, Stefania; Pantazopoulou, Vasiliki; Anagnostopoulos, Athanasios K; Papassideri, Issidora S; Basdra, Efthimia K; Bei, Marianna; Kontakiotis, Evangelos G; Tsangaris, George Th; Stravopodis, Dimitrios J; Anastasiadou, Ema

2018-01-05

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways.

A High-Resolution Proteomic Landscaping of Primary Human Dental Stem Cells: Identification of SHED- and PDLSC-Specific Biomarkers

PubMed Central

Taraslia, Vasiliki; Lymperi, Stefania; Pantazopoulou, Vasiliki; Anagnostopoulos, Athanasios K.; Basdra, Efthimia K.; Bei, Marianna; Kontakiotis, Evangelos G.; Tsangaris, George Th.; Stravopodis, Dimitrios J.; Anastasiadou, Ema

2018-01-01

Dental stem cells (DSCs) have emerged as a promising tool for basic research and clinical practice. A variety of adult stem cell (ASC) populations can be isolated from different areas within the dental tissue, which, due to their cellular and molecular characteristics, could give rise to different outcomes when used in potential applications. In this study, we performed a high-throughput molecular comparison of two primary human adult dental stem cell (hADSC) sub-populations: Stem Cells from Human Exfoliated Deciduous Teeth (SHEDs) and Periodontal Ligament Stem Cells (PDLSCs). A detailed proteomic mapping of SHEDs and PDLSCs, via employment of nano-LC tandem-mass spectrometry (MS/MS) revealed 2032 identified proteins in SHEDs and 3235 in PDLSCs. In total, 1516 proteins were expressed in both populations, while 517 were unique for SHEDs and 1721 were exclusively expressed in PDLSCs. Further analysis of the recorded proteins suggested that SHEDs predominantly expressed molecules that are involved in organizing the cytoskeletal network, cellular migration and adhesion, whereas PDLSCs are highly energy-producing cells, vastly expressing proteins that are implicated in various aspects of cell metabolism and proliferation. Applying the Rho-GDI signaling pathway as a paradigm, we propose potential biomarkers for SHEDs and for PDLSCs, reflecting their unique features, properties and engaged molecular pathways. PMID:29304003

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

PubMed Central

Ivanov, Sergey V.; Kuzmin, Igor; Wei, Ming-Hui; Pack, Svetlana; Geil, Laura; Johnson, Bruce E.; Stanbridge, Eric J.; Lerman, Michael I.

1998-01-01

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth. PMID:9770531

Antibodies to ependymin block the sharpening of the regenerating retinotectal projection in goldfish.

PubMed

Schmidt, J T; Shashoua, V E

1988-04-19

The regenerating optic nerve of goldfish first reestablishes a rough retinotopic map on the tectum, then goes through an activity dependent refinement that appears to involve the elimination of inappropriate branches from early regenerated arbors. Retinotopically appropriate branches and synapses may be stabilized because the normally correlated firing of neighboring ganglion cells could cause summation of their postsynaptic responses, making them more effective. Thus, refinement of the map may be similar in several ways to associative learning. In this study, we therefore tested whether ependymin, a major protein component of the extracellular fluid that has been implicated in synaptic changes thought to be associated with learning a simple task in goldfish, may also be involved in refinement of the retinotopic map. Goldfish that had undergone unilateral optic nerve crush received intraventricular infusion of antiependymin IgG or of control IgG's beginning at 21 days postcrush. Tectal recordings from these fish at 39-56 days postcrush showed that the projection had failed to sharpen, much as in the fish with activity blocked or synchronized; the average size of the multiunit receptive fields was 31 degrees vs 11 degrees normally. The field potentials elicited from these tecta by optic nerve shock were not significantly smaller than in controls, suggesting normal levels of synaptogenesis. Control projections, identically treated but infused with either unrelated IgG or Ringer's alone regenerated normally, giving multiunit receptive fields of 12 degrees. Intact (non-regenerating) projections of the experimental fish were not rendered abnormal by the IgG treatment. Histology showed the retinas and tecta of the infused fish to be normal in appearance. The results show a specific block of sharpening by antiependymin IgG. The ependymal glia of the tectum stain positively for ependymin in normal fish, particularly the cell bodies in the ependymal layer. The tectum, particularly the ependymal layer, stains more intensely during regeneration, which appears to trigger increased synthesis of ependymins in the ependymal glia. This increase and the block of sharpening by specific antibodies to ependymin suggest a possible role for ependymin in activity dependent synaptic stabilization, possibly through its polymerization when calcium is focally depleted at active synapses.

Cell-accurate optical mapping across the entire developing heart.

PubMed

Weber, Michael; Scherf, Nico; Meyer, Alexander M; Panáková, Daniela; Kohl, Peter; Huisken, Jan

2017-12-29

Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca 2+ -mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs.

Cell-accurate optical mapping across the entire developing heart

PubMed Central

Meyer, Alexander M; Panáková, Daniela; Kohl, Peter

2017-01-01

Organogenesis depends on orchestrated interactions between individual cells and morphogenetically relevant cues at the tissue level. This is true for the heart, whose function critically relies on well-ordered communication between neighboring cells, which is established and fine-tuned during embryonic development. For an integrated understanding of the development of structure and function, we need to move from isolated snap-shot observations of either microscopic or macroscopic parameters to simultaneous and, ideally continuous, cell-to-organ scale imaging. We introduce cell-accurate three-dimensional Ca2+-mapping of all cells in the entire electro-mechanically uncoupled heart during the looping stage of live embryonic zebrafish, using high-speed light sheet microscopy and tailored image processing and analysis. We show how myocardial region-specific heterogeneity in cell function emerges during early development and how structural patterning goes hand-in-hand with functional maturation of the entire heart. Our method opens the way to systematic, scale-bridging, in vivo studies of vertebrate organogenesis by cell-accurate structure-function mapping across entire organs. PMID:29286002

National Assessment of Oil and Gas Project: Areas of Historical Oil and Gas Exploration and Production in the United States

USGS Publications Warehouse

Biewick, Laura

2008-01-01

This report contains maps and associated spatial data showing historical oil and gas exploration and production in the United States. Because of the proprietary nature of many oil and gas well databases, the United States was divided into cells one-quarter square mile and the production status of all wells in a given cell was aggregated. Base-map reference data are included, using the U.S. Geological Survey (USGS) National Map, the USGS and American Geological Institute (AGI) Global GIS, and a World Shaded Relief map service from the ESRI Geography Network. A hardcopy map was created to synthesize recorded exploration data from 1859, when the first oil well was drilled in the U.S., to 2005. In addition to the hardcopy map product, the data have been refined and made more accessible through the use of Geographic Information System (GIS) tools. The cell data are included in a GIS database constructed for spatial analysis via the USGS Internet Map Service or by importing the data into GIS software such as ArcGIS. The USGS internet map service provides a number of useful and sophisticated geoprocessing and cartographic functions via an internet browser. Also included is a video clip of U.S. oil and gas exploration and production through time.

{sup 18}F-FLT uptake kinetics in head and neck squamous cell carcinoma: A PET imaging study

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

Liu, Dan, E-mail: dan.liu@oncology.ox.ac.uk; Fenwick, John D.; Chalkidou, Anastasia

2014-04-15

Purpose: To analyze the kinetics of 3{sup ′}-deoxy-3{sup ′}-[F-18]-fluorothymidine (18F-FLT) uptake by head and neck squamous cell carcinomas and involved nodes imaged using positron emission tomography (PET). Methods: Two- and three-tissue compartment models were fitted to 12 tumor time-activity-curves (TACs) obtained for 6 structures (tumors or involved nodes) imaged in ten dynamic PET studies of 1 h duration, carried out for five patients. The ability of the models to describe the data was assessed using a runs test, the Akaike information criterion (AIC) and leave-one-out cross-validation. To generate parametric maps the models were also fitted to TACs of individual voxels.more » Correlations between maps of different parameters were characterized using Pearson'sr coefficient; in particular the phosphorylation rate-constants k{sub 3-2tiss} and k{sub 5} of the two- and three-tissue models were studied alongside the flux parameters K{sub FLT-2tiss} and K{sub FLT} of these models, and standardized uptake values (SUV). A methodology based on expectation-maximization clustering and the Bayesian information criterion (“EM-BIC clustering”) was used to distil the information from noisy parametric images. Results: Fits of two-tissue models 2C3K and 2C4K and three-tissue models 3C5K and 3C6K comprising three, four, five, and six rate-constants, respectively, pass the runs test for 4, 8, 10, and 11 of 12 tumor TACs. The three-tissue models have lower AIC and cross-validation scores for nine of the 12 tumors. Overall the 3C6K model has the lowest AIC and cross-validation scores and its fitted parameter values are of the same orders of magnitude as literature estimates. Maps ofK{sub FLT} and K{sub FLT-2tiss} are strongly correlated (r = 0.85) and also correlate closely with SUV maps (r = 0.72 for K{sub FLT-2tiss}, 0.64 for K{sub FLT}). Phosphorylation rate-constant maps are moderately correlated with flux maps (r = 0.48 for k{sub 3-2tiss} vs K{sub FLT-2tiss} and r = 0.68 for k{sub 5} vs K{sub FLT}); however, neither phosphorylation rate-constant correlates significantly with SUV. EM-BIC clustering reduces the parametric maps to a small number of levels—on average 5.8, 3.5, 3.4, and 1.4 for K{sub FLT-2tiss}, K{sub FLT}, k{sub 3-2tiss}, and k{sub 5.} This large simplification is potentially useful for radiotherapy dose-painting, but demonstrates the high noise in some maps. Statistical simulations show that voxel level noise degrades TACs generated from the 3C6K model sufficiently that the average AIC score, parameter bias, and total uncertainty of 2C4K model fits are similar to those of 3C6K fits, whereas at the whole tumor level the scores are lower for 3C6K fits. Conclusions: For the patients studied here, whole tumor FLT uptake time-courses are represented better overall by a three-tissue than by a two-tissue model. EM-BIC clustering simplifies noisy parametric maps, providing the best description of the underlying information they contain and is potentially useful for radiotherapy dose-painting. However, the clustering highlights the large degree of noise present in maps of the phosphorylation rate-constantsk{sub 5} and k{sub 3-2tiss}, which are conceptually tightly linked to cellular proliferation. Methods must be found to make these maps more robust—either by constraining other model parameters or modifying dynamic imaging protocols.« less

Mycobacterium avium subsp. paratuberculosis (Map) Fatty Acids Profile Is Strain-Dependent and Changes Upon Host Macrophages Infection

PubMed Central

Alonso-Hearn, Marta; Abendaño, Naiara; Ruvira, Maria A.; Aznar, Rosa; Landin, Mariana; Juste, Ramon A.

2017-01-01

Johne's disease is a chronic granulomatous enteritis of ruminants caused by the intracellular bacterium Mycobacterium avium subsp. paratuberculosis (Map). We previously demonstrated that Map isolates from sheep persisted within host macrophages in lower CFUs than cattle isolates after 7 days of infection. In the current study, we hypothesize that these phenotypic differences between Map isolates may be driven be the fatty acids (FAs) present on the phosphadidyl-1-myo-inositol mannosides of the Map cell wall that mediate recognition by the mannose receptors of host macrophages. FAs modifications may influence Map's envelope fluidity ultimately affecting pathogenicity. To test this hypothesis, we investigated the responses of two Map isolates from cattle (K10 isolate) and sheep (2349/06-1) to the bovine and ovine macrophage environment by measuring the FAs content of extracellular and intracellular bacteria. For this purpose, macrophages cell lines of bovine (BOMAC) and ovine (MOCL-4) origin were infected with the two isolates of Map for 4 days at 37°C. The relative FAs composition of the two isolates recovered from infected BOMAC and MOCL-4 cells was determined by gas chromatography and compared with that of extracellular bacteria and that of bacteria grown in Middlebrook 7H9 medium. Using this approach, we demonstrated that the FAs composition of extracellular and 7H9-grown bacteria was highly conserved within each Map isolate, and statistically different from that of intracellular bacteria. Analysis of FAs composition from extracellular bacteria enabled the distinction of the two Map strains based on the presence of the tuberculostearic acid (18:0 10Me) exclusively in the K10 strain of Map. In addition, significant differences in the content of Palmitic acid and cis-7 Palmitoleic acid between both isolates harvested from the extracellular environment were observed. Once the infection established itself in BOMAC and MOCL-4 cells, the FAs profiles of both Map isolates appeared conserved. Our results suggest that the FAs composition of Map might influence its recognition by macrophages and influence the survival of the bacillus within host macrophages. PMID:28377904

Lead-induced changes of cytoskeletal protein is involved in the pathological basis in mice brain.

PubMed

Ge, Yaming; Chen, Lingli; Sun, Xianghe; Yin, Zhihong; Song, Xiaochao; Li, Chong; Liu, Junwei; An, Zhixing; Yang, Xuefeng; Ning, Hongmei

2018-04-01

Lead poisoning is a geochemical disease. On the other hand, lead is highly carcinogenic and exhibits liver and kidney toxicity. This element can also cross the blood-brain barrier, reduce learning and memory ability and damage the structure of the cerebral cortex and hippocampus. To further investigate the mechanism of lead neurotoxicity, 4-week-old Kunming mice were used to explore the effects of different concentrations of Pb 2+ (0, 2.4, 4.8 and 9.6 mM) for 9 days. In this study, pathological and ultrastructural changes in brain cells of the treated group were related to damages to mitochondria, chromatin and the nucleus. Lead content in blood was tested by atomic absorption spectroscopy, which showed high lead concentrations in the blood with increasing doses of lead. Distribution of lead in nerve cells was analysed by transmission electron microscopy with energy dispersive spectroscopy. Data showed the presence of lead in nucleopores, chromatin and nuclear membrane of nerve cells in the treatment groups, whereas lead content increased with increasing doses of lead acetate. Finally, microtubule-associated protein 2 (MAP2) mRNA and protein expression levels were detected by real-time PCR and Western blotting, which showed a reduction in MAP2 expression with increasing lead doses in the mouse brain. These findings suggest that acute lead poisoning can cause significant dose-dependent toxic effects on mouse brain function and can contribute to better understanding of lead-induced toxicity.

TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network

PubMed Central

Usaj, Matej; Tan, Yizhao; Wang, Wen; VanderSluis, Benjamin; Zou, Albert; Myers, Chad L.; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2017-01-01

Providing access to quantitative genomic data is key to ensure large-scale data validation and promote new discoveries. TheCellMap.org serves as a central repository for storing and analyzing quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with the budding yeast Saccharomyces cerevisiae. In particular, TheCellMap.org allows users to easily access, visualize, explore, and functionally annotate genetic interactions, or to extract and reorganize subnetworks, using data-driven network layouts in an intuitive and interactive manner. PMID:28325812

TheCellMap.org: A Web-Accessible Database for Visualizing and Mining the Global Yeast Genetic Interaction Network.

PubMed

Usaj, Matej; Tan, Yizhao; Wang, Wen; VanderSluis, Benjamin; Zou, Albert; Myers, Chad L; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2017-05-05

Providing access to quantitative genomic data is key to ensure large-scale data validation and promote new discoveries. TheCellMap.org serves as a central repository for storing and analyzing quantitative genetic interaction data produced by genome-scale Synthetic Genetic Array (SGA) experiments with the budding yeast Saccharomyces cerevisiae In particular, TheCellMap.org allows users to easily access, visualize, explore, and functionally annotate genetic interactions, or to extract and reorganize subnetworks, using data-driven network layouts in an intuitive and interactive manner. Copyright © 2017 Usaj et al.

Functional toxicogenomic assessment of triclosan in human ...

EPA Pesticide Factsheets

Thousands of chemicals for which limited toxicological data are available are used and then detected in humans and the environment. Rapid and cost-effective approaches for assessing the toxicological properties of chemicals are needed. We used CRISPR-Cas9 functional genomic screening to identify potential molecular mechanism of a widely used antimicrobial triclosan (TCS) in HepG2 cells. Resistant genes (whose knockout gives potential resistance) at IC50 (50% Inhibition concentration of cell viability) were significantly enriched in adherens junction pathway, MAPK signaling pathway and PPAR signaling pathway, suggesting a potential molecular mechanism in TCS induced cytotoxicity. Evaluation of top-ranked resistant genes, FTO (encoding an mRNA demethylase) and MAP2K3 (a MAP kinase kinase family gene), revealed that their loss conferred resistance to TCS. In contrast, sensitive genes (whose knockout enhances potential sensitivity) at IC10 and IC20 were specifically enriched in pathways involved with immune responses, which was concordant with the transcriptomic profiling of TCS at concentrations

An Experimental Approach to Controllably Vary Protein Oxidation While Minimizing Electrode Adsorption for Boron-Doped Diamond Electrochemical Surface Mapping Applications

PubMed Central

McClintock, Carlee S; Hettich, Robert L.

2012-01-01

Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent – hydroxyl radicals – for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins. PMID:23210708

Solubilization methods and reference 2-DE map of cow milk fat globules.

PubMed

Bianchi, Laura; Puglia, Michele; Landi, Claudia; Matteoni, Silvia; Perini, Daniele; Armini, Alessandro; Verani, Margherita; Trombetta, Claudia; Soldani, Patrizia; Roncada, Paola; Greppi, Gianfranco; Pallini, Vitaliano; Bini, Luca

2009-07-21

Milk fat globules (MFGs) are secretory vesicles assembled and secreted by mammary epithelial cells during lactation. They consist of fat globules surrounded by a lipid bilayer membrane which is derived from the apical membrane of the lactating cells. MFGs contain, besides lipids, proteins from the apical plasma membrane and from the cytoplasmatic material. Their peculiar vesicle nature makes them a suitable and easily available source of biological material in monitoring the physiopathological state of the mammary gland. Unfortunately, the conspicuous lipidic component of MFGs consistently limits protein extraction and purification for MFG proteomic investigations. This work deals with the development of a suitable procedure for protein extraction from the cow MFGs in order to qualitatively and quantitatively improve 2-D electropherograms of the MFG. MFGs were purified from raw milk by centrifugation and then delipidated/precipitated. The resulting protein pellets were solubilised using four different 2-D SDS PAGE compatible lysis buffers. Applied methodological procedures for protein extraction and evaluation of the resulting 2-D protein-pattern are presented and discussed. Using these procedures a reference 2-D map of cow milk fat globules is also reported. The majority of the obtained identifications was represented by proteins involved in lipid synthesis or in fat globule secretion.

15-hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes.

PubMed

Kim, Hak-Jae; Lee, Sora; Lee, Haw-Yong; Won, Hansol; Chang, Sung-Hae; Nah, Seong-Su

2015-09-01

15-Hydroxyprostaglandin dehydrogenase (HPGD) is the key enzyme responsible for the metabolic inactivation of prostaglandin E2 (PGE2) catabolism. PGE2 is one of the predominant catabolic factors involved in rheumatoid arthritis (RA). However, the expression and regulation of HPGD in RA fibroblast‑like synoviocyte (FLS) remain to be elucidated. Disease‑modifying anti‑rheumatic drugs (DMARDs) are the most important anti‑arthritic drugs, which reduce the effect of joint injury. The aim of the present study was to assess the expression of HPGD in RA tissues and cells, and normal synovial tissues and cells. The effect of the most popular DMARDs, hydroxychloroquine, on the expression of HPGD in RA‑FLS was also investigated. Western blotting and immunohistochemical analysis demonstrated that the expression levels of HPGD in human synovium were lower in RA synovium compared with the normal and OA synovium. In RA‑FLS, the expression of HPGD was increased following treatment with several DMARDs, including sulfasalazine, methotrexate, and hydroxychloroquine. Hydroxychloroquine (10 µM) treatment induced the phosphorylation of ERK, SAPK/JNK and p38. Hydroxychloroquine induced a decrease in the release of PGE2, which was restored by mitogen‑activated protein (MAP) kinase pathway inhibitors. Hydroxychloroquine may therefore, affect the pathogenesis of RA through the MAP kinase pathway by regulating the expression of HPGD.

15-Hydroxyprostaglandin dehydrogenase is upregulated by hydroxychloroquine in rheumatoid arthritis fibroblast-like synoviocytes

PubMed Central

KIM, HAK-JAE; LEE, SORA; LEE, HAW-YONG; WON, HANSOL; CHANG, SUNG-HAE; NAH, SEONG-SU

2015-01-01

15-Hydroxyprostaglandin dehydrogenase (HPGD) is the key enzyme responsible for the metabolic inactivation of prostaglandin E2 (PGE2) catabolism. PGE2 is one of the predominant catabolic factors involved in rheumatoid arthritis (RA). However, the expression and regulation of HPGD in RA fibroblast-like synoviocyte (FLS) remain to be elucidated. Disease-modifying anti-rheumatic drugs (DMARDs) are the most important anti-arthritic drugs, which reduce the effect of joint injury. The aim of the present study was to assess the expression of HPGD in RA tissues and cells, and normal synovial tissues and cells. The effect of the most popular DMARDs, hydroxychloroquine, on the expression of HPGD in RA-FLS was also investigated. Western blotting and immuno-histochemical analysis demonstrated that the expression levels of HPGD in human synovium were lower in RA synovium compared with the normal and OA synovium. In RA-FLS, the expression of HPGD was increased following treatment with several DMARDs, including sulfasalazine, methotrexate, and hydroxychloroquine. Hydroxychloroquine (10 µM) treatment induced the phosphorylation of ERK, SAPK/JNK and p38. Hydroxychloroquine induced a decrease in the release of PGE2, which was restored by mitogen-activated protein (MAP) kinase pathway inhibitors. Hydroxychloroquine may therefore, affect the pathogenesis of RA through the MAP kinase pathway by regulating the expression of HPGD. PMID:26082314

Glutathione Fine-Tunes the Innate Immune Response toward Antiviral Pathways in a Macrophage Cell Line Independently of Its Antioxidant Properties

PubMed Central

Diotallevi, Marina; Checconi, Paola; Palamara, Anna Teresa; Celestino, Ignacio; Coppo, Lucia; Holmgren, Arne; Abbas, Kahina; Peyrot, Fabienne; Mengozzi, Manuela; Ghezzi, Pietro

2017-01-01

Glutathione (GSH), a major cellular antioxidant, is considered an inhibitor of the inflammatory response involving reactive oxygen species (ROS). However, evidence is largely based on experiments with exogenously added antioxidants/reducing agents or pro-oxidants. We show that depleting macrophages of 99% of GSH does not exacerbate the inflammatory gene expression profile in the RAW264 macrophage cell line or increase expression of inflammatory cytokines in response to the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS); only two small patterns of LPS-induced genes were sensitive to GSH depletion. One group, mapping to innate immunity and antiviral responses (Oas2, Oas3, Mx2, Irf7, Irf9, STAT1, il1b), required GSH for optimal induction. Consequently, GSH depletion prevented the LPS-induced activation of antiviral response and its inhibition of influenza virus infection. LPS induction of a second group of genes (Prdx1, Srxn1, Hmox1, GSH synthase, cysteine transporters), mapping to nrf2 and the oxidative stress response, was increased by GSH depletion. We conclude that the main function of endogenous GSH is not to limit inflammation but to fine-tune the innate immune response to infection. PMID:29033950

Proteomic profiles in hyperandrogenic syndromes.

PubMed

Misiti, S; Stigliano, A; Borro, M; Gentile, G; Michienzi, S; Cerquetti, L; Bucci, B; Argese, N; Brunetti, E; Simmaco, M; Toscano, V

2010-03-01

Polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia (CAH) represent the most common causes of hyperandrogenism. Although the etiopathogeneses of these syndromes are different, they share many clinical and biochemical signs, such as hirsutism, acne, and chronic anovulation. Experimental data have shown that peripheral T-lymphocytes function as molecular sensors, being able to record molecular signals either at staminal and mature cell levels, or hormones at systemic levels. Twenty PCOS women and 10 CAH with 21-hydroxylase deficiency, aged between 18-35 yr, were studied. T-cells purified from all patients and 20 healthy donors have been analyzed by 2-dimensional gel electrophoresis. Silver-stained proteomic map of each patient was compared with a control map obtained by pooling protein samples of the 20 healthy subjects. Spots of interest were identified by peptide mass fingerprint. Computer analysis evidenced several peptidic spots significantly modulated in all patients examined. Some proteins were modulated in both syndromes, others only in PCOS or in CAH. These proteins are involved in many physiological processes as the functional state of immune system, the regulation of the cytoskeleton structure, the oxidative stress, the coagulation process, and the insulin resistance. Identification of the physiological function of these proteins could help to understand ethiopathogenetic mechanisms of hyperandrogenic syndromes and its complications.

Intracellular pH of Mycobacterium avium subsp. paratuberculosis following exposure to antimicrobial compounds monitored at the single cell level.

PubMed

Gaggìa, Francesca; Nielsen, Dennis Sandris; Biavati, Bruno; Siegumfeldt, Henrik

2010-07-31

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease; moreover, it seems to be implicated in the development of Crohn's disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (pH(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long and problematic incubations needed for methods relying upon growth of MAP such as determination of colony forming units. The FRIM results showed that both nisin and the cell-free supernatant from Lactobacillus plantarum PCA 236 affected the pH(i) of MAP within a few hours. However, monitoring the population for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives at farm level. Copyright 2010 Elsevier B.V. All rights reserved.

Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

PubMed

Lu, D; Yang, H; Raizada, M K

1996-12-01

Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity

PubMed Central

Danson, Christopher M.; Pocha, Shirin M.; Bloomberg, Graham B.; Cory, Giles O.

2009-01-01

Summary The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration. PMID:18032787

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity.

PubMed

Danson, Christopher M; Pocha, Shirin M; Bloomberg, Graham B; Cory, Giles O

2007-12-01

The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.

Application of process mapping to understand integration of high risk medicine care bundles within community pharmacy practice.

PubMed

Weir, Natalie M; Newham, Rosemary; Corcoran, Emma D; Ali Atallah Al-Gethami, Ashwag; Mohammed Abd Alridha, Ali; Bowie, Paul; Watson, Anne; Bennie, Marion

2017-11-21

The Scottish Patient Safety Programme - Pharmacy in Primary Care collaborative is a quality improvement initiative adopting the Institute of Healthcare Improvement Breakthrough Series collaborative approach. The programme developed and piloted High Risk Medicine (HRM) Care Bundles (CB), focused on warfarin and non-steroidal anti-inflammatories (NSAIDs), within 27 community pharmacies over 4 NHS Regions. Each CB involves clinical assessment and patient education, although the CB content varies between regions. To support national implementation, this study aims to understand how the pilot pharmacies integrated the HRM CBs into routine practice to inform the development of a generic HRM CB process map. Regional process maps were developed in 4 pharmacies through simulation of the CB process, staff interviews and documentation of resources. Commonalities were collated to develop a process map for each HRM, which were used to explore variation at a national event. A single, generic process map was developed which underwent validation by case study testing. The findings allowed development of a generic process map applicable to warfarin and NSAID CB implementation. Five steps were identified as required for successful CB delivery: patient identification; clinical assessment; pharmacy CB prompt; CB delivery; and documentation. The generic HRM CB process map encompasses the staff and patients' journey and the CB's integration into routine community pharmacy practice. Pharmacist involvement was required only for clinical assessment, indicating suitability for whole-team involvement. Understanding CB integration into routine practice has positive implications for successful implementation. The generic process map can be used to develop targeted resources, and/or be disseminated to facilitate CB delivery and foster whole team involvement. Similar methods could be utilised within other settings, to allow those developing novel services to distil the key processes and consider their integration within routine workflows to effect maximal, efficient implementation and benefit to patient care. Copyright © 2017 Elsevier Inc. All rights reserved.

Functional reorganization of visual cortex maps after ischemic lesions is accompanied by changes in expression of cytoskeletal proteins and NMDA and GABA(A) receptor subunits.

PubMed

Zepeda, Angelica; Sengpiel, Frank; Guagnelli, Miguel Angel; Vaca, Luis; Arias, Clorinda

2004-02-25

Reorganization of cortical representations after focal visual cortex lesions has been documented. It has been suggested that functional reorganization may rely on cellular mechanisms involving modifications in the excitatory/inhibitory neurotransmission balance and on morphological changes of neurons peripheral to the lesion. We explored functional reorganization of cortical retinotopic maps after a focal ischemic lesion in primary visual cortex of kittens using optical imaging of intrinsic signals. After 1, 2, and 5 weeks postlesion (wPL), we addressed whether functional reorganization correlated in time with changes in the expression of MAP-2, GAP-43, GFAP, GABA(A) receptor subunit alpha1 (GABA(A)alpha1), subunit 1 of the NMDA receptor (NMDAR1), and in neurotransmitter levels at the border of the lesion. Our results show that: (1) retinotopic maps reorganize with time after an ischemic lesion; (2) MAP-2 levels increase gradually from 1wPL to 5wPL; (3) MAP-2 upregulation is associated with an increase in dendritic-like structures surrounding the lesion and a decrease in GFAP-positive cells; (4) GAP-43 levels reach the highest point at 2wPL; (5) NMDAR1 and glutamate contents increase in parallel from 1wPL to 5wPL; (6) GABA(A)alpha1 levels increase from 1wPL to 2wPL but do not change after this time point; and (7) GABA contents remain low from 1wPL to 5wPL. This is a comprehensive study showing for the first time that functional reorganization correlates in time with dendritic sprouting and with changes in the excitatory/inhibitory neurotransmission systems previously proposed to participate in cortical remodeling and suggests mechanisms by which plasticity of cortical representations may occur.

Mapping fuels at multiple scales: landscape application of the fuel characteristic classification system.

Treesearch

D. McKenzie; C.L. Raymond; L.-K.B. Kellogg; R.A. Norheim; A.G. Andreu; A.C. Bayard; K.E. Kopper; E. Elman

2007-01-01

Fuel mapping is a complex and often multidisciplinary process, involving remote sensing, ground-based validation, statistical modeling, and knowledge-based systems. The scale and resolution of fuel mapping depend both on objectives and availability of spatial data layers. We demonstrate use of the Fuel Characteristic Classification System (FCCS) for fuel mapping at two...

The Global Evidence Mapping Initiative: scoping research in broad topic areas.

PubMed

Bragge, Peter; Clavisi, Ornella; Turner, Tari; Tavender, Emma; Collie, Alex; Gruen, Russell L

2011-06-17

Evidence mapping describes the quantity, design and characteristics of research in broad topic areas, in contrast to systematic reviews, which usually address narrowly-focused research questions. The breadth of evidence mapping helps to identify evidence gaps, and may guide future research efforts. The Global Evidence Mapping (GEM) Initiative was established in 2007 to create evidence maps providing an overview of existing research in Traumatic Brain Injury (TBI) and Spinal Cord Injury (SCI). The GEM evidence mapping method involved three core tasks:1. Setting the boundaries and context of the map: Definitions for the fields of TBI and SCI were clarified, the prehospital, acute inhospital and rehabilitation phases of care were delineated and relevant stakeholders (patients, carers, clinicians, researchers and policymakers) who could contribute to the mapping were identified. Researchable clinical questions were developed through consultation with key stakeholders and a broad literature search. 2. Searching for and selection of relevant studies: Evidence search and selection involved development of specific search strategies, development of inclusion and exclusion criteria, searching of relevant databases and independent screening and selection by two researchers. 3. Reporting on yield and study characteristics: Data extraction was performed at two levels - 'interventions and study design' and 'detailed study characteristics'. The evidence map and commentary reflected the depth of data extraction. One hundred and twenty-nine researchable clinical questions in TBI and SCI were identified. These questions were then prioritised into high (n = 60) and low (n = 69) importance by the stakeholders involved in question development. Since 2007, 58 263 abstracts have been screened, 3 731 full text articles have been reviewed and 1 644 relevant neurotrauma publications have been mapped, covering fifty-three high priority questions. GEM Initiative evidence maps have a broad range of potential end-users including funding agencies, researchers and clinicians. Evidence mapping is at least as resource-intensive as systematic reviewing. The GEM Initiative has made advancements in evidence mapping, most notably in the area of question development and prioritisation. Evidence mapping complements other review methods for describing existing research, informing future research efforts, and addressing evidence gaps.

The Global Evidence Mapping Initiative: Scoping research in broad topic areas

PubMed Central

2011-01-01

Background Evidence mapping describes the quantity, design and characteristics of research in broad topic areas, in contrast to systematic reviews, which usually address narrowly-focused research questions. The breadth of evidence mapping helps to identify evidence gaps, and may guide future research efforts. The Global Evidence Mapping (GEM) Initiative was established in 2007 to create evidence maps providing an overview of existing research in Traumatic Brain Injury (TBI) and Spinal Cord Injury (SCI). Methods The GEM evidence mapping method involved three core tasks: 1. Setting the boundaries and context of the map: Definitions for the fields of TBI and SCI were clarified, the prehospital, acute inhospital and rehabilitation phases of care were delineated and relevant stakeholders (patients, carers, clinicians, researchers and policymakers) who could contribute to the mapping were identified. Researchable clinical questions were developed through consultation with key stakeholders and a broad literature search. 2. Searching for and selection of relevant studies: Evidence search and selection involved development of specific search strategies, development of inclusion and exclusion criteria, searching of relevant databases and independent screening and selection by two researchers. 3. Reporting on yield and study characteristics: Data extraction was performed at two levels - 'interventions and study design' and 'detailed study characteristics'. The evidence map and commentary reflected the depth of data extraction. Results One hundred and twenty-nine researchable clinical questions in TBI and SCI were identified. These questions were then prioritised into high (n = 60) and low (n = 69) importance by the stakeholders involved in question development. Since 2007, 58 263 abstracts have been screened, 3 731 full text articles have been reviewed and 1 644 relevant neurotrauma publications have been mapped, covering fifty-three high priority questions. Conclusions GEM Initiative evidence maps have a broad range of potential end-users including funding agencies, researchers and clinicians. Evidence mapping is at least as resource-intensive as systematic reviewing. The GEM Initiative has made advancements in evidence mapping, most notably in the area of question development and prioritisation. Evidence mapping complements other review methods for describing existing research, informing future research efforts, and addressing evidence gaps. PMID:21682870

Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

PubMed Central

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma

2012-01-01

Background Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal Findings We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action. PMID:23227207

Screening the budding yeast genome reveals unique factors affecting K2 toxin susceptibility.

PubMed

Servienė, Elena; Lukša, Juliana; Orentaitė, Irma; Lafontaine, Denis L J; Urbonavičius, Jaunius

2012-01-01

Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

MiR-30c regulates cisplatin-induced apoptosis of renal tubular epithelial cells by targeting Bnip3L and Hspa5

PubMed Central

Du, Bin; Dai, Xiao-meng; Li, Shuang; Qi, Guo-long; Cao, Guang-xu; Zhong, Ying; Yin, Pei-di; Yang, Xue-song

2017-01-01

As a common anticancer drug, cisplatin has been widely used for treating tumors in the clinic. However, its side effects, especially its nephrotoxicity, noticeably restrict the application of cisplatin. Therefore, it is imperative to investigate the mechanism of renal injury and explore the corresponding remedies. In this study, we showed the phenotypes of the renal tubules and epithelial cell death as well as elevated cleaved-caspase3- and TUNEL-positive cells in rats intraperitoneally injected with cisplatin. Similar cisplatin-induced cell apoptosis was found in HK-2 and NRK-52E cells exposed to cisplatin as well. In both models of cisplatin-induced apoptosis in vivo and in vitro, quantitative PCR data displayed reductions in miR-30a-e expression levels, indicating that miR-30 might be involved in regulating cisplatin-induced cell apoptosis. This was further confirmed when the effects of cisplatin-induced cell apoptosis were found to be closely correlated with alterations in miR-30c expression, which were manipulated by transfection of either the miR-30c mimic or miR-30c inhibitor in HK-2 and NRK-52E cells. Using bioinformatics tools, including TargetScan and a gene expression database (Gene Expression Omnibus), Adrb1, Bnip3L, Hspa5 and MAP3K12 were predicted to be putative target genes of miR-30c in cisplatin-induced apoptosis. Subsequently, Bnip3L and Hspa5 were confirmed to be the target genes after determining the expression of these putative genes following manipulation of miR-30c expression levels in HK-2 cells. Taken together, our current experiments reveal that miR-30c is certainly involved in regulating the renal tubular cell apoptosis induced by cisplatin, which might supply a new strategy to minimize cisplatin-induced nephrotoxicity. PMID:28796263

MiR-30c regulates cisplatin-induced apoptosis of renal tubular epithelial cells by targeting Bnip3L and Hspa5.

PubMed

Du, Bin; Dai, Xiao-Meng; Li, Shuang; Qi, Guo-Long; Cao, Guang-Xu; Zhong, Ying; Yin, Pei-di; Yang, Xue-Song

2017-08-10

As a common anticancer drug, cisplatin has been widely used for treating tumors in the clinic. However, its side effects, especially its nephrotoxicity, noticeably restrict the application of cisplatin. Therefore, it is imperative to investigate the mechanism of renal injury and explore the corresponding remedies. In this study, we showed the phenotypes of the renal tubules and epithelial cell death as well as elevated cleaved-caspase3- and TUNEL-positive cells in rats intraperitoneally injected with cisplatin. Similar cisplatin-induced cell apoptosis was found in HK-2 and NRK-52E cells exposed to cisplatin as well. In both models of cisplatin-induced apoptosis in vivo and in vitro, quantitative PCR data displayed reductions in miR-30a-e expression levels, indicating that miR-30 might be involved in regulating cisplatin-induced cell apoptosis. This was further confirmed when the effects of cisplatin-induced cell apoptosis were found to be closely correlated with alterations in miR-30c expression, which were manipulated by transfection of either the miR-30c mimic or miR-30c inhibitor in HK-2 and NRK-52E cells. Using bioinformatics tools, including TargetScan and a gene expression database (Gene Expression Omnibus), Adrb1, Bnip3L, Hspa5 and MAP3K12 were predicted to be putative target genes of miR-30c in cisplatin-induced apoptosis. Subsequently, Bnip3L and Hspa5 were confirmed to be the target genes after determining the expression of these putative genes following manipulation of miR-30c expression levels in HK-2 cells. Taken together, our current experiments reveal that miR-30c is certainly involved in regulating the renal tubular cell apoptosis induced by cisplatin, which might supply a new strategy to minimize cisplatin-induced nephrotoxicity.

Ephs and Ephrins in Cancer: Ephrin-A1 Signaling

PubMed Central

Beauchamp, Amanda; Debinski, Waldemar

2011-01-01

Ephrin-A1 and its primary receptor, EphA2, are involved in numerous physiological processes and have been intensely studied for their roles in malignancy. Ephrin-Eph signalling is complex on its own and is also cell-type dependent, making elucidation of the exact role of ephrin-A1 in neoplasia challenging. Multiple oncogenic signalling pathways, such as MAP/ERK and PI3K are affected by ephrin-A1, and in some cases evidence suggests the promotion of a specific pathway in one cell or cancer type and inhibition of the same pathway in another type of cell or cancer. EphrinA1 also plays an integral role in angiogenesis and tumor neovascularization. Until recently, studies investigating ephrins focused on the ligands as GPI-anchored proteins that required membrane anchoring or artificial clustering for Eph receptor activation. However, recent studies have demonstrated a functional role for soluble, monomeric ephrin-A1. This review will focus on various forms of ephrin-A1-specific signalling in human malignancy. PMID:22040911

Transcriptional profiling of Medicago truncatula meristematic root cells

PubMed Central

Holmes, Peta; Goffard, Nicolas; Weiller, Georg F; Rolfe, Barry G; Imin, Nijat

2008-01-01

Background The root apical meristem of crop and model legume Medicago truncatula is a significantly different stem cell system to that of the widely studied model plant species Arabidopsis thaliana. In this study we used the Affymetrix Medicago GeneChip® to compare the transcriptomes of meristem and non-meristematic root to identify root meristem specific candidate genes. Results Using mRNA from root meristem and non-meristem we were able to identify 324 and 363 transcripts differentially expressed from the two regions. With bioinformatics tools developed to functionally annotate the Medicago genome array we could identify significant changes in metabolism, signalling and the differentially expression of 55 transcription factors in meristematic and non-meristematic roots. Conclusion This is the first comprehensive analysis of M. truncatula root meristem cells using this genome array. This data will facilitate the mapping of regulatory and metabolic networks involved in the open root meristem of M. truncatula and provides candidates for functional analysis. PMID:18302802

A linear model fails to predict orientation selectivity of cells in the cat visual cortex.

PubMed Central

Volgushev, M; Vidyasagar, T R; Pei, X

1996-01-01

1. Postsynaptic potentials (PSPs) evoked by visual stimulation in simple cells in the cat visual cortex were recorded using in vivo whole-cell technique. Responses to small spots of light presented at different positions over the receptive field and responses to elongated bars of different orientations centred on the receptive field were recorded. 2. To test whether a linear model can account for orientation selectivity of cortical neurones, responses to elongated bars were compared with responses predicted by a linear model from the receptive field map obtained from flashing spots. 3. The linear model faithfully predicted the preferred orientation, but not the degree of orientation selectivity or the sharpness of orientation tuning. The ratio of optimal to non-optimal responses was always underestimated by the model. 4. Thus non-linear mechanisms, which can include suppression of non-optimal responses and/or amplification of optimal responses, are involved in the generation of orientation selectivity in the primary visual cortex. PMID:8930828

Genome-wide profiling of PRC1 and PRC2 Polycomb chromatin binding in Drosophila melanogaster.

PubMed

Tolhuis, Bas; de Wit, Elzo; Muijrers, Inhua; Teunissen, Hans; Talhout, Wendy; van Steensel, Bas; van Lohuizen, Maarten

2006-06-01

Polycomb group (PcG) proteins maintain transcriptional repression of developmentally important genes and have been implicated in cell proliferation and stem cell self-renewal. We used a genome-wide approach to map binding patterns of PcG proteins (Pc, esc and Sce) in Drosophila melanogaster Kc cells. We found that Pc associates with large genomic regions of up to approximately 150 kb in size, hereafter referred to as 'Pc domains'. Sce and esc accompany Pc in most of these domains. PcG-bound chromatin is trimethylated at histone H3 Lys27 and is generally transcriptionally silent. Furthermore, PcG proteins preferentially bind to developmental genes. Many of these encode transcriptional regulators and key components of signal transduction pathways, including Wingless, Hedgehog, Notch and Delta. We also identify several new putative functions of PcG proteins, such as in steroid hormone biosynthesis. These results highlight the extensive involvement of PcG proteins in the coordination of development through the formation of large repressive chromatin domains.

Structural basis of substrate discrimination and integrin binding by autotaxin

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

Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos

2013-09-25

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates.more » We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.« less

A DNA methylation map of human cancer at single base-pair resolution.

PubMed

Vidal, E; Sayols, S; Moran, S; Guillaumet-Adkins, A; Schroeder, M P; Royo, R; Orozco, M; Gut, M; Gut, I; Lopez-Bigas, N; Heyn, H; Esteller, M

2017-10-05

Although single base-pair resolution DNA methylation landscapes for embryonic and different somatic cell types provided important insights into epigenetic dynamics and cell-type specificity, such comprehensive profiling is incomplete across human cancer types. This prompted us to perform genome-wide DNA methylation profiling of 22 samples derived from normal tissues and associated neoplasms, including primary tumors and cancer cell lines. Unlike their invariant normal counterparts, cancer samples exhibited highly variable CpG methylation levels in a large proportion of the genome, involving progressive changes during tumor evolution. The whole-genome sequencing results from selected samples were replicated in a large cohort of 1112 primary tumors of various cancer types using genome-scale DNA methylation analysis. Specifically, we determined DNA hypermethylation of promoters and enhancers regulating tumor-suppressor genes, with potential cancer-driving effects. DNA hypermethylation events showed evidence of positive selection, mutual exclusivity and tissue specificity, suggesting their active participation in neoplastic transformation. Our data highlight the extensive changes in DNA methylation that occur in cancer onset, progression and dissemination.

A comprehensive map of the mTOR signaling network

PubMed Central

Caron, Etienne; Ghosh, Samik; Matsuoka, Yukiko; Ashton-Beaucage, Dariel; Therrien, Marc; Lemieux, Sébastien; Perreault, Claude; Roux, Philippe P; Kitano, Hiroaki

2010-01-01

The mammalian target of rapamycin (mTOR) is a central regulator of cell growth and proliferation. mTOR signaling is frequently dysregulated in oncogenic cells, and thus an attractive target for anticancer therapy. Using CellDesigner, a modeling support software for graphical notation, we present herein a comprehensive map of the mTOR signaling network, which includes 964 species connected by 777 reactions. The map complies with both the systems biology markup language (SBML) and graphical notation (SBGN) for computational analysis and graphical representation, respectively. As captured in the mTOR map, we review and discuss our current understanding of the mTOR signaling network and highlight the impact of mTOR feedback and crosstalk regulations on drug-based cancer therapy. This map is available on the Payao platform, a Web 2.0 based community-wide interactive process for creating more accurate and information-rich databases. Thus, this comprehensive map of the mTOR network will serve as a tool to facilitate systems-level study of up-to-date mTOR network components and signaling events toward the discovery of novel regulatory processes and therapeutic strategies for cancer. PMID:21179025

The Human Genome Initiative: First Steps.

ERIC Educational Resources Information Center

Newman, Alan R.

1990-01-01

Described is the basic biology involved in mapping chromosomes as presented at a symposium at a recent meeting of the American Chemical Association which focused on the Human Genome Initiative. Different types of gene maps and techniques used to produce gene maps are discussed. (CW)

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway

PubMed Central

Liu, Kuang-Kai; Qiu, Wei-Ru; Naveen Raj, Emmanuel; Liu, Huei-Fang; Huang, Hou-Syun; Lin, Yu-Wei; Chang, Chien-Jen; Chen, Ting-Hua; Chen, Chinpiao; Chang, Huan-Cheng; Hwang, Jenn-Kang; Chao, Jui-I

2017-01-01

ABSTRACT Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes. PMID:27846374