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  1. Juxtaglomerular cell tumor: A case report

    PubMed Central

    YANG, HONGYUAN; WANG, ZUFEI; JI, JIANSONG

    2016-01-01

    The current study reports the case of a 29-year-old female with a long-standing history of hypertension and headaches who presented to the Outpatient Clinic of The Central Hospital of Lishui (Lishui, Zhejiang, China). Abdominal ultrasound and contrast-enhanced computed tomography were performed, which showed a left renal neoplasm, prompting a diagnosis of renal angiomyolipoma or renal cell carcinoma. After a laparoscopic partial nephrectomy was performed, a number of different diagnoses were suggested by several pathologists from eight hospitals. Considering the patient's gender, age, medical history, histopathological features and immunohistochemistry, a final diagnosis of a juxtaglomerular cell tumor (JGCT) was established. The present study therefore indicates that the possibility of a JGCT should be considered when young adults present with renal parenchymatous tumors and high blood pressure. In addition, pathologists must take clinical information into account to form a precise diagnosis. PMID:26893753

  2. Exocytosis and endocytosis in juxtaglomerular cells.

    PubMed

    Friis, U G; Jensen, B L; Hansen, P B; Andreasen, D; Skøtt, O

    2000-01-01

    The cellular events related to secretion of renin are not well understood. Here we review some of the evidence that has led to the current understanding of renin secretion as a process that involves exocytosis as the predominant mode of secretion. This is based on the observation of occasional fusion events between secretory granules and cell membrane and measurement of intermittent secretion of renin from single afferent arterioles, with a renin content of each secretion episode that corresponds to the renin content of one secretory granule. More recently it has been demonstrated that the afferent arterioles lose a large number of renin granules after acute stimulation without changing the average granular volume. Current electrophysiological techniques have now permitted direct measurements of cell membrane capacitance in juxtaglomerular (JG) cells as a measure of net addition (exocytosis) or removal (endocytosis) of membrane material. With this technique we have shown that cAMP, which is a vasodilator and stimulates renin secretion, enhances net exocytosis at low concentrations, while at higher concentrations membrane retrieval processes are also stimulated. We suggest that both exocytosis and endocytosis are regulated processes in the JG-cells and both may be important for the long-term control of renin secretion at the single cell level. PMID:10691785

  3. Juxtaglomerular cell tumor--a rare cause of secondary hypertension.

    PubMed

    Dolezel, Z; Starha, J; Pavlovsky, Z; Skotakova, J; Dostalkova, D

    2010-01-01

    Secondary hypertension (SH) is much more common in children than in adults. We report a 17-year-old girl with severe hypertension, hypokalemia and metabolic alkalosis. Because of these findings, primary or secondary hyperaldosteronism was suspected. Her initial treatment with spironolactone and ACE inhibitor was unsuccessful. With consideration of high plasma renin activity, the renal computed tomography angiography was performed and showed tumor mass in the left kidney. An uncomplicated partial left nephrectomy was performed. Histopathological examination and electron microscopy showed typical features of juxtaglomerular cell tumor (JCT). Imunohistochemistry of tumor was positive for CD34 and CD117 and this finding is effective in the diagnosis of JCT if immunostain for renin is unavailable. After the resection of JCT, the patient's blood pressure and hypokalemia returned to normal range. JCT is a rare renal neoplasm and an unusual cause of SH in children or adolescents (Fig. 2, Ref. 12). PMID:21384734

  4. Magnetic Resonance Imaging Features of a Juxtaglomerular Cell Tumor

    PubMed Central

    Kang, Suhai; Guo, Aitao; Wang, Haiyi; Ma, Lu; Xie, Zongyu; Li, Jinglong; Tonge, Xinyuan; Ye, Huiyi

    2015-01-01

    Objective: To retrospectively determine whether magnetic resonance imaging (MRI) findings can help differentiate a juxtaglomerular cell tumor (JCT) from clear cell renal cell carcinoma (ccRCC). Materials and Methods: Eight patients with JCTs and 24 patients with pathologically proven ccRCC were included for image analysis. All patients underwent unenhanced MRI and dynamic contrast-enhanced MRI. Fat-suppressed T2-weighted imaging (T2WI), diffusion-weighted imaging (DWI), in- and opposed-phase imaging, and fat-suppressed preliver acquisitions with volume acceleration sequences were performed before enhancement. After the administration of contrast, dynamic imaging was performed in the corticomedullary, nephrographic, and excretory phases. Student's t-test, t′-test, Chi-square test, and nonparametric Kruskal–Wallis H-test were used to determine the significance of the difference between the two groups. The sensitivity and specificity of the MRI findings were calculated. Results: In patients with a JCT, a cystic part of the lesion of <10%, isointensity or mild hyperintensity on T2WI, heterogeneous hyperintensity on DWI, less signal drop (<10%) in in- and opposed-phase imaging, and a degree of enhancement <200% in the corticomedullary phase showed statistically significant differences compared with those of ccRCC (P < 0.05). After combining a lower apparent diffusion coefficient (ADC) value (heterogeneous hyperintensity) on DWI and a degree of enhancement <200% in the corticomedullary phase using a parallel test, the sensitivity and specificity were 90.9% and 91.7%, respectively. Conclusions: Isointensity or mild hyperintensity on T2WI, a lower ADC value (heterogeneous hyperintensity) on DWI, and a degree of enhancement <200% in the corticomedullary phase are the major MRI findings for JCTs, combined with relative clinical manifestations and excluding other renal masses. A main solid tumor, less signal drop (<10%) in in- and opposed-phase imaging, and a less

  5. Electrophysiological and molecular characterization of the inward rectifier in juxtaglomerular cells from rat kidney

    PubMed Central

    Leichtle, Anke; Rauch, Ulrich; Albinus, Margitta; Benöhr, Peter; Kalbacher, Hubert; Mack, Andreas F; Veh, Rüdiger W; Quast, Ulrich; Russ, Ulrich

    2004-01-01

    Renin, the key element of the renin–angiotensin–aldosterone system, is mainly produced by and stored in the juxtaglomerular cells in the kidney. These cells are situated in the media of the afferent arteriole close to the vessel pole and can transform into smooth muscle cells and vice versa. In this study, the electrophysiological properties and the molecular identity of the K+ channels responsible for the resting membrane potential (∼−60 mV) of the juxtaglomerular cells were examined. In order to increase the number of juxtaglomerular cells, afferent arterioles from NaCl-depleted rats were used, and > 90% of the afferent arterioles were renin positive at the distal end of the arteriole. Whole-cell and cell-attached single-channel patch-clamp experiments showed that juxtaglomerular cells are endowed with a strongly inwardly rectifying K+ channel (Kir). The channel was highly sensitive to inhibition by Ba2+ (inhibition constant 37 μm at 0 mV), but relatively insensitive to Cs+ and, with 142 mm K+ in the pipette, had a single-channel conductance of 31.5 pS. Immunocytochemical studies showed the presence of Kir2.1 but no signal for Kir2.2 in the media of the afferent arteriole. In PCR analyses using isolated juxtaglomerular cells, the mRNA for Kir2.1 and Kir2.2 was detected. Collectively, the results show that Kir2.1 is the dominant component of the channel. The current carried by these channels plays a decisive role in setting the membrane potential of juxtaglomerular cells. PMID:15284349

  6. Chronic Hypoxia-Inducible Transcription Factor-2 Activation Stably Transforms Juxtaglomerular Renin Cells into Fibroblast-Like Cells In Vivo

    PubMed Central

    Gerl, Katharina; Karger, Christian; Schwarzensteiner, Ilona; Kurtz, Armin

    2015-01-01

    On the basis of previous observations that deletion of the von Hippel–Lindau protein (pVHL) in juxtaglomerular (JG) cells of the kidney suppresses renin and induces erythropoietin expression, this study aimed to characterize the events underlying this striking change of hormone expression. We found that renin cell-specific deletion of pVHL in mice leads to a phenotype switch in JG cells, from a cuboid and multiple vesicle-containing form into a flat and elongated form without vesicles. This shift of cell phenotype was accompanied by the disappearance of marker proteins for renin cells (e.g., aldo-keto reductase family 1, member 7 and connexin 40) and by the appearance of markers of fibroblast-like cells (e.g., collagen I, ecto-5′-nucleotidase, and PDGF receptor-β). Furthermore, hypoxia-inducible transcription factor-2α (HIF-2α) protein constitutively accumulated in these transformed cells. Codeletion of pVHL and HIF-2α in JG cells completely prevented the phenotypic changes. Similar to renin expression in normal JG cells, angiotensin II negatively regulated erythropoietin expression in the transformed cells. In summary, chronic activation of HIF-2 in renal JG cells leads to a reprogramming of the cells into fibroblast-like cells resembling native erythropoietin-producing cells located in the tubulointerstitium. PMID:25071089

  7. Long-term in vivo single-cell tracking reveals the switch of migration patterns in adult-born juxtaglomerular cells of the mouse olfactory bulb.

    PubMed

    Liang, Yajie; Li, Kaizhen; Riecken, Kristoffer; Maslyukov, Anatoliy; Gomez-Nicola, Diego; Kovalchuk, Yury; Fehse, Boris; Garaschuk, Olga

    2016-07-01

    The behavior of adult-born cells can be easily monitored in cell culture or in lower model organisms, but longitudinal observation of individual mammalian adult-born cells in their native microenvironment still proves to be a challenge. Here we have established an approach named optical cell positioning system for long-term in vivo single-cell tracking, which integrates red-green-blue cell labeling with repeated angiography. By combining this approach with in vivo two-photon imaging technique, we characterized the in vivo migration patterns of adult-born neurons in the olfactory bulb. In contrast to the traditional view of mere radial migration of adult-born cells within the bulb, we found that juxtaglomerular cells switch from radial migration to long distance lateral migration upon arrival in their destination layer. This unique long-distance lateral migration has characteristic temporal (stop-and-go) and spatial (migratory, unidirectional or multidirectional) patterns, with a clear cell age-dependent decrease in the migration speed. The active migration of adult-born cells coincides with the time period of initial fate determination and is likely to impact on the integration sites of adult-born cells, their odor responsiveness, as well as their survival rate. PMID:27174051

  8. Endothelin inhibits renin release from juxtaglomerular cells via endothelin receptors A and B via a transient receptor potential canonical‐mediated pathway

    PubMed Central

    Ortiz‐Capisano, M. Cecilia

    2014-01-01

    Abstract Renin is the rate‐limiting step in the production of angiotensin II: a critical element in the regulation of blood pressure and in the pathogenesis of hypertension. Renin release from the juxtaglomerular (JG) cell is stimulated by the second messenger cAMP and inhibited by increases in calcium (Ca). Endothelins (ETs) inhibit renin release in a Ca‐dependent manner. JG cells contain multiple isoforms of canonical transient receptor potential (TRPC) Ca‐permeable channels. The proposed hypothesis is that endothelin inhibits renin release by activating TRPC store‐operated Ca channels. RT‐PCR and immunofluorescence revealed expression of both ETA and ETB receptors in mouse JG cells. Incubation of primary cultures of JG cells with ET‐1 (10 nmol/L) decreased renin release by 28%. Addition of either an ETA or an ETB receptor blocker completely prevented the ET inhibition of renin release. Incubation with the TRPC blocker (SKF 96365, 50 μmol/L) completely reversed the Ca‐mediated inhibition of renin release by ETs. These results suggest that endothelin inhibits renin release from JG cells via both ETA and ETB receptors, which leads to the activation of TRPC store‐operated Ca channels. PMID:25524278

  9. Prostaglandin E2 EP2 and EP4 receptor activation mediates cAMP-dependent hyperpolarization and exocytosis of renin in juxtaglomerular cells.

    PubMed

    Friis, Ulla G; Stubbe, Jane; Uhrenholt, Torben R; Svenningsen, Per; Nüsing, Rolf M; Skøtt, Ole; Jensen, Boye L

    2005-11-01

    PGE(2) and PGI(2) stimulate renin secretion and cAMP accumulation in juxtaglomerular granular (JG) cells. We addressed, at the single-cell level, the receptor subtypes and intracellular transduction mechanisms involved. Patch clamp was used to determine cell capacitance (C(m)), current, and membrane voltage in response to PGE(2), EP2 and EP4 receptor agonists, and an IP receptor agonist. PGE(2) (0.1 micromol/l) increased C(m) significantly, and the increase was abolished by intracellular application of the protein kinase A antagonist Rp-8-CPT-cAMPS. EP2-selective ligands butaprost (1 micromol/l), AE1-259-01 (1 nmol/l), EP4-selective agonist AE1-329 (1 nmol/l), and IP agonist iloprost (1 micromol/l) significantly increased C(m) mediated by PKA. The EP4 antagonist AE3-208 (10 nmol/l) blocked the effect of EP4 agonist but did not alter the response to PGE(2). Application of both EP4 antagonist and EP2-antagonist AH-6809 abolished the effects of PGE(2) on C(m) and current. EP2 and EP4 ligands stimulated cAMP formation in JG cells. PGE(2) rapidly stimulated renin secretion from superfused JG cells and diminished the membrane-adjacent granule pool as determined by confocal microscopy. The membrane potential hyperpolarized significantly after PGE(2), butaprost, AE1-329 and AE1-259 and outward current was augmented in a PKA-dependent fashion. PGE(2)-stimulated outward current, but not C(m) change, was abolished by the BK(Ca) channel inhibitor iberiotoxin (300 nmol/l). EP2 and EP4 mRNA was detected in sampled JG cells, and the preglomerular and glomerular vasculature was immunopositive for EP4. Thus IP, EP2, and EP4 receptors are associated with JG cells, and their activation leads to rapid PKA-mediated exocytotic fusion and release of renin granules. PMID:15985651

  10. Salt restriction leads to activation of adult renal mesenchymal stromal cell-like cells via prostaglandin E2 and E-prostanoid receptor 4.

    PubMed

    Yang, Yanqiang; Gomez, Jose A; Herrera, Marcela; Perez-Marco, Romelia; Repenning, Peter; Zhang, Zhiping; Payne, Alan; Pratt, Richard E; Koller, Beverly; Beierwaltes, William H; Coffman, Thomas; Mirotsou, Maria; Dzau, Victor J

    2015-05-01

    Despite the importance of juxtaglomerular cell recruitment in the pathophysiology of cardiovascular diseases, the mechanisms that underlie renin production under conditions of chronic stimulation remain elusive. We have previously shown that CD44+ mesenchymal-like cells (CD44+ cells) exist in the adult kidney. Under chronic sodium deprivation, these cells are recruited to the juxtaglomerular area and differentiate to new renin-expressing cells. Given the proximity of macula densa to the juxtaglomerular area and the importance of macula densa released prostanoids in renin synthesis and release, we hypothesized that chronic sodium deprivation induces macula densa release of prostanoids, stimulating renal CD44+ cell activation and differentiation. CD44+ cells were isolated from adult kidneys and cocultured with the macula densa cell line, MMDD1, in normal or low-sodium medium. Low sodium stimulated prostaglandin E2 production by MMDD1 and induced migration of CD44+ cells. These effects were inhibited by addition of a cyclooxygenase 2 inhibitor (NS398) or an E-prostanoid receptor 4 antagonist (AH23848) to MMDD1 or CD44+ cells, respectively. Addition of prostaglandin E2 to CD44+ cells increased cell migration and induced renin expression. In vivo activation of renal CD44+ cells during juxtaglomerular recruitment was attenuated in wild-type mice subjected to salt restriction in the presence of cyclooxygenase 2 inhibitor rofecoxib. Similar results were observed in E-prostanoid receptor 4 knockout mice subjected to salt restriction. These results show that the prostaglandin E2/E-prostanoid receptor 4 pathway plays a key role in the activation of renal CD44+ mesenchymal stromal cell-like cells during conditions of juxtaglomerular recruitment; highlighting the importance of this pathway as a key regulatory mechanism of juxtaglomerular recruitment. PMID:25776075

  11. The endomembrane requirement for cell surface repair

    NASA Technical Reports Server (NTRS)

    McNeil, Paul L.; Miyake, Katsuya; Vogel, Steven S.

    2003-01-01

    The capacity to reseal a plasma membrane disruption rapidly is required for cell survival in many physiological environments. Intracellular membrane (endomembrane) is thought to play a central role in the rapid resealing response. We here directly compare the resealing response of a cell that lacks endomembrane, the red blood cell, with that of several nucleated cells possessing an abundant endomembrane compartment. RBC membrane disruptions inflicted by a mode-locked Ti:sapphire laser, even those initially smaller than hemoglobin, failed to reseal rapidly. By contrast, much larger laser-induced disruptions made in sea urchin eggs, fibroblasts, and neurons exhibited rapid, Ca(2+)-dependent resealing. We conclude that rapid resealing is not mediated by simple physiochemical mechanisms; endomembrane is required.

  12. Cell Alignment Required in Differentiation of Myxococcus xanthus

    NASA Astrophysics Data System (ADS)

    Kim, Seung K.; Kaiser, Dale

    1990-08-01

    During fruiting body morphogenesis of Myxococcus xanthus, cell movement is required for transmission of C-factor, a short range intercellular signaling protein necessary for sporulation and developmental gene expression. Nonmotile cells fail to sporulate and to express C-factor-dependent genes, but both defects were rescued by a simple manipulation of cell position that oriented the cells in aligned, parallel groups. A similar pattern of aligned cells normally results from coordinated recruitment of wild-type cells into multicellular aggregates, which later form mature fruiting bodies. It is proposed that directed cell movement establishes critical contacts between adjacent cells, which are required for efficient intercellular C-factor transmission.

  13. Efficient cell migration requires global chromatin condensation

    PubMed Central

    Gerlitz, Gabi; Bustin, Michael

    2010-01-01

    Cell migration is a fundamental process that is necessary for the development and survival of multicellular organisms. Here, we show that cell migration is contingent on global condensation of the chromatin fiber. Induction of directed cell migration by the scratch-wound assay leads to decreased DNaseI sensitivity, alterations in the chromatin binding of architectural proteins and elevated levels of H4K20me1, H3K27me3 and methylated DNA. All these global changes are indicative of increased chromatin condensation in response to induction of directed cell migration. Conversely, chromatin decondensation inhibited the rate of cell migration, in a transcription-independent manner. We suggest that global chromatin condensation facilitates nuclear movement and reshaping, which are important for cell migration. Our results support a role for the chromatin fiber that is distinct from its known functions in genetic processes. PMID:20530575

  14. PCDH10 is required for the tumorigenicity of glioblastoma cells

    SciTech Connect

    Echizen, Kanae; Nakada, Mitsutoshi; Hayashi, Tomoatsu; Sabit, Hemragul; Furuta, Takuya; Nakai, Miyuki; Koyama-Nasu, Ryo; Nishimura, Yukiko; Taniue, Kenzui; Morishita, Yasuyuki; Hirano, Shinji; Terai, Kenta; Todo, Tomoki; Ino, Yasushi; Mukasa, Akitake; Takayanagi, Shunsaku; Ohtani, Ryohei; Saito, Nobuhito; Akiyama, Tetsu

    2014-01-31

    Highlights: • PCDH10 is required for the proliferation, survival and self-renewal of glioblastoma cells. • PCDH10 is required for glioblastoma cell migration and invasion. • PCDH10 is required for the tumorigenicity of glioblastoma cells. • PCDH10 may be a promising target for the therapy of glioblastoma. - Abstract: Protocadherin10 (PCDH10)/OL-protocadherin is a cadherin-related transmembrane protein that has multiple roles in the brain, including facilitating specific cell–cell connections, cell migration and axon guidance. It has recently been reported that PCDH10 functions as a tumor suppressor and that its overexpression inhibits proliferation or invasion of multiple tumor cells. However, the function of PCDH10 in glioblastoma cells has not been elucidated. In contrast to previous reports on other tumors, we show here that suppression of the expression of PCDH10 by RNA interference (RNAi) induces the growth arrest and apoptosis of glioblastoma cells in vitro. Furthermore, we demonstrate that knockdown of PCDH10 inhibits the growth of glioblastoma cells xenografted into immunocompromised mice. These results suggest that PCDH10 is required for the proliferation and tumorigenicity of glioblastoma cells. We speculate that PCDH10 may be a promising target for the therapy of glioblastoma.

  15. Closure of supporting cell scar formations requires dynamic actin mechanisms.

    PubMed

    Hordichok, Andrew J; Steyger, Peter S

    2007-10-01

    In many vertebrate inner ear sensory epithelia, dying sensory hair cells are extruded, and the apices of surrounding supporting cells converge to re-seal the epithelial barrier between the electrochemically-distinct endolymph and perilymph. These cellular mechanisms remain poorly understood. Dynamic microtubular mechanisms have been proposed for hair cell extrusion; while contractile actomyosin-based mechanisms are required for cellular extrusion and closure in epithelial monolayers. The hypothesis that cytoskeletal mechanisms are required for hair cell extrusion and supporting cell scar formation was tested using bullfrog saccules incubated with gentamicin (6h), and allowed to recover (18h). Explants were then fixed, labeled for actin and cytokeratins, and viewed with confocal microscopy. To block dynamic cytoskeletal processes, disruption agents for microtubules (colchicine, paclitaxel) myosin (Y-27632, ML-9) or actin (cytochalasin D, latrunculin A) were added during treatment and recovery. Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion. Actin disruption agents blocked scar formation, and largely prevented hair cell extrusion. These data suggest that actin-based cytoskeletal processes are required for hair cell extrusion and supporting cell scar formation in bullfrog saccules. PMID:17716843

  16. Differential requirement for Nfil3 during NK cell development.

    PubMed

    Seillet, Cyril; Huntington, Nicholas D; Gangatirkar, Pradnya; Axelsson, Elin; Minnich, Martina; Brady, Hugh J M; Busslinger, Meinrad; Smyth, Mark J; Belz, Gabrielle T; Carotta, Sebastian

    2014-03-15

    NK cells can be grouped into distinct subsets that are localized to different organs and exhibit a different capacity to secrete cytokines and mediate cytotoxicity. Despite these hallmarks that reflect tissue-specific specialization in NK cells, little is known about the factors that control the development of these distinct subsets. The basic leucine zipper transcription factor Nfil3 (E4bp4) is essential for bone marrow-derived NK cell development, but it is not clear whether Nfil3 is equally important for all NK cell subsets or how it induces NK lineage commitment. In this article, we show that Nfil3 is required for the formation of Eomes-expressing NK cells, including conventional medullary and thymic NK cells, whereas TRAIL(+) Eomes(-) NK cells develop independently of Nfil3. Loss of Nfil3 during the development of bone marrow-derived NK cells resulted in reduced expression of Eomes and, conversely, restoration of Eomes expression in Nfil3(-/-) progenitors rescued NK cell development and maturation. Collectively, these findings demonstrate that Nfil3 drives the formation of mature NK cells by inducing Eomes expression and reveal the differential requirements of NK cell subsets for Nfil3. PMID:24532575

  17. Testicular Niche Required for Human Spermatogonial Stem Cell Expansion

    PubMed Central

    Smith, James F.; Yango, Pamela; Altman, Eran; Choudhry, Shweta; Poelzl, Andrea; Zamah, Alberuni M.; Rosen, Mitchell; Klatsky, Peter C.

    2014-01-01

    Prepubertal boys treated with high-dose chemotherapy do not have an established means of fertility preservation because no established in vitro technique exists to expand and mature purified spermatogonial stem cells (SSCs) to functional sperm in humans. In this study, we define and characterize the unique testicular cellular niche required for SSC expansion using testicular tissues from men with normal spermatogenesis. Highly purified SSCs and testicular somatic cells were isolated by fluorescence-activated cell sorting using SSEA-4 and THY1 as markers of SSCs and somatic cells. Cells were cultured on various established niches to assess their role in SSC expansion in a defined somatic cellular niche. Of all the niches examined, cells in the SSEA-4 population exclusively bound to adult testicular stromal cells, established colonies, and expanded. Further characterization of these testicular stromal cells revealed distinct mesenchymal markers and the ability to undergo differentiation along the mesenchymal lineage, supporting a testicular multipotent stromal cell origin. In vitro human SSC expansion requires a unique niche provided exclusively by testicular multipotent stromal cells with mesenchymal properties. These findings provide an important foundation for developing methods of inducing SSC growth and maturation in prepubertal testicular tissue, essential to enabling fertility preservation for these boys. PMID:25038247

  18. Graded requirement for the spliceosome in cell cycle progression

    PubMed Central

    Karamysheva, Zemfira; Díaz-Martínez, Laura A; Warrington, Ross; Yu, Hongtao

    2015-01-01

    Genome stability is ensured by multiple surveillance mechanisms that monitor the duplication, segregation, and integrity of the genome throughout the cell cycle. Depletion of components of the spliceosome, a macromolecular machine essential for mRNA maturation and gene expression, has been associated with increased DNA damage and cell cycle defects. However, the specific role for the spliceosome in these processes has remained elusive, as different cell cycle defects have been reported depending on the specific spliceosome subunit depleted. Through a detailed cell cycle analysis after spliceosome depletion, we demonstrate that the spliceosome is required for progression through multiple phases of the cell cycle. Strikingly, the specific cell cycle phenotype observed after spliceosome depletion correlates with the extent of depletion. Partial depletion of a core spliceosome component results in defects at later stages of the cell cycle (G2 and mitosis), whereas a more complete depletion of the same component elicits an early cell cycle arrest in G1. We propose a quantitative model in which different functional dosages of the spliceosome are required for different cell cycle transitions. PMID:25892155

  19. Ammonium Ion Requirement for the Cell Cycle of Mycobacterium avium

    PubMed Central

    McCarthy, Charlotte

    1978-01-01

    Mycobacterium avium has a defined cell cycle in which small cells elongate to about five times their original length and then divide by fragmentation. The nitrogen requirement for production of maximal number of colony-forming units was assessed by varying concentrations and kinds of nitrogen source in the medium. Ferric ammonium citrate at a concentration in 7H10 medium of 0.17 μmol/ml or ammonium chloride at 0.25 μmol/ml as the nitrogen source permitted the cells to elongate and to undergo limited division, with the final culture at 4 × 107 colony-forming units per ml. Ammonium chloride at 2.5 μmol/ml or glutamine at 1.37 μmol/ml supported completion of the cell cycle with final colony-forming units at about 5 × 108/ml. Other amino acids, including glutamic acid, at 2.5 μmol/ml did not support completion of the cell cycle, although in most cases an intermediate number of colony-forming units per milliliter were formed. Limited uptake of [14C]glutamic acid and uptake of [14C]glutamine were not detectable until cell fission began. Cells not limited for nitrogen took up five times as much 35S during fission as limited cells did during the same time. The nonlimited cells contained 10 times as much sulfolipid as the nitrogen-limited cells at the end of the cell cycle. These results demonstrate that rapidly dividing cells of M. avium utilize amino acids and sulfur and also synthesize sulfolipids in events that are apparently separable from metabolic functions of elongating cells. The results are contrasted with those found for other mycobacteria in which no cell cycle has been demonstrated. Images PMID:624592

  20. Identifying Francisella tularensis Genes Required for Growth in Host Cells

    PubMed Central

    Brunton, J.; Steele, S.; Miller, C.; Lovullo, E.; Taft-Benz, S.

    2015-01-01

    Francisella tularensis is a highly virulent Gram-negative intracellular pathogen capable of infecting a vast diversity of hosts, ranging from amoebae to humans. A hallmark of F. tularensis virulence is its ability to quickly grow to high densities within a diverse set of host cells, including, but not limited to, macrophages and epithelial cells. We developed a luminescence reporter system to facilitate a large-scale transposon mutagenesis screen to identify genes required for growth in macrophage and epithelial cell lines. We screened 7,454 individual mutants, 269 of which exhibited reduced intracellular growth. Transposon insertions in the 269 growth-defective strains mapped to 68 different genes. FTT_0924, a gene of unknown function but highly conserved among Francisella species, was identified in this screen to be defective for intracellular growth within both macrophage and epithelial cell lines. FTT_0924 was required for full Schu S4 virulence in a murine pulmonary infection model. The ΔFTT_0924 mutant bacterial membrane is permeable when replicating in hypotonic solution and within macrophages, resulting in strongly reduced viability. The permeability and reduced viability were rescued when the mutant was grown in a hypertonic solution, indicating that FTT_0924 is required for resisting osmotic stress. The ΔFTT_0924 mutant was also significantly more sensitive to β-lactam antibiotics than Schu S4. Taken together, the data strongly suggest that FTT_0924 is required for maintaining peptidoglycan integrity and virulence. PMID:25987704

  1. Shielded Cells D&D and Dismantlement System Requirements

    SciTech Connect

    Witherspoon, R.L.

    1995-03-27

    This document describes the basis for the development of the System for Highly Radioactive Equipment Dismantlement or SHRED. It is the result of a thorough investigation into current and past dismantlement practices at shielded cell facilities around the DOE complex. This information has been used to formulate the development requirements for the SHRED.

  2. Regulatory T cells require TCR signaling for their suppressive function

    PubMed Central

    Schmidt, Amanda M.; Lu, Wen; Sindhava, Vishal J.; Huang, Yanping; Burkhardt, Janis K.; Yang, Enjun; Riese, Matthew J.; Maltzman, Jonathan S.; Jordan, Martha S.; Kambayashi, Taku

    2015-01-01

    Regulatory T cells (Tregs) are a subset of CD4+ T cells that maintain immune tolerance in part by their ability to inhibit the proliferation of conventional CD4+ T cells (Tconvs). The role of the T cell receptor (TCR) and the downstream signaling pathways required for this suppressive function of Tregs are not fully understood. To yield insight into how TCR-mediated signals influence Treg suppressive function, we assessed the ability of Tregs with altered TCR-mediated signaling capacity to inhibit Tconv proliferation. Mature Tregs deficient in SLP-76, an adaptor protein that nucleates the proximal signaling complex downstream of the TCR, were unable to inhibit Tconv proliferation, suggesting that TCR signaling is required for Treg suppressive function. Moreover, Tregs with defective PLCγ activation due to a Y145F mutation of SLP-76 were also defective in their suppressive function. Conversely, enhancement of diacylglycerol-mediated signaling downstream of PLCγ by genetic ablation of a negative regulator of diacylglycerol kinase ζ increased the suppressive ability of Tregs. Since SLP-76 is also important for integrin activation and signaling, we tested the role of integrin activation in Treg-mediated suppression. Tregs lacking the adaptor proteins ADAP or Crk/CrkL, which are required for TCR-mediated integrin activation, inhibited Tconv proliferation to a similar extent as wildtype Tregs. Together, these data suggest that TCR-mediated PLCγ activation but not integrin activation is required for Tregs to inhibit Tconv proliferation. PMID:25821220

  3. B cell priming for extrafollicular antibody responses requires Bcl-6 expression by T cells.

    PubMed

    Lee, Sau K; Rigby, Robert J; Zotos, Dimitra; Tsai, Louis M; Kawamoto, Shimpei; Marshall, Jennifer L; Ramiscal, Roybel R; Chan, Tyani D; Gatto, Dominique; Brink, Robert; Yu, Di; Fagarasan, Sidonia; Tarlinton, David M; Cunningham, Adam F; Vinuesa, Carola G

    2011-07-01

    T follicular helper cells (Tfh cells) localize to follicles where they provide growth and selection signals to mutated germinal center (GC) B cells, thus promoting their differentiation into high affinity long-lived plasma cells and memory B cells. T-dependent B cell differentiation also occurs extrafollicularly, giving rise to unmutated plasma cells that are important for early protection against microbial infections. Bcl-6 expression in T cells has been shown to be essential for the formation of Tfh cells and GC B cells, but little is known about its requirement in physiological extrafollicular antibody responses. We use several mouse models in which extrafollicular plasma cells can be unequivocally distinguished from those of GC origin, combined with antigen-specific T and B cells, to show that the absence of T cell-expressed Bcl-6 significantly reduces T-dependent extrafollicular antibody responses. Bcl-6(+) T cells appear at the T-B border soon after T cell priming and before GC formation, and these cells express low amounts of PD-1. Their appearance precedes that of Bcl-6(+) PD-1(hi) T cells, which are found within the GC. IL-21 acts early to promote both follicular and extrafollicular antibody responses. In conclusion, Bcl-6(+) T cells are necessary at B cell priming to form extrafollicular antibody responses, and these pre-GC Tfh cells can be distinguished phenotypically from GC Tfh cells. PMID:21708925

  4. Reliable in vitro studies require appropriate ovarian cancer cell lines.

    PubMed

    Jacob, Francis; Nixdorf, Sheri; Hacker, Neville F; Heinzelmann-Schwarz, Viola A

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  5. Reliable in vitro studies require appropriate ovarian cancer cell lines

    PubMed Central

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  6. Bistability: Requirements on Cell-Volume, Protein Diffusion, and Thermodynamics

    PubMed Central

    Endres, Robert G.

    2015-01-01

    Bistability is considered wide-spread among bacteria and eukaryotic cells, useful e.g. for enzyme induction, bet hedging, and epigenetic switching. However, this phenomenon has mostly been described with deterministic dynamic or well-mixed stochastic models. Here, we map known biological bistable systems onto the well-characterized biochemical Schlögl model, using analytical calculations and stochastic spatiotemporal simulations. In addition to network architecture and strong thermodynamic driving away from equilibrium, we show that bistability requires fine-tuning towards small cell volumes (or compartments) and fast protein diffusion (well mixing). Bistability is thus fragile and hence may be restricted to small bacteria and eukaryotic nuclei, with switching triggered by volume changes during the cell cycle. For large volumes, single cells generally loose their ability for bistable switching and instead undergo a first-order phase transition. PMID:25874711

  7. Histone Deacetylase 3 Is Required for Efficient T Cell Development

    PubMed Central

    Stengel, Kristy R.; Zhao, Yue; Klus, Nicholas J.; Kaiser, Jonathan F.; Gordy, Laura E.; Joyce, Sebastian

    2015-01-01

    Hdac3 is a key target for Hdac inhibitors that are efficacious in cutaneous T cell lymphoma. Moreover, the regulation of chromatin structure is critical as thymocytes transition from an immature cell with open chromatin to a mature T cell with tightly condensed chromatin. To define the phenotypes controlled by Hdac3 during T cell development, we conditionally deleted Hdac3 using the Lck-Cre transgene. This strategy inactivated Hdac3 in the double-negative stages of thymocyte development and caused a significant impairment at the CD8 immature single-positive (ISP) stage and the CD4/CD8 double-positive stage, with few mature CD4+ or CD8+ single-positive cells being produced. When Hdac3−/− mice were crossed with Bcl-xL-, Bcl2-, or TCRβ-expressing transgenic mice, a modest level of complementation was found. However, when the null mice were crossed with mice expressing a fully rearranged T cell receptor αβ transgene, normal levels of CD4 single-positive cells were produced. Thus, Hdac3 is required for the efficient transit from double-negative stage 4 through positive selection. PMID:26324326

  8. Autophagy is required for ectoplasmic specialization assembly in sertoli cells.

    PubMed

    Liu, Chao; Wang, Hongna; Shang, Yongliang; Liu, Weixiao; Song, Zhenhua; Zhao, Haichao; Wang, Lina; Jia, Pengfei; Gao, Fengyi; Xu, Zhiliang; Yang, Lin; Gao, Fei; Li, Wei

    2016-05-01

    The ectoplasmic specialization (ES) is essential for Sertoli-germ cell communication to support all phases of germ cell development and maturity. Its formation and remodeling requires rapid reorganization of the cytoskeleton. However, the molecular mechanism underlying the regulation of ES assembly is still largely unknown. Here, we show that Sertoli cell-specific disruption of autophagy influenced male mouse fertility due to the resulting disorganized seminiferous tubules and spermatozoa with malformed heads. In autophagy-deficient mouse testes, cytoskeleton structures were disordered and ES assembly was disrupted. The disorganization of the cytoskeleton structures might be caused by the accumulation of a negative cytoskeleton organization regulator, PDLIM1, and these defects could be partially rescued by Pdlim1 knockdown in autophagy-deficient Sertoli cells. Altogether, our works reveal that the degradation of PDLIM1 by autophagy in Sertoli cells is important for the proper assembly of the ES, and these findings define a novel role for autophagy in Sertoli cell-germ cell communication. PMID:26986811

  9. Histone Deacetylase 3 Is Required for Efficient T Cell Development.

    PubMed

    Stengel, Kristy R; Zhao, Yue; Klus, Nicholas J; Kaiser, Jonathan F; Gordy, Laura E; Joyce, Sebastian; Hiebert, Scott W; Summers, Alyssa R

    2015-11-01

    Hdac3 is a key target for Hdac inhibitors that are efficacious in cutaneous T cell lymphoma. Moreover, the regulation of chromatin structure is critical as thymocytes transition from an immature cell with open chromatin to a mature T cell with tightly condensed chromatin. To define the phenotypes controlled by Hdac3 during T cell development, we conditionally deleted Hdac3 using the Lck-Cre transgene. This strategy inactivated Hdac3 in the double-negative stages of thymocyte development and caused a significant impairment at the CD8 immature single-positive (ISP) stage and the CD4/CD8 double-positive stage, with few mature CD4(+) or CD8(+) single-positive cells being produced. When Hdac3(-/-) mice were crossed with Bcl-xL-, Bcl2-, or TCRβ-expressing transgenic mice, a modest level of complementation was found. However, when the null mice were crossed with mice expressing a fully rearranged T cell receptor αβ transgene, normal levels of CD4 single-positive cells were produced. Thus, Hdac3 is required for the efficient transit from double-negative stage 4 through positive selection. PMID:26324326

  10. Temporal requirements for B cells in the establishment of CD4 T cell memory.

    PubMed

    Mollo, Sarah B; Zajac, Allan J; Harrington, Laurie E

    2013-12-15

    CD4 T cell memory generation is shaped by a number of factors, including the strength and duration of TCR signaling, as well as the priming environment, all of which can be modified by B cells. Studies using B cell-deficient mice indicate B cells play a critical role in generating effector and memory CD4 T cells; however, when and how B cells are acting to promote these responses has not yet been ascertained. In this study, we use anti-CD20 Ab depletion of B cells at different times following Listeria monocytogenes infection to show that B cells are necessary for the induction of optimal CD4 T cell memory, but not for the transition and maintenance of this population. Importantly, the prerequisite of B cells early postinfection is partially dependent on their expression of MHC class II. B cells are not only required during the priming phase, but also necessary for the initiation of robust secondary responses by memory CD4 T cells. Interestingly, the requirement during the recall response is independent of B cell Ag presentation. Overall, these studies demonstrate the temporally and functionally distinct roles for B cells in regulating CD4 T cell responses. PMID:24218454

  11. Production of mouse lymphotoxin by phytohemagglutinin-stimulated spleen cells requires two cell fractions.

    PubMed Central

    Aksamit, R R; Leonard, E J

    1982-01-01

    The appearance of lymphotoxin in the culture fluid of phytohemagglutinin (PHA)-stimulated mouse spleen cells required two cell fractions that were separated by adherence to plastic. Upon stimulation with PHA, neither cell fraction alone produced significant amounts of lymphotoxin; however, when the cell fractions were combined and then stimulated with PHA, full activity was produced. Cytotoxic activity was not fully restored by combining PHA-stimulated cultured fluids from adherent and nonadherent cell fractions. This indicated that the cytotoxic activity was not the result of two factors, one produced by each cell fraction, that acted on the target cells, but rather, two cells interacted to produce lymphotoxin. Treatment of the unfractionated spleen cells with monoclonal anti-Thy1.2 and complement before PHA stimulation greatly reduced the production of lymphotoxin and indicated that at least one of the cells was a T cell. Lymphotoxin production was partially restored by the addition of nonadherent cells to the anti-Thy1.2-treated cells, suggesting that the T cell was nonadherent. Treatment of unfractionated cells with either silica or carrageenan had no effect on the subsequent production of lymphotoxin by PHA, suggesting that the adherent cell was not actively phagocytic. PMID:6980190

  12. Schwann cell myelination requires integration of laminin activities.

    PubMed

    McKee, Karen K; Yang, Dong-Hua; Patel, Rajesh; Chen, Zu-Lin; Strickland, Sidney; Takagi, Junichi; Sekiguchi, Kiyotoshi; Yurchenco, Peter D

    2012-10-01

    Laminins promote early stages of peripheral nerve myelination by assembling basement membranes (BMs) on Schwann cell surfaces, leading to activation of β1 integrins and other receptors. The BM composition, structural bonds and ligands needed to mediate this process, however, are not well understood. Mice hypomorphic for laminin γ1-subunit expression that assembled endoneurial BMs with reduced component density exhibited an axonal sorting defect with amyelination but normal Schwann cell proliferation, the latter unlike the null. To identify the basis for this, and to dissect participating laminin interactions, LAMC1 gene-inactivated dorsal root ganglia were treated with recombinant laminin-211 and -111 lacking different architecture-forming and receptor-binding activities, to induce myelination. Myelin-wrapping of axons by Schwann cells was found to require higher laminin concentrations than either proliferation or axonal ensheathment. Laminins that were unable to polymerize through deletions that removed critical N-terminal (LN) domains, or that lacked cell-adhesive globular (LG) domains, caused reduced BMs and almost no myelination. Laminins engineered to bind weakly to α6β1 and/or α7β1 integrins through their LG domains, even though they could effectively assemble BMs, decreased myelination. Proliferation depended upon both integrin binding to LG domains and polymerization. Collectively these findings reveal that laminins integrate scaffold-forming and cell-adhesion activities to assemble an endoneurial BM, with myelination and proliferation requiring additional α6β1/α7β1-laminin LG domain interactions, and that a high BM ligand/structural density is needed for efficient myelination. PMID:22767514

  13. Materials requirements for high-efficiency silicon solar cells

    NASA Technical Reports Server (NTRS)

    Wolf, M.

    1985-01-01

    To achieve higher Si solar cell efficiencies (greater than 20%), better single-crystal Si must be produced. It is believed possible to bring Cz (Czochralski) Si up to the same low recombination level as FZ (Float Zone) Si. It is also desirable that solar cell Si meet the following requirements: long minority carrier lifetime (0.2 ohm-cm p-type with tau less than 500 microsec); repeatedly uniform lifetime (not spread from 50 to 1000 microsec); a lifetime that does not decrease during normal device processing; a silicon wafer sheet that is flat and stays throughout normal device processing; uniform and reasonable mechanical strength; and, manufacture at low cost (less than $50/sq m).

  14. Genes that Confer the Identity of the Renin Cell

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-12-01

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

  16. Requirement of JNK1 for endothelial cell injury in atherogenesis

    PubMed Central

    Amini, Narges; Boyle, Joseph J.; Moers, Britta; Warboys, Christina M.; Malik, Talat H.; Zakkar, Mustafa; Francis, Sheila E.; Mason, Justin C.; Haskard, Dorian O.; Evans, Paul C.

    2014-01-01

    Objective The c-Jun N-terminal kinase (JNK) family regulates fundamental physiological processes including apoptosis and metabolism. Although JNK2 is known to promote foam cell formation during atherosclerosis, the potential role of JNK1 is uncertain. We examined the potential influence of JNK1 and its negative regulator, MAP kinase phosphatase-1 (MKP-1), on endothelial cell (EC) injury and early lesion formation using hypercholesterolemic LDLR−/− mice. Methods and results To assess the function of JNK1 in early atherogenesis, we measured EC apoptosis and lesion formation in LDLR−/− or LDLR−/−/JNK1−/− mice exposed to a high fat diet for 6 weeks. En face staining using antibodies that recognise active, cleaved caspase-3 (apoptosis) or using Sudan IV (lipid deposition) revealed that genetic deletion of JNK1 reduced EC apoptosis and lesion formation in hypercholesterolemic mice. By contrast, although EC apoptosis was enhanced in LDLR−/−/MKP-1−/− mice compared to LDLR−/− mice, lesion formation was unaltered. Conclusion We conclude that JNK1 is required for EC apoptosis and lipid deposition during early atherogenesis. Thus pharmacological inhibitors of JNK may reduce atherosclerosis by preventing EC injury as well as by influencing foam cell formation. PMID:24956536

  17. 9 CFR 113.51 - Requirements for primary cells used for production of biologics.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from...

  18. 9 CFR 113.51 - Requirements for primary cells used for production of biologics.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from...

  19. 9 CFR 113.51 - Requirements for primary cells used for production of biologics.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from...

  20. 9 CFR 113.51 - Requirements for primary cells used for production of biologics.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for primary cells used... VECTORS STANDARD REQUIREMENTS Ingredient Requirements § 113.51 Requirements for primary cells used for production of biologics. Primary cells used to prepare biological products shall be derived from...

  1. IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches.

    PubMed

    Reboldi, Andrea; Arnon, Tal I; Rodda, Lauren B; Atakilit, Amha; Sheppard, Dean; Cyster, Jason G

    2016-05-13

    Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-β receptor (LTβR)-dependent maintenance of DCs. PP DCs augment IgA production by integrin αvβ8-mediated activation of transforming growth factor-β (TGFβ). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGFβ activation and induction of mucosal IgA responses. PMID:27174992

  2. Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals

    PubMed Central

    Wynn, Michelle L.; Rupp, Paul; Trainor, Paul A.; Schnell, Santiago

    2013-01-01

    Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns. PMID:23735560

  3. Discovery of a Splicing Regulator Required for Cell Cycle Progression

    SciTech Connect

    Suvorova, Elena S.; Croken, Matthew; Kratzer, Stella; Ting, Li-Min; Conde de Felipe, Magnolia; Balu, Bharath; Markillie, Lye Meng; Weiss, Louis M.; Kim, Kami; White, Michael W.

    2013-02-01

    In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

  4. Human corneal endothelial cell sheets for transplantation: thermo-responsive cell culture carriers to meet cell-specific requirements.

    PubMed

    Teichmann, J; Valtink, M; Gramm, S; Nitschke, M; Werner, C; Funk, R H W; Engelmann, K

    2013-02-01

    Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options. PMID:23099299

  5. Klf4 expression in conventional dendritic cells is required for T helper 2 cell responses

    PubMed Central

    Tussiwand, Roxane; Everts, Bart; Grajales-Reyes, Gary E.; Kretzer, Nicole M.; Iwata, Arifumi; Bagaitkar, Juhi; Wu, Xiaodi; Wong, Rachel; Anderson, David A.; Murphy, Theresa L.; Pearce, Edward J.; Murphy, Kenneth M.

    2015-01-01

    Summary The two major lineages of classical dendritic cells (cDCs) express and require either IRF8 or IRF4 transcription factors for their development and function. IRF8-dependent cDCs promote anti-viral and T-helper 1 (Th1) cell responses, whereas IRF4-expressing cDCs have been implicated in controlling both Th2 and Th17 cell responses. Here, we have provided evidence that Kruppel-like factor 4 (Klf4) is required in IRF4-expressing cDCs to promote Th2 but not Th17 cell responses in vivo. Conditional Klf4 deletion within cDCs impaired Th2 cell responses during Schistosoma mansoni infection, Schistosoma egg antigen (SEA) immunization, and house dust mite challenge (HDM), without affecting cytotoxic T lymphocyte (CTL), Th1 and Th17 cell responses to herpes simplex virus, Toxoplasma gondii and Citrobacter rodentium infections. Further, Klf4 deletion reduced IRF4 expression in pre-cDCs and resulted in selective loss of IRF4-expressing cDCs subsets in several tissues. These results indicate that Klf4 guides a transcriptional program promoting IRF4-expressing cDCs heterogeneity. PMID:25992862

  6. Adenosinergic Immunosuppression by Human Mesenchymal Stromal Cells Requires Co-Operation with T cells.

    PubMed

    Kerkelä, Erja; Laitinen, Anita; Räbinä, Jarkko; Valkonen, Sami; Takatalo, Maarit; Larjo, Antti; Veijola, Johanna; Lampinen, Milla; Siljander, Pia; Lehenkari, Petri; Alfthan, Kaija; Laitinen, Saara

    2016-03-01

    Mesenchymal stem/stromal cells (MSCs) have the capacity to counteract excessive inflammatory responses. MSCs possess a range of immunomodulatory mechanisms, which can be deployed in response to signals in a particular environment and in concert with other immune cells. One immunosuppressive mechanism, not so well-known in MSCs, is mediated via adenosinergic pathway by ectonucleotidases CD73 and CD39. In this study, we demonstrate that adenosine is actively produced from adenosine 5'-monophosphate (AMP) by CD73 on MSCs and MSC-derived extracellular vesicles (EVs). Our results indicate that although MSCs express CD39 at low level and it colocalizes with CD73 in bulge areas of membranes, the most efficient adenosine production from adenosine 5'-triphosphate (ATP) requires co-operation of MSCs and activated T cells. Highly CD39 expressing activated T cells produce AMP from ATP and MSCs produce adenosine from AMP via CD73 activity. Furthermore, adenosinergic signaling plays a role in suppression of T cell proliferation in vitro. In conclusion, this study shows that adenosinergic signaling is an important immunoregulatory mechanism of MSCs, especially in situations where ATP is present in the extracellular environment, like in tissue injury. An efficient production of immunosuppressive adenosine is dependent on the concerted action of CD39-positive immune cells with CD73-positive cells such as MSCs or their EVs. Stem Cells 2016;34:781-790. PMID:26731338

  7. LSD1 is Required for Hair Cell Regeneration in Zebrafish.

    PubMed

    He, Yingzi; Tang, Dongmei; Cai, Chengfu; Chai, Renjie; Li, Huawei

    2016-05-01

    Lysine-specific demethylase 1 (LSD1/KDM1A) plays an important role in complex cellular processes such as differentiation, proliferation, apoptosis, and cell cycle progression. It has recently been demonstrated that during development, downregulation of LSD1 inhibits cell proliferation, modulates the expression of cell cycle regulators, and reduces hair cell formation in the zebrafish lateral line, which suggests that LSD1-mediated epigenetic regulation plays a key role in the development of hair cells. However, the role of LSD1 in hair cell regeneration after hair cell loss remains poorly understood. Here, we demonstrate the effect of LSD1 on hair cell regeneration following neomycin-induced hair cell loss. We show that the LSD1 inhibitor trans-2-phenylcyclopropylamine (2-PCPA) significantly decreases the regeneration of hair cells in zebrafish after neomycin damage. In addition, immunofluorescent staining demonstrates that 2-PCPA administration suppresses supporting cell proliferation and alters cell cycle progression. Finally, in situ hybridization shows that 2-PCPA significantly downregulates the expression of genes related to Wnt/β-catenin and Fgf activation. Altogether, our data suggest that downregulation of LSD1 significantly decreases hair cell regeneration after neomycin-induced hair cell loss through inactivation of the Wnt/β-catenin and Fgf signaling pathways. Thus, LSD1 plays a critical role in hair cell regeneration and might represent a novel biomarker and potential therapeutic approach for the treatment of hearing loss. PMID:26008620

  8. The RNA-binding protein Musashi is required intrinsically to maintain stem cell identity.

    PubMed

    Siddall, Nicole A; McLaughlin, Eileen A; Marriner, Neisha L; Hime, Gary R

    2006-05-30

    A key goal of regenerative medicine is an understanding of the genetic factors that define the properties of stem cells. However, stem cell research in mammalian tissue has been hampered by a paucity of stem cell-specific markers. Although increasing evidence suggests that members of the Musashi (Msi) family of RNA-binding proteins play important functions in progenitor cells, it remains unclear whether there is a stem cell-autonomous requirement for Msi because of an inability to distinguish stem cells from early-lineage cells in mammalian tissues. Here, using the Drosophila testis as a model system for the study of stem cell regulation, we show specific evidence for a cell-autonomous requirement for Msi family proteins in regulating stem cell differentiation, leading to the identification of an RNA-binding protein required for spermatogonial stem cell maintenance. We found that loss of Msi function disrupts the balance between germ-line stem cell renewal and differentiation, resulting in the premature differentiation of germ-line stem cells. Moreover, we found that, although Msi is expressed in both somatic and germ cells, Msi function is required intrinsically in stem cells for maintenance of stem cell identity. We also discovered a requirement for Msi function in male meiosis, revealing that Msi has distinct roles at different stages of germ cell differentiation. We describe the complementary expression patterns of the murine Msi paralogues Msi1 and Msi2 during spermatogenesis, which support the idea of distinct, evolutionarily conserved roles of Msi. PMID:16717192

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

  11. 40 CFR Table 8 to Subpart IIIii of... - Requirements for Cell Room Monitoring Program

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 14 2012-07-01 2011-07-01 true Requirements for Cell Room Monitoring... Mercury Cell Chlor-Alkali Plants Pt. 63, Subpt. IIIII, Table 8 Table 8 to Subpart IIIII of Part 63—Requirements for Cell Room Monitoring Program As stated in § 63.8192(g)(1), your mercury monitoring system...

  12. 40 CFR Table 8 to Subpart IIIii of... - Requirements for Cell Room Monitoring Program

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 13 2010-07-01 2010-07-01 false Requirements for Cell Room Monitoring... Mercury Cell Chlor-Alkali Plants Pt. 63, Subpt. IIIII, Table 8 Table 8 to Subpart IIIII of Part 63—Requirements for Cell Room Monitoring Program As stated in § 63.8192(g)(1), your mercury monitoring system...

  13. 40 CFR Table 8 to Subpart IIIii of... - Requirements for Cell Room Monitoring Program

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 14 2014-07-01 2014-07-01 false Requirements for Cell Room Monitoring... Mercury Cell Chlor-Alkali Plants Pt. 63, Subpt. IIIII, Table 8 Table 8 to Subpart IIIII of Part 63—Requirements for Cell Room Monitoring Program As stated in § 63.8192(g)(1), your mercury monitoring system...

  14. 40 CFR Table 8 to Subpart IIIii of... - Requirements for Cell Room Monitoring Program

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 14 2013-07-01 2013-07-01 false Requirements for Cell Room Monitoring... Mercury Cell Chlor-Alkali Plants Pt. 63, Subpt. IIIII, Table 8 Table 8 to Subpart IIIII of Part 63—Requirements for Cell Room Monitoring Program As stated in § 63.8192(g)(1), your mercury monitoring system...

  15. 40 CFR Table 8 to Subpart IIIii of... - Requirements for Cell Room Monitoring Program

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 13 2011-07-01 2011-07-01 false Requirements for Cell Room Monitoring... Mercury Cell Chlor-Alkali Plants Pt. 63, Subpt. IIIII, Table 8 Table 8 to Subpart IIIII of Part 63—Requirements for Cell Room Monitoring Program As stated in § 63.8192(g)(1), your mercury monitoring system...

  16. Functional hair cell mechanotransducer channels are required for aminoglycoside ototoxicity.

    PubMed

    Alharazneh, Abdelrahman; Luk, Lauren; Huth, Markus; Monfared, Ashkan; Steyger, Peter S; Cheng, Alan G; Ricci, Anthony J

    2011-01-01

    Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss. PMID:21818312

  17. Functional Hair Cell Mechanotransducer Channels Are Required for Aminoglycoside Ototoxicity

    PubMed Central

    Huth, Markus; Monfared, Ashkan; Steyger, Peter S.; Cheng, Alan G.; Ricci, Anthony J.

    2011-01-01

    Aminoglycosides (AG) are commonly prescribed antibiotics with potent bactericidal activities. One main side effect is permanent sensorineural hearing loss, induced by selective inner ear sensory hair cell death. Much work has focused on AG's initiating cell death processes, however, fewer studies exist defining mechanisms of AG uptake by hair cells. The current study investigated two proposed mechanisms of AG transport in mammalian hair cells: mechanotransducer (MET) channels and endocytosis. To study these two mechanisms, rat cochlear explants were cultured as whole organs in gentamicin-containing media. Two-photon imaging of Texas Red conjugated gentamicin (GTTR) uptake into live hair cells was rapid and selective. Hypocalcemia, which increases the open probability of MET channels, increased AG entry into hair cells. Three blockers of MET channels (curare, quinine, and amiloride) significantly reduced GTTR uptake, whereas the endocytosis inhibitor concanavalin A did not. Dynosore quenched the fluorescence of GTTR and could not be tested. Pharmacologic blockade of MET channels with curare or quinine, but not concanavalin A or dynosore, prevented hair cell loss when challenged with gentamicin for up to 96 hours. Taken together, data indicate that the patency of MET channels mediated AG entry into hair cells and its toxicity. Results suggest that limiting permeation of AGs through MET channel or preventing their entry into endolymph are potential therapeutic targets for preventing hair cell death and hearing loss. PMID:21818312

  18. Sox10 is required for Schwann cell identity and progression beyond the immature Schwann cell stage.

    PubMed

    Finzsch, Markus; Schreiner, Silke; Kichko, Tatjana; Reeh, Peter; Tamm, Ernst R; Bösl, Michael R; Meijer, Dies; Wegner, Michael

    2010-05-17

    Mutations in the transcription factor SOX10 cause neurocristopathies, including Waardenburg-Hirschsprung syndrome and peripheral neuropathies in humans. This is partly attributed to a requirement for Sox10 in early neural crest for survival, maintenance of pluripotency, and specification to several cell lineages, including peripheral glia. As a consequence, peripheral glia are absent in Sox10-deficient mice. Intriguingly, Sox10 continues to be expressed in these cells after specification. To analyze glial functions after specification, we specifically deleted Sox10 in immature Schwann cells by conditional mutagenesis. Mutant mice died from peripheral neuropathy before the seventh postnatal week. Nerve alterations included a thinned perineurial sheath, increased lipid and collagen deposition, and a dramatically altered cellular composition. Nerve conduction was also grossly aberrant, and neither myelinating nor nonmyelinating Schwann cells formed. Instead, axons of different sizes remained unsorted in large bundles. Schwann cells failed to develop beyond the immature stage and were unable to maintain identity. Thus, our study identifies a novel cause for peripheral neuropathies in patients with SOX10 mutations. PMID:20457761

  19. YY1 Is Required for Germinal Center B Cell Development

    PubMed Central

    Vuyyuru, Raja; Jha, Vibha; Hodewadekar, Suchita; Manser, Tim; Atchison, Michael L.

    2016-01-01

    YY1 has been implicated as a master regulator of germinal center B cell development as YY1 binding sites are frequently present in promoters of germinal center-expressed genes. YY1 is known to be important for other stages of B cell development including the pro-B and pre-B cells stages. To determine if YY1 plays a critical role in germinal center development, we evaluated YY1 expression during B cell development, and used a YY1 conditional knock-out approach for deletion of YY1 in germinal center B cells (CRE driven by the immunoglobulin heavy chain γ1 switch region promoter; γ1-CRE). We found that YY1 is most highly expressed in germinal center B cells and is increased 3 fold in splenic B cells activated by treatment with anti-IgM and anti-CD40. In addition, deletion of the yy1 gene by action of γ1-CRE recombinase resulted in significant loss of GC cells in both un-immunized and immunized contexts with corresponding loss of serum IgG1. Our results show a crucial role for YY1 in the germinal center reaction. PMID:27167731

  20. Rab24 is required for normal cell division.

    PubMed

    Militello, Rodrigo D; Munafó, Daniela B; Berón, Walter; López, Luis A; Monier, Solange; Goud, Bruno; Colombo, María I

    2013-05-01

    Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co-localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i.e., chromatin bridges). Furthermore, in CHO cells stably transfected with GFP-Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules. PMID:23387408

  1. Oct4 is required for primordial germ cell survival

    PubMed Central

    Kehler, James; Tolkunova, Elena; Koschorz, Birgit; Pesce, Maurizio; Gentile, Luca; Boiani, Michele; Lomelí, Hilda; Nagy, Andras; McLaughlin, K John; Schöler, Hans R; Tomilin, Alexey

    2004-01-01

    Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline. PMID:15486564

  2. Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary

    PubMed Central

    Ma, Xing; Wang, Su; Do, Trieu; Song, Xiaoqing; Inaba, Mayu; Nishimoto, Yoshiya; Liu, Lu-ping; Gao, Yuan; Mao, Ying; Li, Hui; McDowell, William; Park, Jungeun; Malanowski, Kate; Peak, Allison; Perera, Anoja; Li, Hua; Gaudenz, Karin; Haug, Jeff; Yamashita, Yukiko; Lin, Haifan; Ni, Jian-quan; Xie, Ting

    2014-01-01

    The piRNA pathway plays an important role in maintaining genome stability in the germ line by silencing transposable elements (TEs) from fly to mammals. As a highly conserved piRNA pathway component, Piwi is widely expressed in both germ cells and somatic cells in the Drosophila ovary and is required for piRNA production in both cell types. In addition to its known role in somatic cap cells to maintain germline stem cells (GSCs), this study has demonstrated that Piwi has novel functions in somatic cells and germ cells of the Drosophila ovary to promote germ cell differentiation. Piwi knockdown in escort cells causes a reduction in escort cell (EC) number and accumulation of undifferentiated germ cells, some of which show active BMP signaling, indicating that Piwi is required to maintain ECs and promote germ cell differentiation. Simultaneous knockdown of dpp, encoding a BMP, in ECs can partially rescue the germ cell differentiation defect, indicating that Piwi is required in ECs to repress dpp. Consistent with its key role in piRNA production, TE transcripts increase significantly and DNA damage is also elevated in the piwi knockdown somatic cells. Germ cell-specific knockdown of piwi surprisingly causes depletion of germ cells before adulthood, suggesting that Piwi might control primordial germ cell maintenance or GSC establishment. Finally, Piwi inactivation in the germ line of the adult ovary leads to gradual GSC loss and germ cell differentiation defects, indicating the intrinsic role of Piwi in adult GSC maintenance and differentiation. This study has revealed new germline requirement of Piwi in controlling GSC maintenance and lineage differentiation as well as its new somatic function in promoting germ cell differentiation. Therefore, Piwi is required in multiple cell types to control GSC lineage development in the Drosophila ovary. PMID:24658126

  3. Sphingosine Kinase Activity Is Not Required for Tumor Cell Viability

    PubMed Central

    Brown, Matthew L.; Carlson, Timothy; Coxon, Angela; Fajardo, Flordeliza; Frank, Brendon; Gustin, Darin; Kamb, Alexander; Kassner, Paul D.; Li, Shyun; Li, Yihong; Morgenstern, Kurt; Plant, Matthew; Quon, Kim; Ruefli-Brasse, Astrid; Schmidt, Joanna; Swearingen, Elissa; Walker, Nigel; Wang, Zhulun; Watson, J. E. Vivienne; Wickramasinghe, Dineli; Wong, Mariwil; Xu, Guifen; Wesche, Holger

    2013-01-01

    Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology. PMID:23861887

  4. PEM fuel cell bipolar plate material requirements for transportation applications

    SciTech Connect

    Borup, R.L.; Stroh, K.R.; Vanderborgh, N.E.

    1996-04-01

    Cost effective bipolar plates are currently under development to help make proton exchange membrane (PEM) fuel cells commercially viable. Bipolar plates separate individual cells of the fuel cell stack, and thus must supply strength, be electrically conductive, provide for thermal control of the fuel stack, be a non-porous materials separating hydrogen and oxygen feed streams, be corrosion resistant, provide gas distribution for the feed streams and meet fuel stack cost targets. Candidate materials include conductive polymers and metal plates with corrosion resistant coatings. Possible metals include aluminium, titanium, iron/stainless steel and nickel.

  5. Ets2 is required for trophoblast stem cell self renewal

    PubMed Central

    Wen, Fang; Tynan, John A.; Cecena, Grace; Williams, Roy; Múnera, Jorge; Mavrothalassitis, George; Oshima, Robert G.

    2007-01-01

    The Ets2 transcription factor is essential for the development of the mouse placenta and for generating signals for embryonic mesoderm and axis formation. Using a conditional targeted Ets2 allele, we show that Ets2 is essential for trophoblast stem (TS) cells self renewal. Inactivation of Ets2 results in TS cell slower growth, increased expression of a subset of differentiation associated genes and decreased expression of several genes implicated in TS self renewal. Among the direct TS targets of Ets2 is Cdx2, a key master regulator of TS cell state. Thus Ets2 contributes to the regulation of multiple genes important for maintaining the undifferentiated state of TS cells and as candidate signals for embryonic development. PMID:17977525

  6. Structural requirements for neural cell adhesion molecule-heparin interaction.

    PubMed Central

    Reyes, A A; Akeson, R; Brezina, L; Cole, G J

    1990-01-01

    Two biological domains have been identified in the amino terminal region of the neural cell adhesion molecule (NCAM): a homophilic-binding domain, responsible for NCAM-NCAM interactions, and a heparin-binding domain (HBD). It is not known whether these two domains exist as distinct structural entities in the NCAM molecule. To approach this question, we have further defined the relationship between NCAM-heparin binding and cell adhesion. A putative HBD consisting of two clusters of basic amino acid residues located close to each other in the linear amino acid sequence of NCAM has previously been identified. Synthetic peptides corresponding to this domain were shown to bind both heparin and retinal cells. Here we report the construction of NCAM cDNAs with targeted mutations in the HBD. Mouse fibroblast cells transfected with the mutant cDNAs express NCAM polypeptides with altered HBD (NCAM-102 and NCAM-104) or deleted HBD (HBD-) at levels similar to those of wild-type NCAM. Mutant NCAM polypeptides purified from transfected cell lines have substantially reduced binding to heparin and fail to promote chick retinal cell attachment. Furthermore, whereas a synthetic peptide that contains both basic amino acid clusters inhibits retinal-cell adhesion to NCAM-coated dishes, synthetic peptides in which either one of the two basic regions is altered to contain only neutral amino acids do not inhibit this adhesion. These results confirm that this region of the NCAM polypeptide does indeed mediate not only the large majority of NCAM's affinity for heparin but also a significant portion of the cell-adhesion-mediating capability of NCAM. Images PMID:2078567

  7. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... production of biologics. 113.52 Section 113.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of biologics. When prescribed in an applicable Standard Requirement or in a filed Outline of Production...

  8. A conformational switch controls cell wall remodeling enzymes required for bacterial cell division

    PubMed Central

    Yang, Desirée C.; Tan, Kemin; Joachimiak, Andrzej; Bernhardt, Thomas G.

    2012-01-01

    Summary Remodeling of the peptidoglycan (PG) exoskeleton is intimately tied to the growth and division of bacteria. Enzymes that hydrolyze PG are critical for these processes, but their activities must be tightly regulated to prevent the generation of lethal breaches in the PG matrix. Despite their importance, the mechanisms regulating PG hydrolase activity have remained elusive. Here we investigate the control of cell division hydrolases called amidases (AmiA, AmiB, and AmiC) required for Escherichia coli cell division. Poorly regulated amiB mutants were isolated encoding lytic AmiB variants with elevated basal PG hydrolase activities in vitro. The structure of an AmiB ortholog was also solved, revealing that the active site of AmiB is occluded by a conserved alpha-helix. Strikingly, most of the amino acid substitutions in the lytic AmiB variants mapped to this domain and are predicted to disrupt its interaction with the active site. Our results therefore support a model in which cell separation is stimulated by the reversible relief of amidase auto-inhibition governed by conserved sub-complexes within the cytokinetic ring. Analogous conformational control mechanisms are likely to be part of a general strategy used to control PG hydrolases present within multi-enzyme PG remodeling machines. PMID:22715947

  9. Apoptosis-Inducing-Factor-Dependent Mitochondrial Function Is Required for T Cell but Not B Cell Function.

    PubMed

    Milasta, Sandra; Dillon, Christopher P; Sturm, Oliver E; Verbist, Katherine C; Brewer, Taylor L; Quarato, Giovanni; Brown, Scott A; Frase, Sharon; Janke, Laura J; Perry, S Scott; Thomas, Paul G; Green, Douglas R

    2016-01-19

    The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells. PMID:26795252

  10. PAF-Wnt signaling-induced cell plasticity is required for maintenance of breast cancer cell stemness

    PubMed Central

    Wang, Xin; Jung, Youn-Sang; Jun, Sohee; Lee, Sunhye; Wang, Wenqi; Schneider, Andrea; Sun Oh, Young; Lin, Steven H.; Park, Bum-Joon; Chen, Junjie; Keyomarsi, Khandan; Park, Jae-Il

    2016-01-01

    Cancer stem cells (CSCs) contribute to tumour heterogeneity, therapy resistance and metastasis. However, the regulatory mechanisms of cancer cell stemness remain elusive. Here we identify PCNA-associated factor (PAF) as a key molecule that controls cancer cell stemness. PAF is highly expressed in breast cancer cells but not in mammary epithelial cells (MECs). In MECs, ectopic expression of PAF induces anchorage-independent cell growth and breast CSC marker expression. In mouse models, conditional PAF expression induces mammary ductal hyperplasia. Moreover, PAF expression endows MECs with a self-renewing capacity and cell heterogeneity generation via Wnt signalling. Conversely, ablation of endogenous PAF induces the loss of breast cancer cell stemness. Further cancer drug repurposing approaches reveal that NVP-AUY922 downregulates PAF and decreases breast cancer cell stemness. Our results unveil an unsuspected role of the PAF-Wnt signalling axis in modulating cell plasticity, which is required for the maintenance of breast cancer cell stemness. PMID:26843124

  11. Requirement of Phosphoinositides Containing Stearic Acid To Control Cell Polarity

    PubMed Central

    Laquel, Patricia; Testet, Eric; Tuphile, Karine; Fouillen, Laetitia; Bessoule, Jean-Jacques

    2015-01-01

    Phosphoinositides (PIPs) are present in very small amounts but are essential for cell signaling, morphogenesis, and polarity. By mass spectrometry, we demonstrated that some PIPs with stearic acyl chains were strongly disturbed in a psi1Δ Saccharomyces cerevisiae yeast strain deficient in the specific incorporation of a stearoyl chain at the sn-1 position of phosphatidylinositol. The absence of PIPs containing stearic acid induced disturbances in intracellular trafficking, although the total amount of PIPs was not diminished. Changes in PIPs also induced alterations in the budding pattern and defects in actin cytoskeleton organization (cables and patches). Moreover, when the PSI1 gene was impaired, a high proportion of cells with bipolar cortical actin patches that occurred concomitantly with the bipolar localization of Cdc42p was specifically found among diploid cells. This bipolar cortical actin phenotype, never previously described, was also detected in a bud9Δ/bud9Δ strain. Very interestingly, overexpression of PSI1 reversed this phenotype. PMID:26711260

  12. T-cell activation is required for efficient replication of human herpesvirus 6.

    PubMed Central

    Frenkel, N; Schirmer, E C; Katsafanas, G; June, C H

    1990-01-01

    We have investigated whether T-cell activation is required for the replication of the T-lymphotropic human herpesvirus 6. The virus did not replicate in quiescent peripheral blood lymphocytes but replicated efficiently following exposure of the cells to the polyclonal mitogen phytohemagglutinin (PHA). When purified T cells were treated with PHA in the absence of accessory cells, no virus replication was observed unless exogenous interleukin-2 (IL-2) was added to the medium, promoting cell division. Incubation of peripheral blood lymphocytes in the absence of PHA but in the presence of IL-2 resulted in delayed cell blastogenesis and virus replication. Cell blastogenesis and virus replication did not occur in the purified T-cell cultures incubated with IL-2 alone. Taken together, the results show that human herpesvirus 6 replication requires full progression of the cell cycle. This finding might have implications for the pathogenicity of the virus in the human host. Images PMID:2166835

  13. Secretion is required for late events in the cell-fusion pathway of mating yeast.

    PubMed

    Grote, Eric

    2010-06-01

    Secretory vesicles accumulate adjacent to the contact site between the two cells of a yeast mating pair before they fuse, but there is no direct evidence that secretion is required to complete fusion. In this study, temperature-sensitive secretion (sec(ts)) mutants were used to investigate the role of secretion in yeast cell fusion. Cell fusion arrested less than 5 minutes after inhibiting secretion. This rapid fusion arrest was not an indirect consequence of reduced mating pheromone signaling, mating-pair assembly or actin polarity. Furthermore, secretion was required to complete cell fusion when it was transiently inhibited by addition and removal of the lipophilic styryl dye, FM4-64. These results indicate that ongoing secretion is required for late events in the cell-fusion pathway, which include plasma-membrane fusion and the completion of cell-wall remodeling, and they demonstrate a just-in-time delivery mechanism for the cell-fusion machinery. PMID:20460435

  14. AIP1-mediated actin disassembly is required for postnatal germ cell migration and spermatogonial stem cell niche establishment

    PubMed Central

    Xu, J; Wan, P; Wang, M; Zhang, J; Gao, X; Hu, B; Han, J; Chen, L; Sun, K; Wu, J; Wu, X; Huang, X; Chen, J

    2015-01-01

    In mammals, spermatogonial stem cells (SSCs) arise from early germ cells called gonocytes, which are derived from primordial germ cells during embryogenesis and remain quiescent until birth. After birth, these germ cells migrate from the center of testicular cord, through Sertoli cells, and toward the basement membrane to form the SSC pool and establish the SSC niche architecture. However, molecular mechanisms underlying germ cell migration and niche establishment are largely unknown. Here, we show that the actin disassembly factor actin interacting protein 1 (AIP1) is required in both germ cells and Sertoli cells to regulate this process. Germ cell-specific or Sertoli cell-specific deletion of Aip1 gene each led to significant defects in germ cell migration after postnatal day 4 or 5, accompanied by elevated levels of actin filaments (F-actin) in the affected cells. Furthermore, our data demonstrated that interaction between germ cells and Sertoli cells, likely through E-cadherin-mediated cell adhesion, is critical for germ cells' migration toward the basement membrane. At last, Aip1 deletion in Sertoli cells decreased SSC self-renewal, increased spermatogonial differentiation, but did not affect the expression and secretion levels of growth factors, suggesting that the disruption of SSC function results from architectural changes in the postnatal niche. PMID:26181199

  15. Epithelial cell polarity genes are required for neural tube closure.

    PubMed

    Doudney, Kit; Stanier, Philip

    2005-05-15

    Human neural tube defects (NTD) are a heterogeneous group that exhibit complex inheritance, making it difficult to identify the underlying cause. Due to the uniform genetic background, inbred mouse strains are a more amenable target for genetic studies. We investigated the loop-tail (Lp) mouse as a model for the severe NTD, craniorachischisis. A homozygous point mutation was identified in the transmembrane protein Vangl2, which in Drosophila has been shown to function in the planar cell polarity (PCP) pathway. Morphological analysis of the Lp mice shows that the defect results from an abnormally broad floor plate, most likely through a failure in convergent extension. The elevated neural folds remain too far apart to contact, inhibiting neural tube closure. Recently, two other mouse mutants (crash and circletail) were described with a similar phenotype to Lp and were investigated as potentially new alleles. Mapping studies, however, showed that both mutants segregated to distinct loci. In the crash (Crsh) mouse, a mutation was identified in Celsr1, a seven pass transmembrane receptor that encodes a protein orthologous to Drosophila Flamingo. Like Vangl2, this gene also functions in the PCP pathway. While in circletail, a point mutation was identified introducing a premature stop codon into the apical-basal cell polarity gene scribble (Scrb1). We subsequently demonstrated a genetic interaction between all three genes, where double heterozygotes exhibit the same homozygous NTD phenotype. This strongly suggests both a candidate gene pathway and that interaction between independent recessive alleles may be a possible explanation for the complex inheritance in severe human NTD. PMID:15800847

  16. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts

    PubMed Central

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T.; Lorenzo, Óscar; Revuelta, José L.; McCabe, Paul F.; Arellano, Juan B.

    2014-01-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined. PMID:24723397

  17. Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

    PubMed

    Wingender, Gerhard; Birkholz, Alysia M; Sag, Duygu; Farber, Elisa; Chitale, Sampada; Howell, Amy R; Kronenberg, Mitchell

    2015-10-15

    Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness. PMID:26355152

  18. Fibroblast cluster formation on 3D collagen matrices requires cell contraction dependent fibronectin matrix organization.

    PubMed

    da Rocha-Azevedo, Bruno; Ho, Chin-Han; Grinnell, Frederick

    2013-02-15

    Fibroblasts incubated on 3D collagen matrices in serum or lysophosphatidic acid (LPA)-containing medium self-organize into clusters through a mechanism that requires cell contraction. However, in platelet-derived growth factor (PDGF)-containing medium, cells migrate as individuals and do not form clusters even though they constantly encounter each other. Here, we present evidence that a required function of cell contraction in clustering is formation of fibronectin (FN) fibrillar matrix. We found that in serum or LPA but not in PDGF or basal medium, cells organized FN (both serum and cellular) into a fibrillar, detergent-insoluble matrix. Cell clusters developed concomitant with FN matrix formation. FN fibrils accumulated beneath cells and along the borders of cell clusters in regions of cell-matrix tension. Blocking Rho kinase or myosin II activity prevented FN matrix assembly and cell clustering. Using siRNA silencing and function-blocking antibodies and peptides, we found that cell clustering and FN matrix assembly required α5β1 integrins and fibronectin. Cells were still able to exert contractile force and compact the collagen matrix under the latter conditions, which showed that contraction was not sufficient for cell clustering to occur. Our findings provide new insights into how procontractile (serum/LPA) and promigratory (PDGF) growth factor environments can differentially regulate FN matrix assembly by fibroblasts interacting with collagen matrices and thereby influence mesenchymal cell morphogenetic behavior under physiologic circumstances such as wound repair, morphogenesis and malignancy. PMID:23117111

  19. Calcium requirements for secretion in bovine chromaffin cells.

    PubMed Central

    Augustine, G J; Neher, E

    1992-01-01

    1. Measurements of membrane capacitance and intracellular Ca2+ concentration, [Ca2+]i, were used to examine the Ca2+ dependence of secretion in single adrenal chromaffin cells. 2. Intracellular dialysis of Ca2+, through a patch pipette, promoted secretion; the rate of secretion increased monotonically as [Ca2+]i was elevated, while the total amount of secretion reached a maximum at 1.5 microM-Ca2+ and declined at high [Ca2+]i. 3. Release of Ca2+ from internal stores, using bradykinin or ionomycin, transiently elevated [Ca2+]i and the rate of secretion. 4. Considering responses to both Ca2+ dialysis and release from internal stores, it appears that the rate of secretion increases over a range of [Ca2+]i levels above 0.2 microM and saturates at concentrations greater than 10 microM, if at all. Secretion appears to have a Hill coefficient for Ca2+ of about 2. At [Ca2+]i greater than 1-2 microM, prolonged elevation of [Ca2+]i, via dialysis, produced lower rates of secretion than transient elevation of [Ca2+]i caused by release from internal stores. This may have been caused by a depletion of readily releasable chromaffin granules during prolonged elevation of [Ca2+]i. 5. Brief depolarizing pulses produced transient rises in both [Ca2+]i and the rate of secretion. The ability of these pulses to evoke secretion 'washed out' during prolonged intracellular dialysis, due to both reduced Ca2+ influx and a diminished ability of the cell to secrete in response to a given Ca2+ load. 6. The kinetics of the secretory response depended upon the size of the depolarization-induced Ca2+ load; small rises in [Ca2+]i increased membrane capacitance only during the depolarization, while larger rises in [Ca2+]i produced increases both during and following the depolarization. The secretory responses that outlasted the depolarization appeared to be due to persistent elevation of [Ca2+]i. Secretory responses were sometimes followed by a slower decline in membrane capacitance, probably due to

  20. A Screen for Genetic Loci Required for Hypodermal Cell and Glial-like Cell Development during Caenorhabditis Elegans Embryogenesis

    PubMed Central

    Chanal, P.; Labouesse, M.

    1997-01-01

    The Caenorhabditis elegans lin-26 gene is expressed in all nonneuronal ectodermal cells. To identify genes required to specify the fates of ectodermal cells, we have conducted screens designed to identify loci whose zygotic function would be required for normal lin-26 expression. First, we examined 90 deficiencies covering 75% of the genome; second, we examined the progeny of 3600 genomes after EMS mutagenesis. We identified six loci that appear to be required for normal lin-26 expression. We argue that the deficiency eDf19 deletes a gene involved in specifying hypodermal cell fates. The genes emb-29 (previously known) and ale-1 (newly found) could be involved in a cell cycle function and/or in specifying the fates of some precursors within different lineages that generate hypodermal cells and nonectodermal cells. We argue that the overlapping deficiencies qDf7, qDf8 and qDf9 delete a gene required to limit the number of nonneuronal ectodermal cells. We suggest that the deficiencies ozDf2, itDf2 and nDf42 delete genes required, directly or indirectly, to repress lin-26 expression in cells that normally do not express lin-26. We discuss the implications of these findings concerning the generation of the ectoderm. PMID:9136011

  1. Vaccine-induced tumor regression requires a dynamic cooperation between T cells and myeloid cells at the tumor site.

    PubMed

    Thoreau, Maxime; Penny, HweiXian Leong; Tan, KarWai; Regnier, Fabienne; Weiss, Julia Miriam; Lee, Bernett; Johannes, Ludger; Dransart, Estelle; Le Bon, Agnès; Abastado, Jean-Pierre; Tartour, Eric; Trautmann, Alain; Bercovici, Nadège

    2015-09-29

    Most cancer immunotherapies under present investigation are based on the belief that cytotoxic T cells are the most important anti-tumoral immune cells, whereas intra-tumoral macrophages would rather play a pro-tumoral role. We have challenged this antagonistic point of view and searched for collaborative contributions by tumor-infiltrating T cells and macrophages, reminiscent of those observed in anti-infectious responses. We demonstrate that, in a model of therapeutic vaccination, cooperation between myeloid cells and T cells is indeed required for tumor rejection. Vaccination elicited an early rise of CD11b+ myeloid cells that preceded and conditioned the intra-tumoral accumulation of CD8+ T cells. Conversely, CD8+ T cells and IFNγ production activated myeloid cells were required for tumor regression. A 4-fold reduction of CD8+ T cell infiltrate in CXCR3KO mice did not prevent tumor regression, whereas a reduction of tumor-infiltrating myeloid cells significantly interfered with vaccine efficiency. We show that macrophages from regressing tumors can kill tumor cells in two ways: phagocytosis and TNFα release. Altogether, our data suggest new strategies to improve the efficiency of cancer immunotherapies, by promoting intra-tumoral cooperation between macrophages and T cells. PMID:26337837

  2. Vaccine-induced tumor regression requires a dynamic cooperation between T cells and myeloid cells at the tumor site

    PubMed Central

    Thoreau, Maxime; Penny, HweiXian Leong; Tan, KarWai; Regnier, Fabienne; Weiss, Julia Miriam; Lee, Bernett; Johannes, Ludger; Dransart, Estelle; Le Bon, Agnès; Abastado, Jean-Pierre; Tartour, Eric

    2015-01-01

    Most cancer immunotherapies under present investigation are based on the belief that cytotoxic T cells are the most important anti-tumoral immune cells, whereas intra-tumoral macrophages would rather play a pro-tumoral role. We have challenged this antagonistic point of view and searched for collaborative contributions by tumor-infiltrating T cells and macrophages, reminiscent of those observed in anti-infectious responses. We demonstrate that, in a model of therapeutic vaccination, cooperation between myeloid cells and T cells is indeed required for tumor rejection. Vaccination elicited an early rise of CD11b+ myeloid cells that preceded and conditioned the intra-tumoral accumulation of CD8+ T cells. Conversely, CD8+ T cells and IFNγ production activated myeloid cells were required for tumor regression. A 4-fold reduction of CD8+ T cell infiltrate in CXCR3KO mice did not prevent tumor regression, whereas a reduction of tumor-infiltrating myeloid cells significantly interfered with vaccine efficiency. We show that macrophages from regressing tumors can kill tumor cells in two ways: phagocytosis and TNFα release. Altogether, our data suggest new strategies to improve the efficiency of cancer immunotherapies, by promoting intra-tumoral cooperation between macrophages and T cells. PMID:26337837

  3. Fuel cell systems for passenger cars - opportunities and requirements

    SciTech Connect

    Tachtler, J.; Bourne, C.

    1996-12-31

    From the point of view of energy density, handling and economy, present-day motor fuels are superior to all known alternatives. The internal combustion engine powered by them satisfies the requirements of customers to an excellent degree. The search for alternatives can therefore only be justified if emissions can be avoided totally and non-fossil primary energy sources can be used or at least partially our dependence on mineral oil can be reduced. What was long suspected has been increasingly confirmed, not least by developments at BMW: electricity (stored in batteries) and hydrogen offer the best prerequisites for achieving these goals in the long term. These forms of energy can be produced in sufficient quantities and with relatively little effect on the environment. They promise to produce an absolute minimum of pollutants when used in vehicles. Natural gas, which is very similar to hydrogen, and hybrid systems, that would compensate for battery risks, could perform a valuable function in the transitional phase.

  4. T cell-B cell thymic cross-talk: Maintenance and function of thymic B cells requires cognate CD40-CD40L interaction

    PubMed Central

    Fujihara, Chiharu; Williams, Joy A.; Watanabe, Masashi; Jeon, Hyein; Sharrow, Susan O.; Hodes, Richard J.

    2014-01-01

    Thymic development requires bidirectional interaction or cross-talk between developing T cells and thymic stromal cells, a relationship that has been best characterized for the interaction between thymocytes and thymic epithelial cells (TECs). We have characterized here the requirement for similar cross-talk in the maintenance and function of thymic B cells, another population that plays a role in selection of developing thymic T cells. We found that maintenance of thymic B cells is strongly dependent upon the presence of mature single positive (SP) thymocytes and on the interactions of these T cells with specific antigen ligand. Maintenance of thymic B cell number is strongly dependent upon B cell-autonomous expression of CD40, but not MHCII, indicating that direct engagement of CD40 on thymic B cells is necessary to support their maintenance and proliferation. Thymic B cells can mediate negative selection of superantigen-specific self-reactive SP thymocytes, and we show that CD40 expression on B cells is critical for this negative selection. Cross-talk with thymic T cells is thus required to support the thymic B cell population through a pathway that requires cell-autonomous expression of CD40, and that reciprocally functions in negative selection of autoreactive T cells. PMID:25344473

  5. Epigenetic re-programming of the Germ Cell Nuclear Factor gene is required for proper differentiation of induced pluripotent cells

    PubMed Central

    Wang, Hongran; Wang, Xiaohong; Xu, Xueping; Zwaka, Thomas P.; Cooney, Austin J.

    2013-01-01

    Somatic cells have been reprogrammed into induced pluripotent stem (iPS) cells that recapitulate the pluripotent nature of embryonic stem (ES) cells. Reduced pluripotency and variable differentiation capacities have hampered progress with this technology for applications in regeneration medicine. We have previously shown that Germ Cell Nuclear Factor (Gcnf) is required for the repression of pluripotency genes during ES cell differentiation and embryonic development. Here we report that iPS cell lines, in which the Gcnf gene was properly re-programmed, allowing expression of Gcnf, repress pluripotency genes during subsequent differentiation. In contrast, iPS clones in which the Gcnf gene was not re-programmed maintained pluripotency gene expression during differentiation and did not differentiate properly either in vivo or in vitro. These mal-reprogrammed cells re-capitulated the phenotype of Gcnf knock out (Gcnf−/−) ES cells. Re-introduction of Gcnf into either the Gcnf negative iPS cells or the Gcnf−/− ES cells, rescued repression of Oct4 during differentiation. Our findings establish a key role for Gcnf as a regulator of iPS cell pluripotency gene expression. It also demonstrates that reactivation of the Gcnf gene may serve as a marker to distinguish completely re-programmed iPS cells from incompletely pluripotent cells, which would make therapeutic use of iPS cells safer and more practical as it would reduce the oncogenic potential of iPS cells. PMID:23495137

  6. NK Cell-extrinsic IL-18 Signaling Is Required for Efficient NK Cell Activation to Vaccinia Virus

    PubMed Central

    Brandstadter, Joshua D.; Huang, Xiaopei; Yang, Yiping

    2014-01-01

    Summary NK cells are important for the control of vaccinia virus (VV) in vivo. Recent studies have shown that multiple pathways are required for effective activation of NK cells. These include both TLR-dependent and -independent pathways, as well as the NKG2D activating receptor that recognizes host stress-induced NKG2D ligands. However, it remains largely unknown what controls the upregulation of NKG2D ligands in response to VV infection. In this study, we first showed that IL-18 is critical for NK cell activation and viral clearance. We then demonstrated that IL-18 signaling on both NK cells and DCs is required for efficient NK cell activation upon VV infection in vitro. We further showed in vivo that efficient NK cell activation to VV is dependent on DCs and IL-18 signaling in non-NK cells, suggesting an essential role for NK cell-extrinsic IL-18 signaling in NK cell activation. Mechanistically, IL-18 signaling in DCs promotes expression of Rae-1, an NKG2D ligand. Collectively, our data reveal a previously unrecognized role for NK cell-extrinsic IL-18 signaling in NK cell activation through upregulation of NKG2D ligands. These observations may provide insights into the design of effective NK cell-based therapies for viral infections and cancer. PMID:24846540

  7. Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis

    PubMed Central

    Levring, Trine B.; Kongsbak, Martin; Rode, Anna K. O.; Woetmann, Anders; Ødum, Niels; Bonefeld, Charlotte Menné; Geisler, Carsten

    2015-01-01

    Adaptive immune responses require activation and expansion of antigen-specific T cells. Whereas early T cell activation is independent of exogenous cystine (Cys2), T cell proliferation is dependent of Cys2. However, the exact roles of Cys2 in T cell proliferation still need to be determined. The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys2. Vice-versa, the GSH synthesis inhibitor L-buthionine-sulfoximine (BSO) and inhibition of Cys2 uptake with glutamate inhibited GSH and DNA synthesis in parallel. We further found that thioredoxin (Trx) can partly substitute for GSH during DNA synthesis. Finally, we show that GSH or Trx is required for the activity of ribonucleotide reductase (RNR), the enzyme responsible for generation of the deoxyribonucleotide DNA building blocks. In conclusion, we show that activated human T cells require exogenous Cys2 to proliferate and that this is partly explained by the fact that Cys2 is required for production of GSH, which in turn is required for optimal RNR-mediated deoxyribonucleotide synthesis and DNA replication. PMID:26392411

  8. Hemogenic endothelial cell specification requires c-kit, notch signaling, and p27-mediated cell-cycle control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Delineating the mechanism or mechanisms that regulate the specification of hemogenic endothelial cells from primordial endothelium is critical for optimizing their derivation from human stem cells for clinical therapies. We previously determined that retinoic acid (RA) is required for hemogenic spec...

  9. Serum and growth factor requirements for proliferation of human adrenocortical cells in culture: comparison with bovine adrenocortical cells.

    PubMed

    Hornsby, P J; Sturek, M; Harris, S E; Simonian, M H

    1983-11-01

    Although bovine adrenocortical cells proliferate readily in cell culture, proliferation of fetal or adult human adrenocortical cells has been observed to be limited and preparation of pure proliferating cultures of human adrenocortical cells has not been reported. The growth requirements of fetal human definitive zone adrenocortical cells in culture were compared to the established requirements of bovine adrenocortical cells. The medium used was 1:1 Ham's F12 and Dulbecco's modified Eagle's medium supplemented with transferrin and insulin. Earlier experiments showed that human cells had a greater proliferative response to horse serum than to fetal bovine serum, whereas the opposite was true for bovine cells. When plated on fibronectin-coated dishes and exposed to varying concentrations of horse serum in the presence of 100 ng/ml fibroblast growth factor (FGF), increasing cell growth was observed up to a serum concentration of 50%. When 50% fetal bovine serum was used instead of horse serum proliferation was less. In contrast, bovine adrenocortical cells showed a maximal proliferative response to either fetal bovine serum or horse serum at 10%. Human adrenocortical cells thus have a very high requirement for serum; 50% is the highest level that may be practically used, but the shape of the dose-response curve suggests that this concentration is still suboptimal. Growth was less in the absence of FGF. Epidermal growth factor can partially substitute for FGF. No response to 100 nM placental lactogen was observed. Less growth was observed when dishes were not coated with fibronectin. The factors present in horse serum that are evidently needed in high amounts by human cells are unknown. Despite this lack of knowledge, use of 50% horse serum enabled long-term growth of human adrenocortical cells that are pure by the criterion of retraction in response to ACTH. Nonadrenocortical cells do not show a retraction response. Such long-term cultures may be useful in studies of

  10. Requirements for growth and IL-10 expression of highly purified human T regulatory cells

    PubMed Central

    Bonacci, Benedetta; Edwards, Brandon; Jia, Shuang; Williams, Calvin; Hessner, Martin J.; Gauld, Stephen; Verbsky, James

    2013-01-01

    Human regulatory T cells (TR) cells have potential for the treatment of a variety of immune mediated diseases but the anergic phenotype of these cells makes them difficult to expand in vitro. We have examined the requirements for growth and cytokine expression from highly purified human TR cells, and correlated these findings with the signal transduction events of these cells. We demonstrate that these cells do not proliferate or secrete IL-10 even in the presence of high doses of IL-2. Stimulation with a superagonistic anti-CD28 antibody (clone 9D4) and IL-2 partially reversed the proliferative defect, and this correlated with reversal of the defective calcium mobilization in these cells. Dendritic cells were effective at promoting TR cell proliferation, and under these conditions the proliferative capacity of TR cells was comparable to conventional CD4 lymphocytes. Blocking TGF-β activity abrogated IL-10 expression from these cells, while addition of TGF-β resulted in IL-10 production. These data demonstrate that highly purified populations of TR cells are anergic even in the presence of high doses of IL-2. Furthermore, antigen presenting cells provide proper co-stimulation to overcome the anergic phenotype of TR cells, and under these conditions they are highly sensitive to IL-2. In addition, these data demonstrate for the first time that TGF-β is critical to enable human TR cells to express IL-10. PMID:22562448

  11. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells

    SciTech Connect

    Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J.; Radisky, Derek C.

    2008-06-26

    Cell morphology dictates response to a wide variety of stimuli, controlling cell metabolism, differentiation, proliferation, and death. Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire migratory characteristics, and in the process convert from a 'cuboidal' epithelial structure into an elongated mesenchymal shape. We had shown previously that matrix metalloproteinase-3 (MMP3) can stimulate EMT of cultured mouse mammary epithelial cells through a process that involves increased expression of Rac1b, a protein that stimulates alterations in cytoskeletal structure. We show here that cells treated with MMP-3 or induced to express Rac1b spread to cover a larger surface, and that this induction of cell spreading is a requirement of MMP-3/Rac1b-induced EMT. We find that limiting cell spreading, either by increasing cell density or by culturing cells on precisely defined micropatterned substrata, blocks expression of characteristic markers of EMT in cells treated with MMP-3. These effects are not caused by general disruptions in cell signaling pathways, as TGF-{beta}-induced EMT is not affected by similar limitations on cell spreading. Our data reveal a previously unanticipated cell shape-dependent mechanism that controls this key phenotypic alteration and provide insight into the distinct mechanisms activated by different EMT-inducing agents.

  12. Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans

    PubMed Central

    Flynn, Ryan; Du, Jing; Veenstra, Rachelle G.; Reichenbach, Dawn K.; Panoskaltsis-Mortari, Angela; Taylor, Patricia A.; Freeman, Gordon J.; Serody, Jonathan S.; Murphy, William J.; Munn, David H.; Sarantopoulos, Stefanie; Luznik, Leo; Maillard, Ivan; Koreth, John; Cutler, Corey; Soiffer, Robert J.; Antin, Joseph H.; Ritz, Jerome; Dubovsky, Jason A.; Byrd, John C.; MacDonald, Kelli P.; Hill, Geoff R.; Blazar, Bruce R.

    2014-01-01

    Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD. PMID:24820310

  13. Prkci is required for a non-autonomous signal that coordinates cell polarity during cavitation.

    PubMed

    Mah, In Kyoung; Soloff, Rachel; Izuhara, Audrey K; Lakeland, Daniel L; Wang, Charles; Mariani, Francesca V

    2016-08-01

    Polarized epithelia define boundaries, spaces, and cavities within organisms. Cavitation, a process by which multicellular hollow balls or tubes are produced, is typically associated with the formation of organized epithelia. In order for these epithelial layers to form, cells must ultimately establish a distinct apical-basal polarity. Atypical PKCs have been proposed to be required for apical-basal polarity in diverse species. Here we show that while cells null for the Prkci isozyme exhibit some polarity characteristics, they fail to properly segregate apical-basal proteins, form a coordinated ectodermal epithelium, or participate in normal cavitation. A failure to cavitate could be due to an overgrowth of interior cells or to an inability of interior cells to die. Null cells however, do not have a marked change in proliferation rate and are still capable of undergoing cell death, suggesting that alterations in these processes are not the predominant cause of the failed cavitation. Overexpression of BMP4 or EZRIN can partially rescue the phenotype possibly by promoting cell death, polarity, and differentiation. However, neither is sufficient to provide the required cues to generate a polarized epithelium and fully rescue cavitation. Interestingly, when wildtype and Prkci(-/-) ES cells are mixed together, a polarized ectodermal epithelium forms and cavitation is rescued, likely due to the ability of wildtype cells to produce non-autonomous polarity cues. We conclude that Prkci is not required for cells to respond to these cues, though it is required to produce them. Together these findings indicate that environmental cues can facilitate the formation of polarized epithelia and that cavitation requires the proper coordination of multiple basic cellular processes including proliferation, differentiation, cell death, and apical-basal polarization. PMID:27312576

  14. ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCTED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS

    EPA Science Inventory

    ORIENTATION REQUIREMENT TO DETECT MAGNETIC FIELD-INDUCED ALTERATION OF GAP JUNCTION COMMUNICATION IN EPITHELIAL CELLS.
    OBJECTIVE: We have shown that functional gap junction communication as measured by Lucifer yellow dye transfer (DT) in Clone-9 rat liver epithelial cells, c...

  15. Mapping the polarity and stimulus density requirements for T-cell activation

    NASA Astrophysics Data System (ADS)

    Wei, Xunbin; Krasieva, Tatiana B.; Zhang, Zhanxiang; Negulescu, Paul A.; Sun, Chung-Ho; Berns, Michael W.; Cahalan, Michael D.; Tromberg, Bruce J.

    1998-08-01

    T-cell contact with antigen-presenting cells (APC) initiates an activation cascade which includes an increase in T-cell intracellular calcium [(Ca2+)i] and leads to T-cell proliferation and differentiation. Although T-cell/APC physical contact is required for an immune response, little is known about the patterns of cellular interaction and their relation to activation. We have combined fluorescence spectroscopy and imaging with optical manipulation to investigate the contact requirements for T-cell activation, using optical tweezers to control the orientation of T- cell/APC pairs and fluorescence microscopy to measure the subsequent (Ca2+)i response, detected as an emission shift from the combination of fura-red and oregon- green, two cytoplasmic (Ca2+) indicators. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling (2-5 micrometers /min). T cells contacted with antigen- presenting cells or antibody-coated beads entered a dynamic and reproducible program in the first 10 - 20 mins, including (Ca2+)i increase, changes in shape and motility, engulfment, and stable contact. T cells presented with antigen at the leading edge had a higher probability of responding (85%) and a shorter latency of response (50 secs) than those contacting APCs or beads with their trailing end (APCs: 30%, 150 secs; beads: 6%, 300 secs). Alterations in antibody density, quantified by FACS analysis, and bead size were used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal. Preliminary data show that T cell responses [response percentage, latency and (Ca2+)i pattern] depend on both antibody density and bead size.

  16. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for cell lines used for production of biologics. 113.52 Section 113.52 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS...

  17. Tracking mesenchymal stromal cells using an ultra-bright TAT-functionalized plasmonic-active nanoplatform.

    PubMed

    Yuan, Hsiangkuo; Gomez, Jose A; Chien, Jennifer S; Zhang, Lunan; Wilson, Christy M; Li, Shuqin; Fales, Andrew M; Liu, Yang; Grant, Gerald A; Mirotsou, Maria; Dzau, Victor J; Vo-Dinh, Tuan

    2016-04-01

    High-resolution tracking of stem cells remains a challenging task. An ultra-bright contrast agent with extended intracellular retention is suitable for in vivo high-resolution tracking of stem cells following the implantation. Here, a plasmonic-active nanoplatform was developed for tracking mesenchymal stromal cells (MSCs) in mice. The nanoplatform consisted of TAT peptide-functionalized gold nanostars (TAT-GNS) that emit ultra-bright two-photon photoluminescence capable of tracking MSCs under high-resolution optical imaging. In vitro experiment showed TAT-GNS-labeled MSCs retained a similar differentiability to that of non-labeled MSCs controls. Due to their star shape, TAT-GNS exhibited greater intracellular retention than that of commercial Q-Tracker. In vivo imaging of TAT-GNS-labeled MSCs five days following intra-arterial injections in mice kidneys showed possible MSCs implantation in juxta-glomerular (JG) regions, but non-specifically in glomeruli and afferent arterioles as well. With future design to optimize GNS labeling specificity and clearance, plasmonic-active nanoplatforms may be a useful intracellular tracking tool for stem cell research. An ultra-bright intracellular contrast agent is developed using TAT peptide-functionalized gold nanostars (TAT-GNS). It poses minimal influence on the stem cell differentiability. It exhibits stronger two-photon photoluminescence and superior labeling efficiency than commercial Q-Tracker. Following renal implantation, some TAT-GNS-labeled MSCs permeate blood vessels and migrate to the juxta-glomerular region. PMID:27095616

  18. c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1

    PubMed Central

    Song, Yu; Luciani, Marie-Françoise; Giusti, Corinne; Golstein, Pierre

    2015-01-01

    Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP–induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms. PMID:25518941

  19. Phenotypically distinct helper NK cells are required for gp96-mediated anti-tumor immunity

    PubMed Central

    Sedlacek, Abigail L.; Kinner-Bibeau, Lauren B.; Binder, Robert J.

    2016-01-01

    A number of Heat Shock Proteins (HSPs), in the extracellular environment, are immunogenic. Following cross-presentation of HSP-chaperoned peptides by CD91+ antigen presenting cells (APCs), T cells are primed with specificity for the derivative antigen-bearing cell. Accordingly, tumor-derived HSPs are in clinical trials for cancer immunotherapy. We investigate the role of NK cells in gp96-mediated anti-tumor immune responses given their propensity to lyse tumor cells. We show that gp96-mediated rejection of tumors requires a unique and necessary helper role in NK cells. This helper role occurs during the effector phase of the anti-tumor immune response and is required for T cell and APC function. Gp96 activates NK cells indirectly via APCs to a phenotype distinct from NK cells activated by other mechanisms such as IL-2. While NK cells have both lytic and cytokine producing properties, we show that gp96 selectively activates cytokine production in NK cells, which is important in the HSP anti-tumor immune response, and leaves their cytotoxic capacity unchanged. PMID:27431727

  20. Cutting edge: stage-specific requirement of IL-18 for antiviral NK cell expansion.

    PubMed

    Madera, Sharline; Sun, Joseph C

    2015-02-15

    Although NK cells are considered part of the innate immune system, recent studies have demonstrated the ability of Ag-experienced NK cells to become long-lived and contribute to potent recall responses similar to T and B cells. The precise signals that promote the generation of a long-lived NK cell response are largely undefined. In this article, we demonstrate that NK cells require IL-18 signaling to generate a robust primary response during mouse CMV (MCMV) infection but do not require this signal for memory cell maintenance or recall responses. IL-12 signaling and STAT4 in activated NK cells increased the expression of the adaptor protein MyD88, which mediates signaling downstream of the IL-18 and IL-1 receptors. During MCMV infection, NK cells required MyD88, but not IL-1R, for optimal expansion. Thus, an IL-18-MyD88 signaling axis facilitates the prolific expansion of NK cells in response to primary viral infection, but not recall responses. PMID:25589075

  1. Stage-specific requirement of IL-18 for antiviral NK cell expansion

    PubMed Central

    Madera, Sharline; Sun, Joseph C.

    2014-01-01

    Although natural killer (NK) cells are considered part of the innate immune system, recent studies have demonstrated the ability of antigen-experienced NK cells to become long-lived and contribute to potent recall responses similar to T and B cells. The precise signals that promote the generation of a long-lived NK cell response are largely undefined. Here, we demonstrate that NK cells require interleukin (IL)-18 signaling to generate a robust primary response during mouse cytomegalovirus (MCMV) infection, but do not require this signal for memory cell maintenance or recall responses. IL-12 signaling and STAT4 in activated NK cells increased the expression of the adaptor protein MyD88, which mediates signaling downstream of the IL-18 and IL-1 receptors. During MCMV infection, NK cells required MyD88 but not IL-1 receptor for optimal expansion. Thus, an IL-18-MyD88 signaling axis facilitates the prolific expansion of NK cells in response to primary viral infection, but not recall responses. PMID:25589075

  2. DMRT1 Is Required for Mouse Spermatogonial Stem Cell Maintenance and Replenishment.

    PubMed

    Zhang, Teng; Oatley, Jon; Bardwell, Vivian J; Zarkower, David

    2016-09-01

    Male mammals produce sperm for most of postnatal life and therefore require a robust germ line stem cell system, with precise balance between self-renewal and differentiation. Prior work established doublesex- and mab-3-related transcription factor 1 (Dmrt1) as a conserved transcriptional regulator of male sexual differentiation. Here we investigate the role of Dmrt1 in mouse spermatogonial stem cell (SSC) homeostasis. We find that Dmrt1 maintains SSCs during steady state spermatogenesis, where it regulates expression of Plzf, another transcription factor required for SSC maintenance. We also find that Dmrt1 is required for recovery of spermatogenesis after germ cell depletion. Committed progenitor cells expressing Ngn3 normally do not contribute to SSCs marked by the Id4-Gfp transgene, but do so when spermatogonia are chemically depleted using busulfan. Removal of Dmrt1 from Ngn3-positive germ cells blocks the replenishment of Id4-GFP-positive SSCs and recovery of spermatogenesis after busulfan treatment. Our data therefore reveal that Dmrt1 supports SSC maintenance in two ways: allowing SSCs to remain in the stem cell pool under normal conditions; and enabling progenitor cells to help restore the stem cell pool after germ cell depletion. PMID:27583450

  3. Activation requirements of 2,4-dinitrobenzenesulfonate-primed T suppressor cells

    SciTech Connect

    Fairchild, R.L.; Moorhead, J.W.

    1986-03-05

    Culture of spleen cells from mice tolerized with 2,4-dinitrobenzenesulfonate (DNBS) with DNP-labeled spleen cells (DNP-SC) activates Lyt 2/sup +/ Ts to synthesize and release a suppressor factor (SSF) into the supernatant (SN) which suppresses the transfer of contact sensitivity to 2,4-dinitrofluorobenzene. Activation of the Ts requires recognition of DNP in association with class I MHC determinants. The signals which activate these Ts to produce SSF were examined in greater detail. The SN from cultures of L3T4-depleted tolerant cells and DNP-SC did not contain SSF activity, suggesting that an L3T4/sup +/ cell was required for Ts activation. Similarly, the SN from cultures of DNBS-primed cells and gluteraldehyde-fixed (Glu-) DNP-SC had no SSF activity. Addition of Il-1 restored the ability of the Ts to synthesize and release the factor into the SN in both cases. By contrast to the SN, the soluble cell lysate from cultures of tolerant cells and Glu-DNP-SC, or from L3T4-depleted tolerance cells and DNP-SC did contain the SSF activity. These results indicate that two signals are required to activate DNBS-primed Ts to produce SSF. Recognition of DNP/class I MHC stimulates the Ts to synthesize SSF and a costimulator is required for release of the factor. These results further indicate that the L3T4-bearing cells play no role in the synthesis of SSF. Rather, these cells induce the costimulator which is required for factor release.

  4. Sequential regulation of developmental events during polar morphogenesis in Caulobacter crescentus: assembly of pili on swarmer cells requires cell separation.

    PubMed Central

    Sommer, J M; Newton, A

    1988-01-01

    Pili, along with the flagellum and DNA bacteriophage receptors, are structural markers for polar morphogenesis in Caulobacter crescentus. Pili act as primary receptors for a number of small, C. crescentus-specific DNA and RNA bacteriophages, and the timing of pilus-dependent adsorption of bacteriophage phiCb5 in synchronized cell populations has led to the general conclusion that pili are formed coordinately with the flagellum and other polar surface structures in the predivisional cell. The use of rotary platinum shadow casting and electron microscopy as a direct assay for formation of flagella and pili in synchronous cell cultures now shows, however, that when expressed as fractions of the swarmer cell cycle, flagella are assembled on the predivisional cells at approximately 0.8 and that pili are assembled on the new swarmer cells at approximately 0.1 of the next cell cycle. Adsorption of pilus-specific bacteriophage phiCb5 prevented the loss of pili from swarmer cells during development, which suggests that these structures are retracted at the time of stalk formation. Examination of temperature-sensitive cell division mutants showed that the assembly of pili depends on completion of cell separation. These results indicate that the stage-specific events required for polar morphogenesis in C. crescentus occur sequentially, rather than coordinately in the cell cycle, and that the timing of these events reflects the order of underlying cell cycle steps. Images PMID:2891681

  5. Induction of type I IFN is required for overcoming tumor-specific T-cell tolerance after stem cell transplantation.

    PubMed

    Horkheimer, Ian; Quigley, Michael; Zhu, Jiangao; Huang, Xiaopei; Chao, Nelson J; Yang, Yiping

    2009-05-21

    Tumor-specific T-cell tolerance represents one major mechanism of tumor-induced immune evasion. Myeloablative chemotherapy with stem cell transplantation may offer the best chance of achieving a state of minimal residual disease and, thus, minimize tumor-induced immune evasion. However, studies have shown that tumor-specific T-cell tolerance persists after transplantation. Here, we showed that CD4(+)CD25(+) regulatory T (T(Reg)) cells play a critical role in tumor-specific CD8(+) T-cell tolerance after transplantation. Removal of T(Reg) cells from the donor lymphocyte graft did not overcome this tolerance because of rapid conversion of donor CD4(+)CD25(-) T cells into CD4(+)CD25(+)Foxp3(+) T(Reg) cells in recipients after transplantation, and depletion of T(Reg) cells in recipients was necessary for the reversal of tumor-specific tolerance. These results suggest that strategies capable of overcoming T-cell tolerance in recipients are required to promote antitumor immunity after transplantation. Toward this goal, we showed that dendritic cell (DC) vaccines coadministered with the TLR9 ligand, CpG could effectively overcome tumor-specific tolerance, leading to significant prolongation of tumor-free survival after transplantation. We further showed that CpG-induced type I interferon was critical for the reversal of tumor-specific tolerance in vivo. Collectively, these results may suggest effective immunotherapeutic strategies for treating cancer after stem cell transplantation. PMID:19279333

  6. Longitudinal Requirement for CD4+ T Cell Help for Adenovirus Vector–Elicited CD8+ T Cell Responses

    PubMed Central

    Provine, Nicholas M.; Larocca, Rafael A.; Penaloza-MacMaster, Pablo; Borducchi, Erica N.; McNally, Anna; Parenteau, Lily R.; Kaufman, David R.

    2014-01-01

    Despite the widespread use of replication-incompetent recombinant adenovirus (Ad) vectors as candidate vaccine platforms, the mechanism by which these vectors elicit CD8+ T cell responses remains poorly understood. Our data demonstrate that induction and maintenance of CD8+ T cell responses by Ad vector immunization is longitudinally dependent on CD4+ T cell help for a prolonged period. Depletion of CD4+ T cells in wild type mice within the first 8 d following Ad immunization resulted in dramatically reduced induction of Ag-specific CD8+ T cells, decreased T-bet and eomesodermin expression, impaired KLRG1+ effector differentiation, and atypical expression of the memory markers CD127, CD27, and CD62L. Moreover, these CD8+ T cells failed to protect against a lethal recombinant Listeria monocytogenes challenge. Depletion of CD4+ T cells between weeks 1 and 4 following immunization resulted in increased contraction of memory CD8+ T cells. These data demonstrate a prolonged temporal requirement for CD4+ T cell help for vaccine-elicited CD8+ T cell responses in mice. These findings have important implications in the design of vaccines aimed at eliciting CD8+ T cell responses and may provide insight into the impaired immunogenicity of vaccines in the context of AIDS and other CD4+ T cell immune deficiencies. PMID:24778441

  7. GENETICS OF SOMATIC MAMMALIAN CELLS, X. COMPLEMENTATION ANALYSIS OF GLYCINE-REQUIRING MUTANTS*

    PubMed Central

    Kao, Fa-Ten; Chasin, Lawrence; Puck, Theodore T.

    1969-01-01

    Complementation analysis at the single gene level has been carried out on 13 different, recessive, glycine-requiring mutants produced by action of mutagenic agents on the CHO-K1 Chinese hamster cell. The mutants form four different complementation classes indicating that at least four separate genes can produce a growth requirement for glycine. The four classes of mutants can be distinguished biochemically as well as genetically. Existence of these four mutant classes appears to explain the preponderance of the glycine-requiring forms in the auxotrophs produced by treatment of the parental cell with mutagenic agents. PMID:5271752

  8. A Src-Tks5 Pathway Is Required for Neural Crest Cell Migration during Embryonic Development

    PubMed Central

    Murphy, Danielle A.; Tsai, Jeff H.; Kawakami, Yasuhiko; Maurer, Jochen; Stewart, Rodney A.; Izpisúa-Belmonte, Juan Carlos; Courtneidge, Sara A.

    2011-01-01

    In the adult organism, cell migration is required for physiological processes such as angiogenesis and immune surveillance, as well as pathological events such as tumor metastasis. The adaptor protein and Src substrate Tks5 is necessary for cancer cell migration through extracellular matrix in vitro and tumorigenicity in vivo. However, a role for Tks5 during embryonic development, where cell migration is essential, has not been examined. We used morpholinos to reduce Tks5 expression in zebrafish embryos, and observed developmental defects, most prominently in neural crest-derived tissues such as craniofacial structures and pigmentation. The Tks5 morphant phenotype was rescued by expression of mammalian Tks5, but not by a variant of Tks5 in which the Src phosphorylation sites have been mutated. We further evaluated the role of Tks5 in neural crest cells and neural crest-derived tissues and found that loss of Tks5 impaired their ventral migration. Inhibition of Src family kinases also led to abnormal ventral patterning of neural crest cells and their derivatives. We confirmed that these effects were likely to be cell autonomous by shRNA-mediated knockdown of Tks5 in a murine neural crest stem cell line. Tks5 was required for neural crest cell migration in vitro, and both Src and Tks5 were required for the formation of actin-rich structures with similarity to podosomes. Additionally, we observed that neural crest cells formed Src-Tks5-dependent cell protrusions in 3-D culture conditions and in vivo. These results reveal an important and novel role for the Src-Tks5 pathway in neural crest cell migration during embryonic development. Furthermore, our data suggests that this pathway regulates neural crest cell migration through the generation of actin-rich pro-migratory structures, implying that similar mechanisms are used to control cell migration during embryogenesis and cancer metastasis. PMID:21799874

  9. Sox2 Is Required for Maintenance and Differentiation of Bronchiolar Clara, Ciliated, and Goblet Cells

    PubMed Central

    Tompkins, David H.; Besnard, Valérie; Lange, Alexander W.; Wert, Susan E.; Keiser, Angela R.; Smith, April N.; Lang, Richard; Whitsett, Jeffrey A.

    2009-01-01

    The bronchioles of the murine lung are lined by a simple columnar epithelium composed of ciliated, Clara, and goblet cells that together mediate barrier function, mucociliary clearance and innate host defense, vital for pulmonary homeostasis. In the present work, we demonstrate that expression of Sox2 in Clara cells is required for the differentiation of ciliated, Clara, and goblet cells that line the bronchioles of the postnatal lung. The gene was selectively deleted in Clara cells utilizing Scgb1a1-Cre, causing the progressive loss of Sox2 in the bronchioles during perinatal and postnatal development. The rate of bronchiolar cell proliferation was decreased and associated with the formation of an undifferentiated, cuboidal-squamous epithelium lacking the expression of markers of Clara cells (Scgb1a1), ciliated cells (FoxJ1 and α-tubulin), and goblet cells (Spdef and Muc5AC). By adulthood, bronchiolar cell numbers were decreased and Sox2 was absent in extensive regions of the bronchiolar epithelium, at which time residual Sox2 expression was primarily restricted to selective niches of CGRP staining neuroepithelial cells. Allergen-induced goblet cell differentiation and mucus production was absent in the respiratory epithelium lacking Sox2. In vitro, Sox2 activated promoter-luciferase reporter constructs for differentiation markers characteristic of Clara, ciliated, and goblet cells, Scgb1a1, FoxJ1, and Agr2, respectively. Sox2 physically interacted with Smad3 and inhibited TGF-β1/Smad3-mediated transcriptional activity in vitro, a pathway that negatively regulates proliferation. Sox2 is required for proliferation and differentiation of Clara cells that serve as the progenitor cells from which Clara, ciliated, and goblet cells are derived. PMID:20011520

  10. Ki-67 is required for maintenance of cancer stem cells but not cell proliferation

    PubMed Central

    Cidado, Justin; Wong, Hong Yuen; Rosen, D. Marc; Cimino-Mathews, Ashley; Garay, Joseph P.; Fessler, Abigail G.; Rasheed, Zeshaan A.; Hicks, Jessica; Cochran, Rory L.; Croessmann, Sarah; Zabransky, Daniel J.; Mohseni, Morassa; Beaver, Julia A.; Chu, David; Cravero, Karen; Christenson, Eric S.; Medford, Arielle; Mattox, Austin; De Marzo, Angelo M.; Argani, Pedram; Chawla, Ajay; Hurley, Paula J.; Lauring, Josh; Park, Ben Ho

    2016-01-01

    Ki-67 expression is correlated with cell proliferation and is a prognostic marker for various cancers; however, its function is unknown. Here we demonstrate that genetic disruption of Ki-67 in human epithelial breast and colon cancer cells depletes the cancer stem cell niche. Ki-67 null cells had a proliferative disadvantage compared to wildtype controls in colony formation assays and displayed increased sensitivity to various chemotherapies. Ki-67 null cancer cells showed decreased and delayed tumor formation in xenograft assays, which was associated with a reduction in cancer stem cell markers. Immunohistochemical analyses of human breast cancers revealed that Ki-67 expression is maintained at equivalent or greater levels in metastatic sites of disease compared to matched primary tumors, suggesting that maintenance of Ki-67 expression is associated with metastatic/clonogenic potential. These results elucidate Ki-67's role in maintaining the cancer stem cell niche, which has potential diagnostic and therapeutic implications for human malignancies. PMID:26823390

  11. Activation-specific metabolic requirements for NK cell IFN-γ production1

    PubMed Central

    Keppel, Molly P.; Topcagic, Nermina; Mah, Annelise Y.; Vogel, Tiphanie P.; Cooper, Megan A.

    2014-01-01

    There has been increasing recognition of the importance of cellular metabolism and metabolic substrates for the function and differentiation of immune cells. Here, for the first time, we investigate the metabolic requirements for production of IFN-γ by freshly isolated NK cells. Primary murine NK cells mainly utilize mitochondrial oxidative phosphorylation at rest and with short-term activation. Remarkably, we discovered significant differences in the metabolic requirements of murine NK cell IFN-γ production depending upon the activation signal. Stimulation of NK cell IFN-γ production was independent of glycolysis or mitochondrial oxidative phosphorylation when cells were activated with IL-12+IL-18. By contrast, stimulation via activating NK receptors required glucose-driven oxidative phosphorylation. Prolonged treatment with high-dose, but not low dose, IL-15 eliminated the metabolic requirement for receptor stimulation. In summary, this study demonstrates that metabolism provides an essential second signal for induction of IFN-γ production by activating NK cell receptors that can be reversed with prolonged high-dose IL-15 treatment. PMID:25595780

  12. Requirements for dE2F function in proliferating cells and in post-mitotic differentiating cells.

    PubMed Central

    Brook, A; Xie, J E; Du, W; Dyson, N

    1996-01-01

    The transcription factor E2F is a target of the retinoblastoma tumor suppressor protein (pRB) and may mediate pRB regulation of S phase entry in mammalian cells. The recent identification of mutant alleles of the Drosophila E2F gene (dE2F) has shown that dE2F is required for embryogenesis. dE2F-mutant embryos lack a co-ordinated program of gene expression which accompanies S phase entry and DNA synthesis declines to levels that are barely detectable. We have investigated the role of the dE2F gene at later stages of development. dE2F is expressed in several larval tissues and is required for cell proliferation in the eye imaginal disc. Surprisingly, dE2F expression persists in post-mitotic cells of the eye disc of third-instar larvae. The loss of dE2F function in these cells causes a novel phenotype, characterized by loss of photoreceptors and abnormal rhabdomere cell morphology. These results show that dE2F is required at multiple stages of development and suggest that E2F may have an important function in post-mitotic cells in addition to its role during cell proliferation. Images PMID:8670871

  13. FRIZZLED7 Is Required for Tumor Inititation and Metastatic Growth of Melanoma Cells

    PubMed Central

    Tiwary, Shweta; Xu, Lei

    2016-01-01

    Metastases are thought to arise from cancer stem cells and their tumor initiating abilities are required for the establishment of metastases. Nevertheless, in metastatic melanoma, the nature of cancer stem cells is under debate and their contribution to metastasis formation remains unknown. Using an experimental metastasis model, we discovered that high levels of the WNT receptor, FZD7, correlated with enhanced metastatic potentials of melanoma cell lines. Knocking down of FZD7 in a panel of four melanoma cell lines led to a significant reduction in lung metastases in animal models, arguing that FZD7 plays a causal role during metastasis formation. Notably, limiting dilution analyses revealed that FZD7 is essential for the tumor initiation of melanoma cells and FZD7 knockdown impeded the early expansion of metastatic melanoma cells shortly after seeding, in accordance with the view that tumor initiating ability of cancer cells is required for metastasis formation. FZD7 activated JNK in melanoma cell lines in vitro and the expression of a dominant negative JNK suppressed metastasis formation in vivo, suggesting that FZD7 may promote metastatic growth of melanoma cells via activation of JNK. Taken together, our findings uncovered a signaling pathway that regulates the tumor initiation of melanoma cells and contributes to metastasis formation in melanoma. PMID:26808375

  14. ABCC4 is required for cell proliferation and tumorigenesis in non-small cell lung cancer

    PubMed Central

    Zhao, Xiaoting; Guo, Yinan; Yue, Wentao; Zhang, Lina; Gu, Meng; Wang, Yue

    2014-01-01

    Background Multidrug resistance protein 4 (MRP4), also known as ATP-cassette binding protein 4 (ABCC4), is a member of the MRP/ABCC subfamily of ATP-binding cassette transporters, which are capable of pumping a wide variety of drugs out of the cell. However, little is known about the function of ABCC4 in the proliferation of lung cancer cells. Methods ABCC4 mRNA and protein levels in lung cancer cell lines were measured by real-time polymerase chain reaction and Western blot, respectively. A lentivirus-mediated RNA interference technique was used to inhibit ABCC4 mRNA expression in A549 and 801D cells. The function of ABCC4 in cell growth was investigated by MTS and colony formation assays. The role of ABCC4 in cell cycle progression was evaluated by flow cytometry and Western blot analysis. ABCC4 mRNA levels in 30 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients were detected by real-time polymerase chain reaction. Results ABCC4 was highly expressed in lung cancer cell lines. ABCC4 expression was markedly downregulated in A549 and 801D cells using the RNA interference technique. Suppression of ABCC4 expression inhibited cell growth. The percentage of cells in G1 phase was increased when ABCC4 expression was suppressed. Phosphorylation of retinoblastoma protein was weakened, originating in the downregulation of ABCC4. ABCC4 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines. Conclusion ABCC4 may play an important role in the control of A549 and 801D cell growth. ABCC4 is a potential target for lung cancer therapy. PMID:24591841

  15. NASA specification for manufacturing and performance requirements of NASA standard aerospace nickel-cadmium cells

    NASA Technical Reports Server (NTRS)

    1988-01-01

    On November 25, 1985, the NASA Chief Engineer established a NASA-wide policy to maintain and to require the use of the NASA standard for aerospace nickel-cadmium cells and batteries. The Associate Administrator for Safety, Reliability, Maintainability, and Quality Assurance stated on December 29, 1986, the intent to retain the NASA standard cell usage policy established by the Office of the Chief Engineer. The current NASA policy is also to incorporate technological advances as they are tested and proven for spaceflight applications. This policy will be implemented by modifying the existing standard cells or by developing new NASA standards and their specifications in accordance with the NASA's Aerospace Battery Systems Program Plan. This NASA Specification for Manufacturing and Performance Requirements of NASA Standard Aerospace Nickel-Cadmium Cells is prepared to provide requirements for the NASA standard nickel-cadmium cell. It is an interim specification pending resolution of the separator material availability. This specification has evolved from over 15 years of nickel-cadmium cell experience by NASA. Consequently, considerable experience has been collected and cell performance has been well characterized from many years of ground testing and from in-flight operations in both geosynchronous (GEO) and low earth orbit (LEO) applications. NASA has developed and successfully used two standard flight qualified cell designs.

  16. Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell-cell interaction.

    PubMed

    Pietilä, Mika; Lehenkari, Petri; Kuvaja, Paula; Kaakinen, Mika; Kaul, Sunil C; Wadhwa, Renu; Uemura, Toshimasa

    2013-11-01

    The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells. PMID:23928292

  17. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila.

    PubMed

    Giansanti, Maria Grazia; Vanderleest, Timothy E; Jewett, Cayla E; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C; Brill, Julie A; Loerke, Dinah; Fuller, Margaret T; Blankenship, J Todd

    2015-11-01

    Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

  18. Rubisco Activity in Guard Cells Compared with the Solute Requirement for Stomatal Opening 1

    PubMed Central

    Reckmann, Udo; Scheibe, Renate; Raschke, Klaus

    1990-01-01

    We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the [14C]carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to 14CO2 for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19 femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant. Images Figure 1 PMID:16667255

  19. Exocyst-Dependent Membrane Addition Is Required for Anaphase Cell Elongation and Cytokinesis in Drosophila

    PubMed Central

    Giansanti, Maria Grazia; Vanderleest, Timothy E.; Jewett, Cayla E.; Sechi, Stefano; Frappaolo, Anna; Fabian, Lacramioara; Robinett, Carmen C.; Brill, Julie A.; Loerke, Dinah; Fuller, Margaret T.; Blankenship, J. Todd

    2015-01-01

    Mitotic and cytokinetic processes harness cell machinery to drive chromosomal segregation and the physical separation of dividing cells. Here, we investigate the functional requirements for exocyst complex function during cell division in vivo, and demonstrate a common mechanism that directs anaphase cell elongation and cleavage furrow progression during cell division. We show that onion rings (onr) and funnel cakes (fun) encode the Drosophila homologs of the Exo84 and Sec8 exocyst subunits, respectively. In onr and fun mutant cells, contractile ring proteins are recruited to the equatorial region of dividing spermatocytes. However, cytokinesis is disrupted early in furrow ingression, leading to cytokinesis failure. We use high temporal and spatial resolution confocal imaging with automated computational analysis to quantitatively compare wild-type versus onr and fun mutant cells. These results demonstrate that anaphase cell elongation is grossly disrupted in cells that are compromised in exocyst complex function. Additionally, we observe that the increase in cell surface area in wild type peaks a few minutes into cytokinesis, and that onr and fun mutant cells have a greatly reduced rate of surface area growth specifically during cell division. Analysis by transmission electron microscopy reveals a massive build-up of cytoplasmic astral membrane and loss of normal Golgi architecture in onr and fun spermatocytes, suggesting that exocyst complex is required for proper vesicular trafficking through these compartments. Moreover, recruitment of the small GTPase Rab11 and the PITP Giotto to the cleavage site depends on wild-type function of the exocyst subunits Exo84 and Sec8. Finally, we show that the exocyst subunit Sec5 coimmunoprecipitates with Rab11. Our results are consistent with the exocyst complex mediating an essential, coordinated increase in cell surface area that potentiates anaphase cell elongation and cleavage furrow ingression. PMID:26528720

  20. Rubisco activity in guard cells compared with the solute requirement for stomatal opening. [Pisum sativum

    SciTech Connect

    Reckmann, U.; Scheibe, R.; Raschke, K. )

    1990-01-01

    We investigated whether the reductive pentose phosphate path in guard cells of Pisum sativum had the capacity to contribute significantly to the production of osmotica during stomatal opening in the light. Amounts of ribulose 1,5-bisphophate carboxylase/oxygenase (Rubisco) were determined by the ({sup 14}C) carboxyarabinitol bisphosphate assay. A guard cell contained about 1.2 and a mesophyll cell about 324 picograms of the enzyme; the ratio was 1:270. The specific activities of Rubisco in guard cells and in mesophyll cells were equal; there was no indication of a specific inhibitor of Rubisco in guard cells. Rubisco activity was 115 femtomol per guard-cell protoplast and hour. This value was different from zero with a probability of 0.99. After exposure of guard-cell protoplasts to {sup 14}CO{sub 2} for 2 seconds in the light, about one-half of the radioactivity was in phosphorylated compounds and <10% in malate. Guard cells in epidermal strips produced a different labelling pattern; in the light, <10% of the label was in phosphorylated compounds and about 60% in malate. The rate of solute accumulation in intact guard cells was estimated to have been 900 femto-osmol per cell and hour. If Rubisco operated at full capacity in guard cells, and hexoses were produced as osmotica, solutes could be supplied at a rate of 19femto-osmol per cell and hour, which would constitute 2% of the estimated requirement. The capacity of guard-cell Rubisco to meet the solute requirement for stomatal opening in leaves of Pisum sativum is insignificant.

  1. Regulated Expression of miR-155 is Required for iNKT Cell Development

    PubMed Central

    Burocchi, Alessia; Pittoni, Paola; Tili, Esmerina; Rigoni, Alice; Costinean, Stefan; Croce, Carlo Maria; Colombo, Mario Paolo

    2015-01-01

    Invariant natural killer T cells (iNKT cells) are CD1d-restricted, lipid antigen-reactive T lymphocytes with immunoregulatory functions. iNKT cell development in the thymus proceeds through subsequent stages, defined by the expression of CD44 and NK1.1, and is dictated by a unique gene expression program, including microRNAs. Here, we investigated whether miR-155, a microRNA involved in differentiation of most hematopoietic cells, played any role in iNKT cell development. To this end, we assessed the expression of miR-155 along iNKT cell maturation in the thymus, and studied the effects of miR-155 on iNKT cell development using Lck-miR-155 transgenic mice, which over express miR-155 in T cell lineage under the lymphocyte-specific protein tyrosine kinase (Lck) promoter. We show that miR-155 is expressed by newly selected immature wild-type iNKT cells and turned off along iNKT cells differentiation. In transgenic mice, miR-155 over-expression resulted in a substantial block of iNKT cell maturation at Stage 2, in the thymus toward an overall reduction of peripheral iNKT cells, unlike mainstream T cells. Furthermore, the effects of miR-155 over-expression on iNKT cell differentiation were cell autonomous. Finally, we identified Ets1 and ITK transcripts as relevant targets of miR-155 in iNKT cell differentiation. Altogether, these results demonstrate that a tight control of miR-155 expression is required for the development of iNKT cells. PMID:25870598

  2. Requirement for CD4 T Cell Help in Generating Functional CD8 T Cell Memory

    NASA Astrophysics Data System (ADS)

    Shedlock, Devon J.; Shen, Hao

    2003-04-01

    Although primary CD8 responses to acute infections are independent of CD4 help, it is unknown whether a similar situation applies to secondary responses. We show that depletion of CD4 cells during the recall response has minimal effect, whereas depletion during the priming phase leads to reduced responses by memory CD8 cells to reinfection. Memory CD8 cells generated in CD4+/+ mice responded normally when transferred into CD4-/- hosts, whereas memory CD8 cells generated in CD4-/- mice mounted defective recall responses in CD4+/+ adoptive hosts. These results demonstrate a previously undescribed role for CD4 help in the development of functional CD8 memory.

  3. Communication between Human Dendritic Cell Subsets in Tuberculosis: Requirements for Naive CD4+ T Cell Stimulation

    PubMed Central

    Lozza, Laura; Farinacci, Maura; Bechtle, Marina; Stäber, Manuela; Zedler, Ulrike; Baiocchini, Andrea; del Nonno, Franca; Kaufmann, Stefan H. E.

    2014-01-01

    Human primary dendritic cells (DCs) are heterogeneous by phenotype, function, and tissue localization and distinct from inflammatory monocyte-derived DCs. Current information regarding the susceptibility and functional role of primary human DC subsets to Mycobacterium tuberculosis (Mtb) infection is limited. Here, we dissect the response of different primary DC subsets to Mtb infection. Myeloid CD11c+ cells and pDCs (C-type lectin 4C+ cells) were located in human lymph nodes (LNs) of tuberculosis (TB) patients by histochemistry. Rare CD141hi DCs (C-type lectin 9A+ cells) were also identified. Infection with live Mtb revealed a higher responsiveness of myeloid CD1c+ DCs compared to CD141hi DCs and pDCs. CD1c+ DCs produced interleukin (IL)-6, tumor necrosis factor α, and IL-1β but not IL-12p70, a cytokine important for Th1 activation and host defenses against Mtb. Yet, CD1c+ DCs were able to activate autologous naïve CD4+ T cells. By combining cell purification with fluorescence-activated cell sorting and gene expression profiling on rare cell populations, we detected in responding CD4+ T cells, genes related to effector-cytolytic functions and transcription factors associated with Th1, Th17, and Treg polarization, suggesting multifunctional properties in our experimental conditions. Finally, immunohistologic analyses revealed contact between CD11c+ cells and pDCs in LNs of TB patients and in vitro data suggest that cooperation between Mtb-infected CD1c+ DCs and pDCs favors stimulation of CD4+ T cells. PMID:25071784

  4. Signals required for programming effector and memory development by CD8+ T cells.

    PubMed

    Mescher, Matthew F; Curtsinger, Julie M; Agarwal, Pujya; Casey, Kerry A; Gerner, Michael; Hammerbeck, Christopher D; Popescu, Flavia; Xiao, Zhengguo

    2006-06-01

    Stimulation of naïve CD8+ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin-12 (IL-12) or interferon-alpha (IFN-alpha). CD4+ T cells condition dendritic cells (DCs) to effectively present antigen to CD8+ T cells, and this conditioning involves, at least in part, CD40-dependent upregulation of the production of these signal 3 cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation-induced non-responsiveness (AINR), a form of split anergy characterized by an inability to produce IL-2 to support continued expansion. If antigen remains present, IL-2 provided by CD4+ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL-2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8+ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations. PMID:16824119

  5. Programmed cell cycle arrest is required for infection of corn plants by the fungus Ustilago maydis.

    PubMed

    Castanheira, Sónia; Mielnichuk, Natalia; Pérez-Martín, José

    2014-12-01

    Ustilago maydis is a plant pathogen that requires a specific structure called infective filament to penetrate the plant tissue. Although able to grow, this filament is cell cycle arrested on the plant surface. This cell cycle arrest is released once the filament penetrates the plant tissue. The reasons and mechanisms for this cell cycle arrest are unknown. Here, we have tried to address these questions. We reached three conclusions from our studies. First, the observed cell cycle arrest is the result of the cooperation of at least two distinct mechanisms: one involving the activation of the DNA damage response (DDR) cascade; and the other relying on the transcriptional downregulation of Hsl1, a kinase that modulates the G2/M transition. Second, a sustained cell cycle arrest during the infective filament step is necessary for the virulence in U. maydis, as a strain unable to arrest the cell cycle was severely impaired in its ability to infect corn plants. Third, production of the appressorium, a structure required for plant penetration, is incompatible with an active cell cycle. The inability to infect plants by strains defective in cell cycle arrest seems to be caused by their failure to induce the appressorium formation process. In summary, our findings uncover genetic circuits to arrest the cell cycle during the growth of this fungus on the plant surface, thus allowing the penetration into plant tissue. PMID:25411209

  6. FoxO1-mediated autophagy is required for NK cell development and innate immunity.

    PubMed

    Wang, Shuo; Xia, Pengyan; Huang, Guanling; Zhu, Pingping; Liu, Jing; Ye, Buqing; Du, Ying; Fan, Zusen

    2016-01-01

    Natural killer (NK) cells exert a crucial role in early immune responses as a major innate effector component. However, the underlying mechanisms of NK cell development remain largely elusive. Here we show that robust autophagy appears in the stage of immature NK cells (iNKs), which is required for NK cell development. Autophagy defects result in damaged mitochondria and accumulation of reactive oxygen species (ROS) that leads to apoptosis of NK cells. Autophagy protects NK cell viability during development through removal of damaged mitochondria and intracellular ROS. Phosphorylated Forkhead box O (FoxO)1 is located to the cytoplasm of iNKs and interacts with Atg7, leading to induction of autophagy. FoxO1 deficiency or an inactive FoxO1(AAA) mutant abrogates autophagy initiation in iNKs and impairs NK cell development and viral clearance. Therefore we conclude that FoxO1-mediated autophagy is required for NK cell development and NK cell-induced innate immunity. PMID:27010363

  7. FoxO1-mediated autophagy is required for NK cell development and innate immunity

    PubMed Central

    Wang, Shuo; Xia, Pengyan; Huang, Guanling; Zhu, Pingping; Liu, Jing; Ye, Buqing; Du, Ying; Fan, Zusen

    2016-01-01

    Natural killer (NK) cells exert a crucial role in early immune responses as a major innate effector component. However, the underlying mechanisms of NK cell development remain largely elusive. Here we show that robust autophagy appears in the stage of immature NK cells (iNKs), which is required for NK cell development. Autophagy defects result in damaged mitochondria and accumulation of reactive oxygen species (ROS) that leads to apoptosis of NK cells. Autophagy protects NK cell viability during development through removal of damaged mitochondria and intracellular ROS. Phosphorylated Forkhead box O (FoxO)1 is located to the cytoplasm of iNKs and interacts with Atg7, leading to induction of autophagy. FoxO1 deficiency or an inactive FoxO1AAA mutant abrogates autophagy initiation in iNKs and impairs NK cell development and viral clearance. Therefore we conclude that FoxO1-mediated autophagy is required for NK cell development and NK cell-induced innate immunity. PMID:27010363

  8. Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells

    SciTech Connect

    Zhan, Yu; Abi Saab, Widian F.; Modi, Nidhi; Stewart, Amanda M.; Liu, Jinsong; Chadee, Deborah N.

    2012-08-15

    Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells. -- Highlights: Black-Right-Pointing-Pointer Ovarian cancer cell lines have high levels of total and phosphorylated MLK3. Black-Right-Pointing-Pointer MLK3 is required for MMP expression and activity in ovarian cancer cells. Black-Right-Pointing-Pointer MLK3 is required for invasion of SKOV3 and HEY1B ovarian cancer cells. Black-Right-Pointing-Pointer MLK3-dependent regulation of MMP-2 and MMP-9 activities requires ERK and JNK.

  9. Efficient T-cell priming and activation requires signaling through prostaglandin E2 (EP) receptors.

    PubMed

    Sreeramkumar, Vinatha; Hons, Miroslav; Punzón, Carmen; Stein, Jens V; Sancho, David; Fresno, Manuel; Cuesta, Natalia

    2016-01-01

    Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings. PMID:26051593

  10. Tolerance induction in memory CD4 T cells requires two rounds of antigen-specific activation.

    PubMed

    David, Alexandria; Crawford, Frances; Garside, Paul; Kappler, John W; Marrack, Philippa; MacLeod, Megan

    2014-05-27

    A major goal for immunotherapy is to tolerize the immune cells that coordinate tissue damage in autoimmune and alloantigen responses. CD4 T cells play a central role in many of these conditions and improved antigen-specific regulation or removal of these cells could revolutionize current treatments. A confounding factor is that little is known about whether and how tolerance is induced in memory CD4 T cells. We used MHC class II tetramers to track and analyze a population of endogenous antigen-specific memory CD4 T cells exposed to soluble peptide in the absence of adjuvant. We found that such memory T cells proliferated and reentered the memory pool apparently unperturbed by the incomplete activation signals provided by the peptide. Upon further restimulation in vivo, CD4 memory T cells that had been previously exposed to peptide proliferated, provided help to primary responding B cells, and migrated to inflamed sites. However, these reactivated memory cells failed to survive. The reduction in T-cell number was marked by low expression of the antiapoptotic molecule B cell lymphoma 2 (Bcl2) and increased expression of activated caspase molecules. Consequently, these cells failed to sustain a delayed-type hypersensitivity response. Moreover, following two separate exposures to soluble antigen, no T-cell recall response and no helper activity for B cells could be detected. These results suggest that the induction of tolerance in memory CD4 T cells is possible but that deletion and permanent removal of the antigen-specific T cells requires reactivation following exposure to the tolerogenic antigen. PMID:24821788

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

    PubMed

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

    2015-10-01

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

  12. Smad4 is required to regulate the fate of cranial neural crest cells

    PubMed Central

    Ko, Seung O; Chung, Il Hyuk; Xu, Xun; Oka, Shoji; Zhao, Hu; Cho, Eui Sic; Deng, Chuxia; Chai, Yang

    2009-01-01

    Smad4 is the central mediator for TGF-β/BMP signals, which are involved in regulating cranial neural crest (CNC) cell formation, migration, proliferation and fate determination. It is unclear whether TGF-β/BMP signals utilize Smad-dependent or –independent pathways to control the development of CNC cells. To investigate the functional significance of Smad4 in regulating CNC cells, we generated mice with neural crest specific inactivation of the Smad4 gene. Our study shows that Smad4 is not required for the migration of CNC cells, but is required in neural crest cells for the development of the cardiac outflow tract. Smad4 is essential in mediating BMP signaling in the CNC-derived ectomesenchyme during early stages of tooth development because conditional inactivation of Smad4 in neural crest derived cells results in incisor and molar development arrested at the dental lamina stage. Furthermore, Smad-mediated TGF-β/BMP signaling controls the homeobox gene patterning of oral/aboral and proximal/distal domains within the first branchial arch. At the cellular level, a Smad4-mediated downstream target gene(s) is required for the survival of CNC cells in the proximal domain of the first branchial arch. Smad4 mutant mice show underdevelopment of the first branchial arch and midline fusion defects. Taken together, our data show that TGF-β/BMP signals rely on Smad-dependent pathways in the ectomesenchyme to mediate epithelial-mesenchymal interactions that control craniofacial organogenesis. PMID:17964566

  13. Interleukin-6 is required for pristane-induced plasma cell hyperplasia in mice.

    PubMed

    Dedera, D A; Urashima, M; Chauhan, D; LeBrun, D P; Bronson, R T; Anderson, K C

    1996-07-01

    Intraperitoneal injection of pristane induces production of interleukin-6 (IL-6) and either plasmacytosis or plasmacytoma in mice, depending upon the genetic background. Pristane does not induce plasmacytoma in IL-6 knockout (IL-6-/-) mice, suggesting that IL-6 is required for this process. In the present study we determined whether IL-6 is also required for pristane-induced hyperplasia of normal plasma cells. Pristane was injected intraperitoneally into IL-6-/- and IL-6 wild-type (IL-6+/+) mice. Overall there were more deaths in IL-6+/+ mice (85%) than in IL-6-/- mice (40%), P = 0.024. Hyperplastic lymph node and spleen weight did not differ (P = 0.82 and P = 0.15, respectively) in IL-6-/- versus IL-6+/+ mice. Lymphocytosis with similar patterns of expression of B-cell (B220) and T-cell (Thy-1) antigens was noted in both IL-6-/- and IL-6+/+ mice. However, morphological studies, dual fluorescent staining for Syn-1 and B220 antigens (syn-1+ B220+ cells), and intracytoplasmic Ig staining revealed plasma cell hyperplasia in lymph node and spleen from IL-6+/+, but not IL-6-/-, mice. These plasma cells from IL-6+/+ mice were polyclonal and unable to induce tumour formation in severe combined immunodeficient mice. These data demonstrate that IL-6 is required for pristane-induced hyperplasia of polyclonal plasma cells in mice. PMID:8757508

  14. JNK Controls the Onset of Mitosis in Planarian Stem Cells and Triggers Apoptotic Cell Death Required for Regeneration and Remodeling

    PubMed Central

    Almuedo-Castillo, María; Crespo, Xenia; Seebeck, Florian; Bartscherer, Kerstin; Salò, Emili; Adell, Teresa

    2014-01-01

    Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun–NH2–kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal. PMID:24922054

  15. Androgen Receptor Coactivator ARID4B Is Required for the Function of Sertoli Cells in Spermatogenesis.

    PubMed

    Wu, Ray-Chang; Zeng, Yang; Pan, I-Wen; Wu, Mei-Yi

    2015-09-01

    Defects in spermatogenesis, a process that produces spermatozoa inside seminiferous tubules of the testis, result in male infertility. Spermatogenic progression is highly dependent on a microenvironment provided by Sertoli cells, the only somatic cells and epithelium of seminiferous tubules. However, genes that regulate such an important activity of Sertoli cells are poorly understood. Here, we found that AT-rich interactive domain 4B (ARID4B), is essential for the function of Sertoli cells to regulate spermatogenesis. Specifically, we generated Sertoli cell-specific Arid4b knockout (Arid4bSCKO) mice, and showed that the Arid4bSCKO male mice were completely infertile with impaired testis development and significantly reduced testis size. Importantly, severe structural defects accompanied by loss of germ cells and Sertoli cell-only phenotype were found in many seminiferous tubules of the Arid4bSCKO testes. In addition, maturation of Sertoli cells was significantly delayed in the Arid4bSCKO mice, associated with delayed onset of spermatogenesis. Spermatogenic progression was also defective, showing an arrest at the round spermatid stage in the Arid4bSCKO testes. Interestingly, we showed that ARID4B functions as a "coactivator" of androgen receptor and is required for optimal transcriptional activation of reproductive homeobox 5, an androgen receptor target gene specifically expressed in Sertoli cells and critical for spermatogenesis. Together, our study identified ARID4B to be a key regulator of Sertoli cell function important for male germ cell development. PMID:26258622

  16. The Zebrafish G12 Gene is required for Nuclear Positioning and Cell Migrations during Early Development

    NASA Technical Reports Server (NTRS)

    Reinsch, S. S.; Conway, G. C.

    2003-01-01

    After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.

  17. p73 is required for ependymal cell maturation and neurogenic SVZ cytoarchitecture.

    PubMed

    Gonzalez-Cano, L; Fuertes-Alvarez, S; Robledinos-Anton, N; Bizy, A; Villena-Cortes, A; Fariñas, I; Marques, M M; Marin, Maria C

    2016-07-01

    The adult subventricular zone (SVZ) is a highly organized microenvironment established during the first postnatal days when radial glia cells begin to transform into type B-cells and ependymal cells, all of which will form regenerative units, pinwheels, along the lateral wall of the lateral ventricle. Here, we identify p73, a p53 homologue, as a critical factor controlling both cell-type specification and structural organization of the developing mouse SVZ. We describe that p73 deficiency halts the transition of the radial glia into ependymal cells, leading to the emergence of immature cells with abnormal identities in the ventricle and resulting in loss of the ventricular integrity. p73-deficient ependymal cells have noticeably impaired ciliogenesis and they fail to organize into pinwheels, disrupting SVZ niche structure and function. Therefore, p73 is essential for appropriate ependymal cell maturation and the establishment of the neurogenic niche architecture. Accordingly, lack of p73 results in impaired neurogenesis. Moreover, p73 is required for translational planar cell polarity establishment, since p73 deficiency results in profound defects in cilia organization in individual cells and in intercellular patch orientation. Thus, our data reveal a completely new function of p73, independent of p53, in the neurogenic architecture of the SVZ of rodent brain and in the establishment of ependymal planar cell polarity with important implications in neurogenesis. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 730-747, 2016. PMID:26482843

  18. Metabolic requirements for the maintenance of self-renewing stem cells

    PubMed Central

    Ito, Keisuke; Suda, Toshio

    2014-01-01

    A distinctive feature of stem cells is their capacity to self-renew to maintain pluripotency. Studies of genetically-engineered mouse models and recent advances in metabolomic analysis, particularly in haematopoietic stem cells, have deepened our understanding of the contribution made by metabolic cues to the regulation of stem cell self-renewal. Many types of stem cells heavily rely on anaerobic glycolysis, and stem cell function is also regulated by bioenergetic signalling, the AKT–mTOR pathway, Gln metabolism and fatty acid metabolism. As maintenance of a stem cell pool requires a finely-tuned balance between self-renewal and differentiation, investigations into the molecular mechanisms and metabolic pathways underlying these decisions hold great therapeutic promise. PMID:24651542

  19. Metabolic requirements for the maintenance of self-renewing stem cells.

    PubMed

    Ito, Keisuke; Suda, Toshio

    2014-04-01

    A distinctive feature of stem cells is their capacity to self-renew to maintain pluripotency. Studies of genetically-engineered mouse models and recent advances in metabolomic analysis, particularly in haematopoietic stem cells, have deepened our understanding of the contribution made by metabolic cues to the regulation of stem cell self-renewal. Many types of stem cells heavily rely on anaerobic glycolysis, and stem cell function is also regulated by bioenergetic signalling, the AKT-mTOR pathway, Gln metabolism and fatty acid metabolism. As maintenance of a stem cell pool requires a finely-tuned balance between self-renewal and differentiation, investigations into the molecular mechanisms and metabolic pathways underlying these decisions hold great therapeutic promise. PMID:24651542

  20. Collective cell migration requires vesicular trafficking for chemoattractant delivery at the trailing edge

    PubMed Central

    Kriebel, Paul W.; Barr, Valarie A.; Rericha, Erin C.; Zhang, Guofeng; Parent, Carole A.

    2008-01-01

    Chemoattractant signaling induces the polarization and directed movement of cells secondary to the activation of multiple effector pathways. In addition, chemotactic signals can be amplified and relayed to proximal cells via the synthesis and secretion of additional chemoattractant. The mechanisms underlying such remarkable features remain ill defined. We show that the asymmetrical distribution of adenylyl cyclase (ACA) at the back of Dictyostelium discoideum cells, an essential determinant of their ability to migrate in a head-to-tail fashion, requires vesicular trafficking. This trafficking results in a local accumulation of ACA-containing intracellular vesicles and involves intact actin, microtubule networks, and de novo protein synthesis. We also show that migrating cells leave behind ACA-containing vesicles, likely secreted as multivesicular bodies and presumably involved in the formation of head-to-tail arrays of migrating cells. We propose that similar compartmentalization and shedding mechanisms exist in mammalian cells during embryogenesis, wound healing, neuron growth, and metastasis. PMID:19047467

  1. Ligand-independent requirements of steroid receptors EcR and USP for cell survival.

    PubMed

    Mansilla, A; Martín, F A; Martín, D; Ferrús, A

    2016-03-01

    The active form of the Drosophila steroid hormone ecdysone, 20-hydroxyecdysone (20E), binds the heterodimer EcR/USP nuclear receptor to regulate target genes that elicit proliferation, cell death and differentiation during insect development. Although the 20E effects are relatively well known, the physiological relevance of its receptors remains poorly understood. We show here that the prothoracic gland (PG), the major steroid-producing organ of insect larvae, requires EcR and USP to survive in a critical period previous to metamorphosis, and that this requirement is 20E-independent. The cell death induced by the downregulation of these receptors involves the activation of the JNK-encoding basket gene and it can be rescued by upregulating EcR isoforms which are unable to respond to 20E. Also, while PG cell death prevents ecdysone production, blocking hormone synthesis or secretion in normal PG does not lead to cell death, demonstrating further the ecdysone-independent nature of the receptor-deprivation cell death. In contrast to PG cells, wing disc or salivary glands cells do not require these receptors for survival, revealing their cell and developmental time specificity. Exploring the potential use of this feature of steroid receptors in cancer, we assayed tumor overgrowth induced by altered yorkie signaling. This overgrowth is suppressed by EcR downregulation in PG, but not in wing disc, cells. The mechanism of all these cell death features is based on the transcriptional regulation of reaper. These novel and context-dependent functional properties for EcR and USP receptors may help to understand the heterogeneous responses to steroid-based therapies in human pathologies. PMID:26250909

  2. The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila melanogaster.

    PubMed

    Bandura, Jennifer L; Jiang, Huaqi; Nickerson, Derek W; Edgar, Bruce A

    2013-01-01

    The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. However, the full complement of mechanisms necessary to restrain E2F/DP and Cyclin/Cdk activities in differentiating cells are not known. Here, we have performed a genetic screen in Drosophila melanogaster, designed to identify genes required for cell cycle exit. This screen utilized a PCNA-miniwhite(+) reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, we propose that Cdh1/Fzr is a client protein of Hsp83. Our results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. PMID:24086162

  3. Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

    PubMed Central

    Mouneimne, Ghassan; Soon, Lilian; DesMarais, Vera; Sidani, Mazen; Song, Xiaoyan; Yip, Shu-Chin; Ghosh, Mousumi; Eddy, Robert; Backer, Jonathan M.; Condeelis, John

    2004-01-01

    The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF. PMID:15337778

  4. Tim-1-Mediated T Cell Activation Requires Recruitment and Activation of PI 3-Kinase

    PubMed Central

    de Souza, Anjali J.; Oak, Jean S.; Jordanhazy, Ryan; DeKruyff, Rosemarie H.; Fruman, David A.; Kane, Lawrence P.

    2009-01-01

    Ligation of the transmembrane protein Tim-1 can co-stimulate T cell activation. Agonistic antibodies to Tim-1 are also capable of inducing T cell activation without additional stimuli. However, little is known about the biochemical mechanisms underlying T cell stimulation or co-stimulation through Tim-1. We show that a tyrosine in Tim-1 becomes phosphorylated in an lck-dependent manner, whereupon it can directly recruit p85 adaptor subunits of PI 3-kinase. This results in PI3K activation, which is required for Tim-1 function. We also provide genetic evidence that p85 expression is required for optimal Tim-1 function. Thus, we describe a pathway from Tim-1 tyrosine phosphorylation to the PI3K signaling pathway, which appears to be a major effector of Tim-1-mediated T cell activation. PMID:18453570

  5. Drosophila PI4KIIIalpha is required in follicle cells for oocyte polarization and Hippo signaling

    PubMed Central

    Yan, Yan; Denef, Natalie; Tang, Charm; Schüpbach, Trudi

    2011-01-01

    In a genetic screen we isolated mutations in CG10260, which encodes a phosphatidylinositol 4-kinase (PI4KIIIalpha), and found that PI4KIIIalpha is required for Hippo signaling in Drosophila ovarian follicle cells. PI4KIIIalpha mutations in the posterior follicle cells lead to oocyte polarization defects similar to those caused by mutations in the Hippo signaling pathway. PI4KIIIalpha mutations also cause misexpression of well-established Hippo signaling targets. The Merlin-Expanded-Kibra complex is required at the apical membrane for Hippo activity. In PI4KIIIalpha mutant follicle cells, Merlin fails to localize to the apical domain. Our analysis of PI4KIIIalpha mutants provides a new link in Hippo signal transduction from the cell membrane to its core kinase cascade. PMID:21429988

  6. ATMIN is required for maintenance of genomic stability and suppression of B cell lymphoma.

    PubMed

    Loizou, Joanna I; Sancho, Rocio; Kanu, Nnennaya; Bolland, Daniel J; Yang, Fengtang; Rada, Cristina; Corcoran, Anne E; Behrens, Axel

    2011-05-17

    Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMIN(ΔB/ΔB)) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMIN(ΔB/ΔB) cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells. PMID:21575860

  7. Ezrin and Moesin Are Required for Efficient T Cell Adhesion and Homing to Lymphoid Organs

    PubMed Central

    Chen, Emily J. H.; Shaffer, Meredith H.; Williamson, Edward K.; Huang, Yanping; Burkhardt, Janis K.

    2013-01-01

    T cell trafficking between the blood and lymphoid organs is a complex, multistep process that requires several highly dynamic and coordinated changes in cyto-architecture. Members of the ezrin, radixin and moesin (ERM) family of actin-binding proteins have been implicated in several aspects of this process, but studies have yielded conflicting results. Using mice with a conditional deletion of ezrin in CD4+ cells and moesin-specific siRNA, we generated T cells lacking ERM proteins, and investigated the effect on specific events required for T cell trafficking. ERM-deficient T cells migrated normally in multiple in vitro and in vivo assays, and could undergo efficient diapedesis in vitro. However, these cells were impaired in their ability to adhere to the β1 integrin ligand fibronectin, and to polarize appropriately in response to fibronectin and VCAM-1 binding. This defect was specific for β1 integrins, as adhesion and polarization in response to ICAM-1 were normal. In vivo, ERM-deficient T cells showed defects in homing to lymphoid organs. Taken together, these results show that ERM proteins are largely dispensable for T cell chemotaxis, but are important for β1 integrin function and homing to lymphoid organs. PMID:23468835

  8. FAK is required for tension-dependent organization of collective cell movements in Xenopus mesendoderm

    PubMed Central

    Bjerke, Maureen A.; Dzamba, Bette; Wang, Chong; DeSimone, Douglas W.

    2014-01-01

    Collective cell movements are integral to biological processes such as embryonic development and wound healing and also have a prominent role in some metastatic cancers. In migrating Xenopus mesendoderm, traction forces are generated by cells through integrin-based adhesions and tension transmitted across cadherin adhesions. This is accompanied by assembly of a mechanoresponsive cadherin adhesion complex containing keratin intermediate filaments and the catenin-family member plakoglobin. We demonstrate that focal adhesion kinase (FAK), a major component of integrin adhesion complexes, is required for normal morphogenesis at gastrulation, closure of the anterior neural tube, axial elongation and somitogenesis. Depletion of zygotically expressed FAK results in disruption of mesendoderm tissue polarity similar to that observed when expression of keratin or plakoglobin is inhibited. Both individual and collective migrations of mesendoderm cells from FAK depleted embryos are slowed, cell protrusions are disordered, and cell spreading and traction forces are decreased. Additionally, keratin filaments fail to organize at the rear of cells in the tissue and association of plakoglobin with cadherin is diminished. These findings suggest that FAK is required for the tension-dependent assembly of the cadherin adhesion complex that guides collective mesendoderm migration, perhaps by modulating the dynamic balance of substrate traction forces and cell cohesion needed to establish cell polarity. PMID:25127991

  9. The SWI/SNF ATPases Are Required for Triple Negative Breast Cancer Cell Proliferation.

    PubMed

    Wu, Qiong; Madany, Pasil; Akech, Jacqueline; Dobson, Jason R; Douthwright, Stephen; Browne, Gillian; Colby, Jennifer L; Winter, Georg E; Bradner, James E; Pratap, Jitesh; Sluder, Greenfield; Bhargava, Rohit; Chiosea, Simion I; van Wijnen, Andre J; Stein, Janet L; Stein, Gary S; Lian, Jane B; Nickerson, Jeffrey A; Imbalzano, Anthony N

    2015-11-01

    The Brahma (BRM) and Brahma-related Gene 1 (BRG1) ATPases are highly conserved homologs that catalyze the chromatin remodeling functions of the multi-subunit human SWI/SNF chromatin remodeling enzymes in a mutually exclusive manner. SWI/SNF enzyme subunits are mutated or missing in many cancer types, but are overexpressed without apparent mutation in other cancers. Here, we report that both BRG1 and BRM are overexpressed in most primary breast cancers independent of the tumor's receptor status. Knockdown of either ATPase in a triple negative breast cancer cell line reduced tumor formation in vivo and cell proliferation in vitro. Fewer cells in S phase and an extended cell cycle progression time were observed without any indication of apoptosis, senescence, or alterations in migration or attachment properties. Combined knockdown of BRM and BRG1 showed additive effects in the reduction of cell proliferation and time required for completion of cell cycle, suggesting that these enzymes promote cell cycle progression through independent mechanisms. Knockout of BRG1 or BRM using CRISPR/Cas9 technology resulted in the loss of viability, consistent with a requirement for both enzymes in triple negative breast cancer cells. PMID:25808524

  10. Drosophila male and female germline stem cell niches require the nuclear lamina protein Otefin.

    PubMed

    Barton, Lacy J; Lovander, Kaylee E; Pinto, Belinda S; Geyer, Pamela K

    2016-07-01

    The nuclear lamina is an extensive protein network that underlies the inner nuclear envelope. This network includes the LAP2-emerin-MAN1-domain (LEM-D) protein family, proteins that share an association with the chromatin binding protein Barrier-to-autointegration factor (BAF). Loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Mechanisms associated with laminopathies are not yet understood. Here we present our studies of one of the Drosophila nuclear lamina LEM-D proteins, Otefin (Ote), a homologue of emerin. Previous studies have shown that Ote is autonomously required for the survival of female germline stem cells (GSCs). We demonstrate that Ote is also required for survival of somatic cells in the ovarian niche, with loss of Ote causing a decrease in cap cell number and altered signal transduction. We show germ cell-restricted expression of Ote rescues these defects, revealing a non-autonomous function for Ote in niche maintenance and emphasizing that GSCs contribute to the maintenance of their own niches. Further, we investigate the requirement of Ote in the male fertility. We show that ote mutant males become prematurely sterile as they age. Parallel to observations in females, this sterility is associated with GSC loss and changes in somatic cells of the niche, phenotypes that are largely rescued by germ cell-restricted Ote expression. Taken together, our studies demonstrate that Ote is required autonomously for survival of two stem cell populations, as well as non-autonomously for maintenance of two somatic niches. Finally, our data add to growing evidence that LEM-D proteins have critical roles in stem cell survival and tissue homeostasis. PMID:27174470

  11. Cell-to-cell transmission of HIV-1 is required to trigger pyroptotic death of lymphoid tissue-derived CD4 T cells

    PubMed Central

    Monroe, Kathryn M.; Yang, Zhiyuan; Muñoz-Arias, Isa; Levy, David N; Greene, Warner C.

    2015-01-01

    The progressive depletion of CD4 T cells underlies clinical progression to AIDS in untreated HIV-infected subjects. Most dying CD4 T cells correspond to resting nonpermissive cells residing in lymphoid tissues. Death is due to an innate immune response against the incomplete cytosolic viral DNA intermediates accumulating in these cells. The viral DNA is detected by the IFI16 sensor leading to inflammasome assembly, caspase 1 activation, and the induction of pyroptosis, a highly inflammatory form of programmed cell death. We now show that cell-to-cell transmission of HIV is obligatorily required for activation of this death pathway. Cell-free HIV-1 virions, even when added in large quantities, fail to activate pyroptosis. These findings underscore the infected CD4 T cells as the major killing units promoting progression to AIDS and highlight a previously unappreciated role for the virological synapse in HIV pathogenesis. PMID:26321639

  12. GRP78 is required for cell proliferation and protection from apoptosis in chicken embryo fibroblast cells.

    PubMed

    Jeon, M; Choi, H; Lee, S I; Kim, J S; Park, M; Kim, K; Lee, S; Byun, S J

    2016-05-01

    Chicken serum has been suggested as a supplement to promote chicken cell proliferation and development. However, the molecular mechanisms by which chicken serum stimulates chicken cell proliferation remain unknown. Here, we evaluated the effects of chicken serum supplementation on chicken embryo fibroblast (CEF) and DF-1 cell proliferation. We also sought to elucidate the molecular pathways involved in mediating the effects of chicken serum on fibroblasts and DF-1 cells by overexpression of chicken 78 kDa glucose-regulated protein (chGRP78), which is important for cell growth and the prevention of apoptosis. Our data demonstrated that the addition of 5% chicken serum significantly enhanced fibroblast proliferation. Moreover, knockdown of chGRP78 using siRNA decreased fibroblast proliferation and increased apoptosis. Based on these results, we suggest that the chGRP78-mediated signaling pathway plays a critical role in chicken serum-stimulated fibroblast survival and anti-apoptosis. Therefore, our findings have important implications for the maintenance of chicken fibroblast cells through the inhibition of apoptosis and may lead to the development of new treatments for avian disease. PMID:26944959

  13. In vivo collective cell migration requires an LPAR2-dependent increase in tissue fluidity

    PubMed Central

    Kuriyama, Sei; Theveneau, Eric; Benedetto, Alexandre; Parsons, Maddy; Tanaka, Masamitsu; Charras, Guillaume; Kabla, Alexandre

    2014-01-01

    Collective cell migration (CCM) and epithelial–mesenchymal transition (EMT) are common to cancer and morphogenesis, and are often considered to be mutually exclusive in spite of the fact that many cancer and embryonic cells that have gone through EMT still cooperate to migrate collectively. Here we use neural crest (NC) cells to address the question of how cells that have down-regulated cell–cell adhesions can migrate collectively. NC cell dissociation relies on a qualitative and quantitative change of the cadherin repertoire. We found that the level of cell–cell adhesion is precisely regulated by internalization of N-cadherin downstream of lysophosphatidic acid (LPA) receptor 2. Rather than promoting the generation of single, fully mesenchymal cells, this reduction of membrane N-cadherin only triggers a partial mesenchymal phenotype. This intermediate phenotype is characterized by an increase in tissue fluidity akin to a solid-like–to–fluid-like transition. This change of plasticity allows cells to migrate under physical constraints without abolishing cell cooperation required for collectiveness. PMID:25002680

  14. Unexpected requirement for ELMO1 in clearance of apoptotic germ cells in vivo.

    PubMed

    Elliott, Michael R; Zheng, Shuqiu; Park, Daeho; Woodson, Robin I; Reardon, Michael A; Juncadella, Ignacio J; Kinchen, Jason M; Zhang, Jun; Lysiak, Jeffrey J; Ravichandran, Kodi S

    2010-09-16

    Apoptosis and the subsequent clearance of dying cells occurs throughout development and adult life in many tissues. Failure to promptly clear apoptotic cells has been linked to many diseases. ELMO1 is an evolutionarily conserved cytoplasmic engulfment protein that functions downstream of the phosphatidylserine receptor BAI1, and, along with DOCK1 and the GTPase RAC1, promotes internalization of the dying cells. Here we report the generation of ELMO1-deficient mice, which we found to be unexpectedly viable and grossly normal. However, they had a striking testicular pathology, with disrupted seminiferous epithelium, multinucleated giant cells, uncleared apoptotic germ cells and decreased sperm output. Subsequent in vitro and in vivo analyses revealed a crucial role for ELMO1 in the phagocytic clearance of apoptotic germ cells by Sertoli cells lining the seminiferous epithelium. The engulfment receptor BAI1 and RAC1 (upstream and downstream of ELMO1, respectively) were also important for Sertoli-cell-mediated engulfment. Collectively, these findings uncover a selective requirement for ELMO1 in Sertoli-cell-mediated removal of apoptotic germ cells and make a compelling case for a relationship between engulfment and tissue homeostasis in vivo. PMID:20844538

  15. HDAC6 activity is not required for basal autophagic flux in metastatic prostate cancer cells.

    PubMed

    Watson, Gregory W; Wickramasekara, Samanthi; Fang, Yufeng; Maier, Claudia S; Williams, David E; Dashwood, Roderick H; Perez, Viviana I; Ho, Emily

    2016-06-01

    Histone deacetylase 6 is a multifunctional lysine deacetylase that is recently emerging as a central facilitator of response to stress and may play an important role in cancer cell proliferation. The histone deacetylase 6-inhibitor tubacin has been shown to slow the growth of metastatic prostate cancer cells and sensitize cancer cells to chemotherapeutic agents. However, the proteins histone deacetylase 6 interacts with, and thus its role in cancer cells, remains poorly characterized. Histone deacetylase 6 deacetylase activity has recently been shown to be required for efficient basal autophagic flux. Autophagy is often dysregulated in cancer cells and may confer stress resistance and allow for cell maintenance and a high proliferation rate. Tubacin may therefore slow cancer cell proliferation by decreasing autophagic flux. We characterized the histone deacetylase 6-interacting proteins in LNCaP metastatic prostate cancer cells and found that histone deacetylase 6 interacts with proteins involved in several cellular processes, including autophagy. Based on our interaction screen, we assessed the impact of the histone deacetylase 6-inhibitor tubacin on autophagic flux in two metastatic prostate cancer cell lines and found that tubacin does not influence autophagic flux. Histone deacetylase 6 therefore influences cell proliferation through an autophagy-independent mechanism. PMID:26643866

  16. Spen is required for pigment cell survival during pupal development in Drosophila.

    PubMed

    Querenet, Matthieu; Goubard, Valerie; Chatelain, Gilles; Davoust, Nathalie; Mollereau, Bertrand

    2015-06-15

    Apoptosis is required during development to eliminate superfluous cells and sculpt tissues; spatial and timed control of apoptosis ensures that the necessary number of cells is eliminated at a precise time in a given tissue. The elimination of supernumerary pigment or inter-ommatidial cells (IOCs) depends on cell-cell communication and is necessary for the formation of the honeycomb-like structure of the Drosophila eye. However, the mechanisms occurring during pupal development and controlling apoptosis of superfluous IOC in space and time remain unclear. Here, we found that split-ends (spen) is required for IOC survival at the time of removal of superfluous IOCs. Loss of spen function leads to abnormal removal of IOCs by apoptosis. We show that spen is required non-autonomously in cone cells for the survival of IOCs by positively regulating the Spitz/EGFR pathway. We propose that Spen is an important survival factor that ensures spatial control of the apoptotic wave that is necessary for the correct patterning and formation of the Drosophila eye. PMID:25872184

  17. Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

    PubMed

    Curto, M-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M-Henar

    2014-04-01

    The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  18. The HLH protein Extramacrochaetae is required for R7 cell and cone cell fates in the Drosophila eye

    PubMed Central

    Bhattacharya, Abhishek; Baker, Nicholas E.

    2010-01-01

    Summary Notch signaling is one of the most important pathways in development and homeostasis, and is altered in multiple pathologies. Study of Drosophila eye development shows that Notch signaling depends on the HLH protein Extramacrochaetae. Null mutant clones show that extramacrochaetae is required for multiple aspects of eye development that depend on Notch signaling, including morphogenetic furrow progression, differentiation of R4, R7 and cone cell types, and rotation of ommatidial clusters. Detailed analysis of R7 and cone cell specification reveals that extramacrochaetae acts cell autonomously and epistatically to Notch, and is required for normal expression of bHLH genes encoded by the E(spl)-C which are effectors of most Notch signaling. A model is proposed in which Extramacrochaetae acts in parallel to or as a feed-forward regulator of the E(spl)-Complex to promote Notch signaling in particular cellular contexts. PMID:19118542

  19. Murine Gammaherpesvirus 68 Reactivation from B Cells Requires IRF4 but Not XBP-1

    PubMed Central

    Matar, Caline G.; Rangaswamy, Udaya Shankari; Wakeman, Brian S.; Iwakoshi, Neal

    2014-01-01

    ABSTRACT Gammaherpesviruses display tropism for B cells and, like all known herpesviruses, exhibit distinct lytic and latent life cycles. One well-established observation among members of the gammaherpesvirus family is the link between viral reactivation from latently infected B cells and plasma cell differentiation. Importantly, a number of studies have identified a potential role for a CREB/ATF family member, X-box binding protein 1 (XBP-1), in trans-activating the immediate early BZLF-1 or BRLF1/gene 50 promoters of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), respectively. XBP-1 is required for the unfolded protein response and has been identified as a critical transcription factor in plasma cells. Here, we demonstrate that XBP-1 is capable of trans-activating the murine gammaherpesvirus 68 (MHV68) RTA promoter in vitro, consistent with previous observations for EBV and KSHV. However, we show that in vivo there does not appear to be a requirement for XBP-1 expression in B cells for virus reactivation. The MHV68 M2 gene product under some experimental conditions plays an important role in virus reactivation from B cells. M2 has been shown to drive B cell differentiation to plasma cells, as well as interleukin-10 (IL-10) production, both of which are dependent on M2 induction of interferon regulatory factor 4 (IRF4) expression. IRF4 is required for plasma cell differentiation, and consistent with a role for plasma cells in MHV68 reactivation from B cells, we show that IRF4 expression in B cells is required for efficient reactivation of MHV68 from splenocytes. Thus, the latter analyses are consistent with previous studies linking plasma cell differentiation to MHV68 reactivation from B cells. The apparent independence of MHV68 reactivation from XBP-1 expression in plasma cells may reflect redundancy among CREB/ATF family members or the involvement of other plasma cell-specific transcription factors. Regardless, these findings

  20. Scribble is required for normal epithelial cell-cell contacts and lumen morphogenesis in the mammalian lung.

    PubMed

    Yates, Laura L; Schnatwinkel, Carsten; Hazelwood, Lee; Chessum, Lauren; Paudyal, Anju; Hilton, Helen; Romero, M Rosario; Wilde, Jonathan; Bogani, Debora; Sanderson, Jeremy; Formstone, Caroline; Murdoch, Jennifer N; Niswander, Lee A; Greenfield, Andy; Dean, Charlotte H

    2013-01-15

    During lung development, proper epithelial cell arrangements are critical for the formation of an arborized network of tubes. Each tube requires a lumen, the diameter of which must be tightly regulated to enable optimal lung function. Lung branching and lumen morphogenesis require close epithelial cell-cell contacts that are maintained as a result of adherens junctions, tight junctions and by intact apical-basal (A/B) polarity. However, the molecular mechanisms that maintain epithelial cohesion and lumen diameter in the mammalian lung are unknown. Here we show that Scribble, a protein implicated in planar cell polarity (PCP) signalling, is necessary for normal lung morphogenesis. Lungs of the Scrib mouse mutant Circletail (Crc) are abnormally shaped with fewer airways, and these airways often lack a visible, 'open' lumen. Mechanistically we show that Scrib genetically interacts with the core PCP gene Vangl2 in the developing lung and that the distribution of PCP pathway proteins and Rho mediated cytoskeletal modification is perturbed in Scrib(Crc/Crc) lungs. However A/B polarity, which is disrupted in Drosophila Scrib mutants, is largely unaffected. Notably, we find that Scrib mediates functions not attributed to other PCP proteins in the lung. Specifically, Scrib localises to both adherens and tight junctions of lung epithelia and knockdown of Scrib in lung explants and organotypic cultures leads to reduced cohesion of lung epithelial cells. Live imaging of Scrib knockdown lungs shows that Scrib does not affect bud bifurcation, as previously shown for the PCP protein Celsr1, but is required to maintain epithelial cohesion. To understand the mechanism leading to reduced cell-cell association, we show that Scrib associates with β-catenin in embryonic lung and the sub-cellular distribution of adherens and tight junction proteins is perturbed in mutant lung epithelia. Our data reveal that Scrib is required for normal lung epithelial organisation and lumen

  1. Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells

    PubMed Central

    Ronquist, Karl Göran; Sanchez, Claire; Dubois, Louise; Chioureas, Dimitris; Fonseca, Pedro; Larsson, Anders; Ullén, Anders; Yachnin, Jeffrey; Ronquist, Gunnar; Panaretakis, Theocharis

    2016-01-01

    Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process. PMID:26955882

  2. Differential requirements of two insect cell lines for growth in serum-free medium.

    PubMed

    Vaughn, J L; Fan, F

    1997-06-01

    The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 x 10(6) TCID50 extracellular virus and 4.4 x 10(6) polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects. PMID:9201517

  3. Mesoderm is required for coordinated cell movements within zebrafish neural plate in vivo

    PubMed Central

    2014-01-01

    Background Morphogenesis of the zebrafish neural tube requires the coordinated movement of many cells in both time and space. A good example of this is the movement of the cells in the zebrafish neural plate as they converge towards the dorsal midline before internalizing to form a neural keel. How these cells are regulated to ensure that they move together as a coherent tissue is unknown. Previous work in other systems has suggested that the underlying mesoderm may play a role in this process but this has not been shown directly in vivo. Results Here we analyze the roles of subjacent mesoderm in the coordination of neural cell movements during convergence of the zebrafish neural plate and neural keel formation. Live imaging demonstrates that the normal highly coordinated movements of neural plate cells are lost in the absence of underlying mesoderm and the movements of internalization and neural tube formation are severely disrupted. Despite this, neuroepithelial polarity develops in the abnormal neural primordium but the resulting tissue architecture is very disorganized. Conclusions We show that the movements of cells in the zebrafish neural plate are highly coordinated during the convergence and internalization movements of neurulation. Our results demonstrate that the underlying mesoderm is required for these coordinated cell movements in the zebrafish neural plate in vivo. PMID:24755297

  4. Bmi1 Is Required for Hepatic Progenitor Cell Expansion and Liver Tumor Development

    PubMed Central

    Wang, Chunmei; Tao, Junyan; Ho, Coral; Jiang, Lijie; Gui, Bing; Huang, Shiang; Evert, Matthias; Calvisi, Diego F.; Chen, Xin

    2012-01-01

    Bmi1 is a polycomb group transcriptional repressor and it has been implicated in regulating self-renewal and proliferation of many types of stem or progenitor cells. In addition, Bmi1 has been shown to function as an oncogene in multiple tumor types. In this study, we investigated the functional significance of Bmi1 in regulating hepatic oval cells, the major type of bipotential progenitor cells in adult liver, as well as the role of Bmi1 during hepatocarcinogenesis using Bmi1 knockout mice. We found that loss of Bmi1 significantly restricted chemically induced oval cell expansion in the mouse liver. Concomitant deletion of Ink4a/Arf in Bmi1 deficient mice completely rescued the oval cell expansion phenotype. Furthermore, ablation of Bmi1 delayed hepatocarcinogenesis induced by AKT and Ras co-expression. This antineoplastic effect was accompanied by the loss of hepatic oval cell marker expression in the liver tumor samples. In summary, our data demonstrated that Bmi1 is required for hepatic oval cell expansion via deregulating the Ink4a/Arf locus in mice. Our study also provides the evidence, for the first time, that Bmi1 expression is required for liver cancer development in vivo, thus representing a promising target for innovative treatments against human liver cancer. PMID:23029524

  5. Heme exporter FLVCR is required for T cell development and peripheral survival.

    PubMed

    Philip, Mary; Funkhouser, Scott A; Chiu, Edison Y; Phelps, Susan R; Delrow, Jeffrey J; Cox, James; Fink, Pamela J; Abkowitz, Janis L

    2015-02-15

    All aerobic cells and organisms must synthesize heme from the amino acid glycine and the tricarboxylic acid cycle intermediate succinyl CoA for incorporation into hemoproteins, such as the cytochromes needed for oxidative phosphorylation. Most studies on heme regulation have been done in erythroid cells or hepatocytes; however, much less is known about heme metabolism in other cell types. The feline leukemia virus subgroup C receptor (FLVCR) is a 12-transmembrane domain surface protein that exports heme from cells, and it was shown to be required for erythroid development. In this article, we show that deletion of Flvcr in murine hematopoietic precursors caused a complete block in αβ T cell development at the CD4(+)CD8(+) double-positive stage, although other lymphoid lineages were not affected. Moreover, FLVCR was required for the proliferation and survival of peripheral CD4(+) and CD8(+) T cells. These studies identify a novel and unexpected role for FLVCR, a major facilitator superfamily metabolite transporter, in T cell development and suggest that heme metabolism is particularly important in the T lineage. PMID:25582857

  6. Chemokine-dependent T cell migration requires aquaporin-3–mediated hydrogen peroxide uptake

    PubMed Central

    Chikuma, Shunsuke; Sugiyama, Yoshinori; Kabashima, Kenji; Verkman, Alan S.; Inoue, Shintaro; Miyachi, Yoshiki

    2012-01-01

    Chemokine-dependent trafficking is indispensable for the effector function of antigen-experienced T cells during immune responses. In this study, we report that the water/glycerol channel aquaporin-3 (AQP3) is expressed on T cells and regulates their trafficking in cutaneous immune reactions. T cell migration toward chemokines is dependent on AQP3-mediated hydrogen peroxide (H2O2) uptake but not the canonical water/glycerol transport. AQP3-mediated H2O2 transport is essential for the activation of the Rho family GTPase Cdc42 and the subsequent actin dynamics. Coincidentally, AQP3-deficient mice are defective in the development of hapten-induced contact hypersensitivity, which is attributed to the impaired trafficking of antigen-primed T cells to the hapten-challenged skin. We therefore suggest that AQP3-mediated H2O2 uptake is required for chemokine-dependent T cell migration in sufficient immune response. PMID:22927550

  7. TCR ITAM multiplicity is required for the generation of follicular helper T-cells

    PubMed Central

    Hwang, SuJin; Palin, Amy C.; Li, LiQi; Song, Ki-Duk; Lee, Jan; Herz, Jasmin; Tubo, Noah; Chu, Hamlet; Pepper, Marion; Lesourne, Renaud; Zvezdova, Ekaterina; Pinkhasov, Julia; Jenkins, Marc K.; McGavern, Dorian; Love, Paul E.

    2015-01-01

    The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated ‘knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR–ligand interactions, but is not essential for ‘general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire. PMID:25959494

  8. TCR ITAM multiplicity is required for the generation of follicular helper T-cells.

    PubMed

    Hwang, SuJin; Palin, Amy C; Li, LiQi; Song, Ki-Duk; Lee, Jan; Herz, Jasmin; Tubo, Noah; Chu, Hamlet; Pepper, Marion; Lesourne, Renaud; Zvezdova, Ekaterina; Pinkhasov, Julia; Jenkins, Marc K; McGavern, Dorian; Love, Paul E

    2015-01-01

    The T-cell antigen receptor (TCR) complex contains 10 copies of a di-tyrosine Immunoreceptor-Tyrosine-based-Activation-Motif (ITAM) that initiates TCR signalling by recruiting protein tyrosine kinases. ITAM multiplicity amplifies TCR signals, but the importance of this capability for T-cell responses remains undefined. Most TCR ITAMs (6 of 10) are contributed by the CD3ζ subunits. We generated 'knock-in' mice that express non-signalling CD3ζ chains in lieu of wild-type CD3ζ. Here we demonstrate that ITAM multiplicity is important for the development of innate-like T-cells and follicular helper T-cells, events that are known to require strong/sustained TCR-ligand interactions, but is not essential for 'general' T-cell responses including proliferation and cytokine production or for the generation of a diverse antigen-reactive TCR repertoire. PMID:25959494

  9. Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC.

    PubMed

    Wang, C; Jung, D; Cao, Z; Chung, C Y

    2015-09-25

    The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC(-) cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC(-) cells. racC(-) cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 μm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 μm/min was maintained in racC(-) cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell. PMID:26315268

  10. TRPM7 is required for ovarian cancer cell growth, migration and invasion

    SciTech Connect

    Wang, Jing; Liao, Qian-jin; Zhang, Yi; Zhou, Hui; Luo, Chen-hui; Tang, Jie; Wang, Ying; Tang, Yan; Zhao, Min; Zhao, Xue-heng; Zhang, Qiong-yu; Xiao, Ling

    2014-11-28

    Highlights: • Silence of TRPM7 in ovarian cancer cells inhibits cell proliferation, migration and invasion. • Silence of TRPM7 decreases phosphorylation levels of Akt, Src and p38 in ovarian cancer cells. • Silence of TRPM7 increases expression of filamentous actin and number of focal adhesions in ovarian cancer cells. - Abstract: Our previous study demonstrated that the melastatin-related transient receptor potential channel 7 (TRPM7) was highly expressed in ovarian carcinomas and its overexpression was significantly associated with poor prognosis in ovarian cancer patients. However, the function of TRPM7 in ovarian cancer is mostly unknown. In this study, we examined the roles of TRPM7 in ovarian cancer cell proliferation, migration and invasion. We found that short hairpin RNA interference-mediated silence of TRPM7 significantly inhibited cell proliferation, colony formation, migration and invasion in multiple ovarian cancer cell lines. Mechanistic investigation revealed that silence of TRPM7 decreased phosphorylation levels of Akt, Src and p38 and increased filamentous actin and focal adhesion number in ovarian cancer cells. Thus, our results suggest that TRPM7 is required for proliferation, migration and invasion of ovarian cancer cells through regulating multiple signaling transduction pathways and the formation of focal adhesions.

  11. BMP signaling is required for the generation of primordial germ cells in an insect

    PubMed Central

    Donoughe, Seth; Nakamura, Taro; Ewen-Campen, Ben; Green, Delbert A.; Henderson, Lory; Extavour, Cassandra G.

    2014-01-01

    Two modes of germ cell formation are known in animals. Specification through maternally inherited germ plasm occurs in many well-characterized model organisms, but most animals lack germ plasm by morphological and functional criteria. The only known alternative mechanism is induction, experimentally described only in mice, which specify germ cells through bone morphogenetic protein (BMP) signal-mediated induction of a subpopulation of mesodermal cells. Until this report, no experimental evidence of an inductive germ cell signal for specification has been available outside of vertebrates. Here we provide functional genetic experimental evidence consistent with a role for BMP signaling in germ cell formation in a basally branching insect. We show that primordial germ cells of the cricket Gryllus bimaculatus transduce BMP signals and require BMP pathway activity for their formation. Moreover, increased BMP activity leads to ectopic and supernumerary germ cells. Given the commonality of BMP signaling in mouse and cricket germ cell induction, we suggest that BMP-based germ cell formation may be a shared ancestral mechanism in animals. PMID:24591634

  12. Release of endogenous opioids from duodenal enteroendocrine cells requires Trpm5

    PubMed Central

    Kokrashvili, Zaza; Rodriguez, Deniliz; Yevshayeva, Valeriya; Zhou, Hang; Margolskee, Robert F

    2009-01-01

    Background & Aims Enteroendocrine cells, the largest and most diverse population of mammalian endocrine cells, comprise a number of different cell types in the gut mucosa that produce, store, and secrete small molecules, peptides and/or larger proteins that regulate many aspects of gut physiology. Little is known about less-typical endocrine cells in the intestinal mucosa that do not contain secretory granules, such as brush or caveolated cells. We studied a subset of these enteroendocrine cells in duodenum that produce several peptides, including endogenous opioids, and that also express the Trpm5 cation channel. Methods We studied expression patterns of Trpm5 and other molecules by immunohistochemical and ELISA analyses of intestinal tissues from transgenic mice that express green fluorescent protein from theTrpm5 promoter, as well as wild-type and Trpm5-null mice. Results We describe a type of enteroendocrine cell in mouse duodenum that is defined by the presence of the Trpm5, that does not contain typical secretory granules, yet expresses endogenous opioids (β-endorphin and Met-enkephalin) and uroguanylin in apical compartments close to the lumen of the gut. Conclusion Solitary chemosensory cells that co-express β-endorphin, Met-enkephalin, uroguanylin and Trpm5 exist in mouse duodenum. These cells are likely to secrete the bioactive peptides into the intestinal lumen in response to dietary factors; release of the opioid peptides requires the Trpm5 ion channel. PMID:19272386

  13. Breast cancer lung metastasis requires expression of chemokine receptor CCR4 and regulatory T cells.

    PubMed

    Olkhanud, Purevdorj B; Baatar, Dolgor; Bodogai, Monica; Hakim, Fran; Gress, Ronald; Anderson, Robin L; Deng, Jie; Xu, Mai; Briest, Susanne; Biragyn, Arya

    2009-07-15

    Cancer metastasis is a leading cause of cancer morbidity and mortality. More needs to be learned about mechanisms that control this process. In particular, the role of chemokine receptors in metastasis remains controversial. Here, using a highly metastatic breast cancer (4T1) model, we show that lung metastasis is a feature of only a proportion of the tumor cells that express CCR4. Moreover, the primary tumor growing in mammary pads activates remotely the expression of TARC/CCL17 and MDC/CCL22 in the lungs. These chemokines acting through CCR4 attract both tumor and immune cells. However, CCR4-mediated chemotaxis was not sufficient to produce metastasis, as tumor cells in the lung were efficiently eliminated by natural killer (NK) cells. Lung metastasis required CCR4(+) regulatory T cells (Treg), which directly killed NK cells using beta-galactoside-binding protein. Thus, strategies that abrogate any part of this process should improve the outcome through activation of effector cells and prevention of tumor cell migration. We confirm this prediction by killing CCR4(+) cells through delivery of TARC-fused toxins or depleting Tregs and preventing lung metastasis. PMID:19567680

  14. BMP signaling is required for the generation of primordial germ cells in an insect.

    PubMed

    Donoughe, Seth; Nakamura, Taro; Ewen-Campen, Ben; Green, Delbert A; Henderson, Lory; Extavour, Cassandra G

    2014-03-18

    Two modes of germ cell formation are known in animals. Specification through maternally inherited germ plasm occurs in many well-characterized model organisms, but most animals lack germ plasm by morphological and functional criteria. The only known alternative mechanism is induction, experimentally described only in mice, which specify germ cells through bone morphogenetic protein (BMP) signal-mediated induction of a subpopulation of mesodermal cells. Until this report, no experimental evidence of an inductive germ cell signal for specification has been available outside of vertebrates. Here we provide functional genetic experimental evidence consistent with a role for BMP signaling in germ cell formation in a basally branching insect. We show that primordial germ cells of the cricket Gryllus bimaculatus transduce BMP signals and require BMP pathway activity for their formation. Moreover, increased BMP activity leads to ectopic and supernumerary germ cells. Given the commonality of BMP signaling in mouse and cricket germ cell induction, we suggest that BMP-based germ cell formation may be a shared ancestral mechanism in animals. PMID:24591634

  15. Discrete Notch signaling requirements in the specification of hematopoietic stem cells

    PubMed Central

    Kim, Albert D; Melick, Chase H; Clements, Wilson K; Stachura, David L; Distel, Martin; Panáková, Daniela; MacRae, Calum; Mork, Lindsey A; Crump, J Gage; Traver, David

    2014-01-01

    Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium. PMID:25230933

  16. CD34 EXPRESSION BY HAIR FOLLICLE STEM CELLS IS REQUIRED FOR SKIN TUMOR DEVELOPMENT IN MICE

    EPA Science Inventory

    We used knockout mice to show that a cell surface protein called CD34 is required for skin tumor formation in mice. Wild type mice treated with 7-12-Dimethylbenz(a)anthracene (DMBA) and a tumor promoter developed papillomas. When we treated CD34 knockout (KO) mice the same way, n...

  17. Math5 is required for retinal ganglion cell and optic nerve formation

    PubMed Central

    Brown, Nadean L.; Patel, Sima; Brzezinski, Joseph; Glaser, Tom

    2006-01-01

    SUMMARY The vertebrate retina contains seven major neuronal and glial cell types in an interconnected network that collects, processes and sends visual signals through the optic nerve to the brain. Retinal neuron differentiation is thought to require both intrinsic and extrinsic factors, yet few intrinsic gene products have been identified that direct this process. Math5 (Atoh7) encodes a basic helix-loop-helix (bHLH) transcription factor that is specifically expressed by mouse retinal progenitors. Math5 is highly homologous to atonal, which is critically required for R8 neuron formation during Drosophila eye development. Like R8 cells in the fly eye, retinal ganglion cells (RGCs) are the first neurons in the vertebrate eye. Here we show that Math5 mutant mice are fully viable, yet lack RGCs and optic nerves. Thus, two evolutionarily diverse eye types require atonal gene family function for the earliest stages of retinal neuron formation. At the same time, the abundance of cone photoreceptors is significantly increased in Math5−/− retinae, suggesting a binary change in cell fate from RGCs to cones. A small number of nascent RGCs are detected during embryogenesis, but these fail to develop further, suggesting that committed RGCs may also require Math5 function. PMID:11493566

  18. MiR-24 is required for hematopoietic differentiation of mouse embryonic stem cells.

    PubMed

    Roy, Lynn; Bikorimana, Emmanuel; Lapid, Danica; Choi, Hyewon; Nguyen, Tan; Dahl, Richard

    2015-01-01

    Overexpression of miRNA, miR-24, in mouse hematopoietic progenitors increases monocytic/ granulocytic differentiation and inhibits B cell development. To determine if endogenous miR-24 is required for hematopoiesis, we antagonized miR-24 in mouse embryonic stem cells (ESCs) and performed in vitro differentiations. Suppression of miR-24 resulted in an inability to produce blood and hematopoietic progenitors (HPCs) from ESCs. The phenotype is not a general defect in mesoderm production since we observe production of nascent mesoderm as well as mesoderm derived cardiac muscle and endothelial cells. Results from blast colony forming cell (BL-CFC) assays demonstrate that miR-24 is not required for generation of the hemangioblast, the mesoderm progenitor that gives rise to blood and endothelial cells. However, expression of the transcription factors Runx1 and Scl is greatly reduced, suggesting an impaired ability of the hemangioblast to differentiate. Lastly, we observed that known miR-24 target, Trib3, is upregulated in the miR-24 antagonized embryoid bodies (EBs). Overexpression of Trib3 alone in ESCs was able to decrease HPC production, though not as great as seen with miR-24 knockdown. These results demonstrate an essential role for miR-24 in the hematopoietic differentiation of ESCs. Although many miRNAs have been implicated in regulation of hematopoiesis, this is the first miRNA observed to be required for the specification of mammalian blood progenitors from early mesoderm. PMID:25634354

  19. iNKT cells require TSC1 for terminal maturation and effector lineage fate decisions

    PubMed Central

    Wu, Jinhong; Yang, Jialong; Yang, Kai; Wang, Hongxia; Gorentla, Balachandra; Shin, Jinwook; Qiu, Yurong; Que, Loretta G.; Foster, W. Michael; Xia, Zhenwei; Chi, Hongbo; Zhong, Xiao-Ping

    2014-01-01

    Terminal maturation of invariant NKT (iNKT) cells from stage 2 (CD44+NK1.1–) to stage 3 (CD44+NK1.1+) is accompanied by a functional acquisition of a predominant IFN-γ–producing (iNKT-1) phenotype; however, some cells develop into IL-17–producing iNKT (iNKT-17) cells. iNKT-17 cells are rare and restricted to a CD44+NK1.1– lineage. It is unclear how iNKT terminal maturation is regulated and what factors mediate the predominance of iNKT-1 compared with iNKT-17. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling, which regulates T cell differentiation, function, and trafficking. Here, we determined that mice lacking TSC1 exhibit a developmental block of iNKT differentiation at stage 2 and skew from a predominantly iNKT-1 population toward a predominantly iNKT-17 population, leading to enhanced airway hypersensitivity. Evaluation of purified iNKT cells revealed that TSC1 promotes T-bet, which regulates iNKT maturation, but downregulates ICOS expression in iNKT cells by inhibiting mTOR complex 1 (mTORC1). Furthermore, mice lacking T-bet exhibited both a terminal maturation defect of iNKT cells and a predominance of iNKT-17 cells, and increased ICOS expression was required for the predominance of iNKT-17 cells in the population of TSC1-deficient iNKT cells. Our data indicate that TSC1-dependent control of mTORC1 is crucial for terminal iNKT maturation and effector lineage decisions, resulting in the predominance of iNKT-1 cells. PMID:24614103

  20. Fast Adaptation in Vestibular Hair Cells Requires Myosin-1c Activity

    PubMed Central

    Stauffer, Eric A.; Scarborough, John D.; Hirono, Moritoshi; Miller, Emilie D.; Shah, Kavita; Mercer, John A.; Holt, Jeffrey R.; Gillespie, Peter G.

    2009-01-01

    Summary In sensory hair cells of the inner ear, mechanical amplification of small stimuli requires fast adaptation, the rapid closing of mechanically activated transduction channels. In frog and mouse vestibular hair cells, we found that the rate of fast adaptation depends on both channel opening and stimulus size and that it is modeled well as a release of a mechanical element in series with the transduction apparatus. To determine whether myosin-1c molecules of the adaptation motor are responsible for the release, we introduced the Y61G mutation into the Myo1c locus and generated mice homozygous for this sensitized allele. Measuring transduction and adaptation in the presence of NMB-ADP, an allele-specific inhibitor, we found that the inhibitor not only blocked slow adaptation, as demonstrated previously in transgenic mice, but also inhibited fast adaptation. These results suggest that mechanical activity of myosin-1c is required for fast adaptation in vestibular hair cells. PMID:16102537

  1. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... MOTOR VEHICLES Fuel Economy Labeling § 600.304-12 Fuel economy label—special requirements for hydrogen fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  2. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... MOTOR VEHICLES Fuel Economy Labeling § 600.304-12 Fuel economy label—special requirements for hydrogen fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  3. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... MOTOR VEHICLES Fuel Economy Labeling § 600.304-12 Fuel economy label—special requirements for hydrogen fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  4. Prox1 Is Required for Oligodendrocyte Cell Identity in Adult Neural Stem Cells of the Subventricular Zone.

    PubMed

    Bunk, Eva C; Ertaylan, Gökhan; Ortega, Felipe; Pavlou, Maria A; Gonzalez Cano, Laura; Stergiopoulos, Athanasios; Safaiyan, Shima; Völs, Sandra; van Cann, Marianne; Politis, Panagiotis K; Simons, Mikael; Berninger, Benedikt; Del Sol, Antonio; Schwamborn, Jens C

    2016-08-01

    Adult neural stem cells with the ability to generate neurons and glia cells are active throughout life in both the dentate gyrus (DG) and the subventricular zone (SVZ). Differentiation of adult neural stem cells is induced by cell fate determinants like the transcription factor Prox1. Evidence has been provided for a function of Prox1 as an inducer of neuronal differentiation within the DG. We now show that within the SVZ Prox1 induces differentiation into oligodendrocytes. Moreover, we find that loss of Prox1 expression in vivo reduces cell migration into the corpus callosum, where the few Prox1 deficient SVZ-derived remaining cells fail to differentiate into oligodendrocytes. Thus, our work uncovers a novel function of Prox1 as a fate determinant for oligodendrocytes in the adult mammalian brain. These data indicate that the neurogenic and oligodendrogliogenic lineages in the two adult neurogenic niches exhibit a distinct requirement for Prox1, being important for neurogenesis in the DG but being indispensable for oligodendrogliogenesis in the SVZ. Stem Cells 2016;34:2115-2129. PMID:27068685

  5. TAF4b is required for mouse spermatogonial stem cell development

    PubMed Central

    Lovasco, Lindsay A.; Gustafson, Eric A.; Seymour, Kimberly A.; de Rooij, Dirk G.; Freiman, Richard N.

    2014-01-01

    Long-term mammalian spermatogenesis requires proper development of spermatogonial stem cells (SSCs) that replenish the testis with germ cell progenitors during adult life. TAF4b is a gonadal-enriched component of the general transcription factor complex, TFIID, which is required for the maintenance of spermatogenesis in the mouse. Successful germ cell transplantation assays into adult TAF4b-deficient host testes suggested that TAF4b performs an essential germ cell autonomous function in SSC establishment and/or maintenance. To elucidate the SSC function of TAF4b, we characterized the initial gonocyte pool and rounds of spermatogenic differentiation in the context of the Taf4b-deficient mouse testis. Here we demonstrate a significant reduction in the late embryonic gonocyte pool and a deficient expansion of this pool soon after birth. Resulting from this reduction of germ cell progenitors is a developmental delay in meiosis initiation, as compared to age-matched controls. While GFRα1+ spermatogonia are appropriately present as Asingle and Apaired in wild type testes, TAF4b-deficient testes display an increased proportion of long and clustered chains of GFRα1+ cells. In the absence of TAF4b, seminiferous tubules in the adult testis either lack germ cells altogether or are found to have missing generations of spermatogenic progenitor cells. Together these data indicate that TAF4b-deficient spermatogenic progenitor cells display a tendency for differentiation at the expense of self-renewal and a renewing pool of SSCs fail to establish during the critical window of SSC development. PMID:25727968

  6. SHP-2 phosphatase activity is required for PECAM-1-dependent cell motility.

    PubMed

    Zhu, Jing-Xu; Cao, Gaoyuan; Williams, James T; Delisser, Horace M

    2010-10-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1) has been implicated in endothelial cell motility during angiogenesis. Although there is evidence that SHP-2 plays a role in PECAM-1-dependent cell motility, the molecular basis of the activity of SHP-2 in this process has not been defined. To investigate the requirement of SHP-2 in PECAM-1-dependent cell motility, studies were done in which various constructs of SHP-2 were expressed in cell transfectants expressing PECAM-1. We observed that the levels of PECAM-1 tyrosine phosphorylation and SHP-2 association with PECAM-1 were significantly increased in cells expressing a phosphatase-inactive SHP-2 mutant, suggesting that the level of PECAM-1 tyrosine phosphorylation, and thus SHP-2 binding are regulated in part by bound, catalytically active SHP-2. We subsequently found that expression of PECAM-1 stimulated wound-induced migration and the formation of filopodia (a morphological feature of motile cells). These activities were associated with increased mitogen-activated protein kinase (MAPK) activation and the dephosphorylation of paxillin (an event implicated in the activation of MAPK). The phosphatase-inactive SHP-2 mutant, however, suppressed these PECAM-1-dependent phenomena, whereas the activity of PECAM-1 expressing cells was not altered by expression of wild-type SHP-2 or SHP-2 in which the scaffold/adaptor function had been disabled. Pharmacological inhibition of SHP-2 phosphatase activity also suppressed PECAM-1-dependent motility. Furthermore, PECAM-1 expression also stimulates tube formation, but none of the SHP-2 constructs affected this process. These findings therefore suggest a model for the involvement of SHP-2 in PECAM-1-dependent motility in which SHP-2, recruited by its interaction with PECAM-1, targets paxillin to ultimately activate the MAPK pathway and downstream events required for cell motility. PMID:20631249

  7. Long-term maintenance of hematopoietic stem cells does not require contact with embryo-derived stromal cells in cocultures.

    PubMed

    Oostendorp, Robert A J; Robin, Catherine; Steinhoff, Christine; Marz, Stefanie; Bräuer, Rosalinde; Nuber, Ulrike A; Dzierzak, Elaine A; Peschel, Christian

    2005-01-01

    We recently established that two midgestation-derived stromal clones--UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived--support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly-6C- side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which factors might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed 21 genes at a higher level, including the putative secreted factors fibroblast growth factor-7, insulin-like growth factor-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support. PMID:15917480

  8. KOJAK encodes a cellulose synthase-like protein required for root hair cell morphogenesis in Arabidopsis

    PubMed Central

    Favery, Bruno; Ryan, Eoin; Foreman, Julia; Linstead, Paul; Boudonck, Kurt; Steer, Martin; Shaw, Peter; Dolan, Liam

    2001-01-01

    The cell wall is an important determinant of plant cell form. Here we define a class of Arabidopsis root hair mutants with defective cell walls. Plants homozygous for kojak (kjk) mutations initiate root hairs that rupture at their tip soon after initiation. The KJK gene was isolated by positional cloning, and its identity was confirmed by the molecular complementation of the Kjk− phenotype and the sequence of three kjk mutant alleles. KOJAK encodes a cellulose synthase-like protein, AtCSLD3. KOJAK/AtCSLD3 is the first member of this subfamily of proteins to be shown to have a function in cell growth. Subcellular localization of the KOJAK/AtCSLD3 protein using a GFP fusion shows that KOJAK/AtCSLD3 is located on the endoplasmic reticulum, indicating that KOJAK/AtCSLD3 is required for the synthesis of a noncellulosic wall polysaccharide. Consistent with the cell specific defect in the roots of kjk mutants, KOJAK/AtCSDL3 is preferentially expressed in hair cells of the epidermis. The Kjk− phenotype and the pattern of KOJAK/AtCSLD3 expression suggest that this gene acts early in the process of root hair outgrowth. These results suggest that KOJAK/AtCSLD3 is involved in the biosynthesis of β-glucan-containing polysaccharides that are required during root hair elongation. PMID:11156607

  9. The Host Defense Peptide Cathelicidin Is Required for NK Cell-Mediated Suppression of Tumor Growth

    PubMed Central

    Büchau, Amanda S.; Morizane, Shin; Trowbridge, Janet; Schauber, Jürgen; Kotol, Paul; Bui, Jack D.; Gallo, Richard L.

    2010-01-01

    Tumor surveillance requires the interaction of multiple molecules and cells that participate in innate and the adaptive immunity. Cathelicidin was initially identified as an antimicrobial peptide, although it is now clear that it fulfills a variety of immune functions beyond microbial killing. Recent data have suggested contrasting roles for cathelicidin in tumor development. Because its role in tumor surveillance is not well understood, we investigated the requirement of cathelicidin in controlling transplantable tumors in mice. Cathelicidin was observed to be abundant in tumor-infiltrating NK1.1+ cells in mice. The importance of this finding was demonstrated by the fact that cathelicidin knockout mice (Camp−/−) permitted faster tumor growth than wild type controls in two different xenograft tumor mouse models (B16.F10 and RMA-S). Functional in vitro analyses found that NK cells derived from Camp−/− versus wild type mice showed impaired cytotoxic activity toward tumor targets. These findings could not be solely attributed to an observed perforin deficiency in freshly isolated Camp−/− NK cells, because this deficiency could be partially restored by IL-2 treatment, whereas cytotoxic activity was still defective in IL-2-activated Camp−/− NK cells. Thus, we demonstrate a previously unrecognized role of cathelicidin in NK cell antitumor function. PMID:19949065

  10. A mex3 homolog is required for differentiation during planarian stem cell lineage development

    PubMed Central

    Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

    2015-01-01

    Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment. DOI: http://dx.doi.org/10.7554/eLife.07025.001 PMID:26114597

  11. A mex3 homolog is required for differentiation during planarian stem cell lineage development.

    PubMed

    Zhu, Shu Jun; Hallows, Stephanie E; Currie, Ko W; Xu, ChangJiang; Pearson, Bret J

    2015-01-01

    Neoblasts are adult stem cells (ASCs) in planarians that sustain cell replacement during homeostasis and regeneration of any missing tissue. While numerous studies have examined genes underlying neoblast pluripotency, molecular pathways driving postmitotic fates remain poorly defined. In this study, we used transcriptional profiling of irradiation-sensitive and irradiation-insensitive cell populations and RNA interference (RNAi) functional screening to uncover markers and regulators of postmitotic progeny. We identified 32 new markers distinguishing two main epithelial progenitor populations and a planarian homolog to the MEX3 RNA-binding protein (Smed-mex3-1) as a key regulator of lineage progression. mex3-1 was required for generating differentiated cells of multiple lineages, while restricting the size of the stem cell compartment. We also demonstrated the utility of using mex3-1(RNAi) animals to identify additional progenitor markers. These results identified mex3-1 as a cell fate regulator, broadly required for differentiation, and suggest that mex3-1 helps to mediate the balance between ASC self-renewal and commitment. PMID:26114597

  12. Notch signal reception is required in vascular smooth muscle cells for ductus arteriosus closure.

    PubMed

    Krebs, Luke T; Norton, Christine R; Gridley, Thomas

    2016-02-01

    The ductus arteriosus is an arterial vessel that shunts blood flow away from the lungs during fetal life, but normally occludes after birth to establish the adult circulation pattern. Failure of the ductus arteriosus to close after birth is termed patent ductus arteriosus, and is one of the most common congenital heart defects. Our previous work demonstrated that vascular smooth muscle cell expression of the Jag1 gene, which encodes a ligand for Notch family receptors, is essential for postnatal closure of the ductus arteriosus in mice. However, it was not known what cell population was responsible for receiving the Jag1-mediated signal. Here we show, using smooth muscle cell-specific deletion of the Rbpj gene, which encodes a transcription factor that mediates all canonical Notch signaling, that Notch signal reception in the vascular smooth muscle cell compartment is required for ductus arteriosus closure. These data indicate that homotypic vascular smooth muscle cell interactions are required for proper contractile smooth muscle cell differentiation and postnatal closure of the ductus arteriosus in mice. PMID:26742650

  13. MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration.

    PubMed

    Aranda, Juan F; Reglero-Real, Natalia; Kremer, Leonor; Marcos-Ramiro, Beatriz; Ruiz-Sáenz, Ana; Calvo, María; Enrich, Carlos; Correas, Isabel; Millán, Jaime; Alonso, Miguel A

    2011-04-15

    Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes. PMID:21325632

  14. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling

    PubMed Central

    Sena, Laura A.; Li, Sha; Jairaman, Amit; Prakriya, Murali; Ezponda, Teresa; Hildeman, David A.; Wang, Chyung-Ru; Schumacker, Paul T.; Licht, Jonathan D.; Perlman, Harris; Bryce, Paul J.; Chandel, Navdeep S.

    2013-01-01

    SUMMARY It is widely appreciated that T cells increase glycolytic flux during activation, however the role of mitochondrial flux is unclear. Here we have shown that mitochondrial metabolism, in the absence of glucose metabolism, was sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs−/− mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, however Uqcrfs1−/− T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1−/− T cells were not lacking bioenergetically, but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through production of complex III ROS. PMID:23415911

  15. Cdc42 is required for EGF-stimulated protrusion and motility in MTLn3 carcinoma cells

    PubMed Central

    El-Sibai, Mirvat; Nalbant, Peri; Pang, Huan; Flinn, Rory J.; Sarmiento, Corina; Macaluso, Frank; Cammer, Michael; Condeelis, John S.; Hahn, Klaus M.; Backer, Jonathan M.

    2014-01-01

    Summary Cdc42 plays a central role in regulating the actin cytoskeleton and maintaining cell polarity. Here, we show that Cdc42 is crucial for epidermal growth factor (EGF)-stimulated protrusion in MTLn3 carcinoma cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the protruding edge of the cells. siRNA-mediated knockdown of Cdc42 expression caused a decrease in EGF-stimulated protrusion and reduced cell motility in time-lapse studies. These changes were correlated with a decrease in barbed-end formation and Arp2/3 localization at the cell edge, and a marked defect in actin filament branching, as revealed by rotary-shadowing scanning electron microscopy. Upstream of Arp2/3, Cdc42 knockdown inhibited EGF-stimulated activation of PI 3-kinase at early (within 1 minute) but not late (within 3 minutes) time points. Membrane targeting of N-WASP, WAVE2 and IRSp53 were also inhibited. Effects on WAVE2 were not owing to Rac1 inhibition, because WAVE2 recruitment is unaffected by Rac1 knockdown. Our data suggest that Cdc42 activation is crucial for the regulation of actin polymerization in carcinoma cells, and required for both EGF-stimulated protrusion and cell motility independently of effects on Rac. PMID:17855387

  16. Identification of Genes Required for Normal Pheromone-Induced Cell Polarization in Saccharomyces Cerevisiae

    PubMed Central

    Chenevert, J.; Valtz, N.; Herskowitz, I.

    1994-01-01

    In response to mating pheromones, cells of the yeast Saccharomyces cerevisiae adopt a polarized ``shmoo'' morphology, in which the cytoskeleton and proteins involved in mating are localized to a cell-surface projection. This polarization is presumed to reflect the oriented morphogenesis that occurs between mating partners to facilitate cell and nuclear fusion. To identify genes involved in pheromone-induced cell polarization, we have isolated mutants defective in mating to an enfeebled partner and studied a subset of these mutants. The 34 mutants of interest are proficient for pheromone production, arrest in response to pheromone, mate to wild-type strains, and exhibit normal cell polarity during vegetative growth. The mutants were divided into classes based on their morphological responses to mating pheromone. One class is unable to localize cell-surface growth in response to mating factor and instead enlarges in a uniform manner. These mutants harbor special alleles of genes required for cell polarization during vegetative growth, BEM1 and CDC24. Another class of mutants forms bilobed, peanut-like shapes when treated with pheromone and defines two genes, PEA1 and PEA2. PEA1 is identical to SPA2. A third class forms normally shaped but tiny shmoos and defines the gene TNY1. A final group of mutants exhibits apparently normal shmoo morphology. The nature of their mating defect is yet to be determined. We discuss the possible roles of these gene products in establishing cell polarity during mating. PMID:8013906

  17. Requirement for HIV-1 TAR sequences for Tat activation in rodent cells.

    PubMed

    Sutton, J A; Braddock, M; Kingsman, A J; Kingsman, S M

    1995-01-10

    HIV-1 gene expression is activated via an interaction between the virally encoded Tat protein and a target RNA, TAR. TAR is located at the immediate 5' end of all viral mRNAs and comprises a partially base-paired stem with a tripyrimidine bulge in the upper stem and a hexanucleotide loop. In vitro, Tat binds specifically to the bulge and upper stem region with no requirement for the loop. In contrast, when Tat activation is analyzed in primate cells, mutations in the loop abolish activation, suggesting a critical role for loop binding cellular factors. However, in rodent cells the reverse is true. Messages with a mutation in the TAR loop are activated whereas messages harboring a wild-type TAR sequence are not activated. By testing the effect of mutations in the bulge and stem in the context of mutation in the loop we now show that this loop-independent activation by Tat in rodent cells requires the critical bulge-stem sequences needed for Tat binding in vitro. These data suggest that in rodent cells, as in vitro, Tat does not require a loop binding cofactor. In rodent cells containing human chromosome 12 (CHO12), however, Tat activation is both bulge and loop dependent. It appears that rodent cells, but not CHO12 cells, are refractory to the normal Tat/TAR activation pathway not by virtue of lacking a loop binding cofactor, but rather by the presence of a loop binding inhibitor of either Tat binding or the activation process. PMID:7530399

  18. Actopaxin (α-parvin) phosphorylation is required for matrix degradation and cancer cell invasion.

    PubMed

    Pignatelli, Jeanine; LaLonde, Sara E; LaLonde, David P; Clarke, Dominic; Turner, Christopher E

    2012-10-26

    Dysregulation of cell adhesion and motility is known to be an important factor in the development of tumor malignancy. Actopaxin (α-parvin) is a paxillin, integrin-linked kinase, and F-actin binding focal adhesion protein with several serine phosphorylation sites in the amino terminus that contribute to the regulation of cell spreading and migration. Here, phosphorylation of actopaxin is shown to contribute to the regulation of matrix degradation and cell invasion. Osteosarcoma cells stably expressing wild type (WT), nonphosphorylatable (Quint), and phosphomimetic (S4D/S8D) actopaxin demonstrate that actopaxin phosphorylation is necessary for efficient Src and matrix metalloproteinase-driven degradation of extracellular matrix. Rac1 was found to be required for actopaxin-induced matrix degradation whereas inhibition of myosin contractility promoted degradation in the phosphomutant-expressing Quint cells, indicating that a balance of Rho GTPase signaling and regulation of cellular tension are important for the process. Furthermore, actopaxin forms a complex with the Rac1/Cdc42 GEF β-PIX and Rac1/Cdc42 effector PAK1, to regulate actopaxin-dependent matrix degradation. Actopaxin phosphorylation is elevated in the invasive breast cancer cell line MDA-MB-231 compared with normal breast epithelial MCF10A cells. Expression of the nonphosphorylatable Quint actopaxin in MDA-MB-231 cells inhibits cell invasion whereas overexpression of WT actopaxin promotes invasion in MCF10A cells. Taken together, this study demonstrates a new role for actopaxin phosphorylation in matrix degradation and cell invasion via regulation of Rho GTPase signaling. PMID:22955285

  19. Behaviour of human immunoregulatory cells in culture. I. Variables requiring consideration for clinical studies.

    PubMed Central

    Dwyer, J M; Johnson, C; Desaules, M

    1979-01-01

    The suppressor function of lymphocytes stimulated with concanavalin A (Con A) provides a potential method for examining disorders of immunoregulation. Clinical application, however, requires definition of the culture conditions that influence the expression of normal suppressor cell activity. In the present studies culture conditions were modified until a sensitive assay for non-specific suppressor cell function was reproducible utilizing the response to varying doses of phytohaemagglutinin (PHA) as an indicator system. Practical conclusions included (1) that sensitivity was not lost if the suppressor cells and responder cells were allogenic; (2) that fresh responder cells were as sensitive as precultured responder cells; (3) that a wide range of Con A concentrations could induce suppressor activity; and (4) that the sensitivity of the assay was much enhanced by using suboptimal mitogen doses of PHA. Twelve percent of normal subjects gave false negative results but these could be avoided by studying cells at more than one time point after stimulation with Con A. Cells resting in culture for 7 days could be induced to suppress after stimulation with Con A and these suppressor cells were very sensitive to pharmacological doses of dexamethasone. Studies utilizing different times of cell pre-incubation before Con A stimulation and different periods of exposure to Con A revealed fluctuation in the induction of suppression that may represent alternating periods of suppression and amplifying activity among stimulated cells in vitro. Such variations will need to be taken into account in the application of this type of assay to clinical studies seeking disordered immunoregulation. PMID:161214

  20. The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells

    PubMed Central

    Shields, Alicia R.; Spence, Allyson C.; Yamashita, Yukiko M.; Davies, Erin L.; Fuller, Margaret T.

    2014-01-01

    Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. Here, we demonstrate that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia. PMID:24346697

  1. Mutant IDH1 is required for IDH1 mutated tumor cell growth.

    PubMed

    Jin, Genglin; Pirozzi, Christopher J; Chen, Lee H; Lopez, Giselle Y; Duncan, Christopher G; Feng, Jie; Spasojevic, Ivan; Bigner, Darell D; He, Yiping; Yan, Hai

    2012-08-01

    Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy. However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown. Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1(R132C) heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential. Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target. PMID:22885298

  2. Innate lymphoid cell development requires TOX-dependent generation of a common ILC progenitor

    PubMed Central

    Seehus, Corey R.; Aliahmad, Parinaz; de la Torre, Brian; Iliev, Iliyan D.; Spurka, Lindsay; Funari, Vincent A.; Kaye, Jonathan

    2015-01-01

    Diverse innate lymphoid cell (ILC) subtypes have been defined, based on effector function and transcription factor expression. ILCs derive from common lymphoid progenitors, although the transcriptional pathways leading to ILC lineage specification remain poorly characterized. Here we demonstrate that transcriptional regulator TOX is required for the in vivo differentiation of common lymphoid progenitors to ILC lineage-restricted cells. In vitro modeling demonstrates that TOX deficiency results in early defects in progenitor cell survival or expansion as well as later stage ILC differentiation. In addition, comparative transcriptome analysis of bone marrow progenitors reveals that TOX-deficient cells fail to upregulate many aspects of the ILC gene program, including Notch gene targets, implicating TOX as a key determinant of early ILC lineage specification. PMID:25915732

  3. Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation☆

    PubMed Central

    Jaber-Hijazi, Farah; Lo, Priscilla J.K.P.; Mihaylova, Yuliana; Foster, Jeremy M.; Benner, Jack S.; Tejada Romero, Belen; Chen, Chen; Malla, Sunir; Solana, Jordi; Ruzov, Alexey; Aziz Aboobaker, A.

    2013-01-01

    Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5mC) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation. PMID:24063805

  4. ATM kinase is required for telomere elongation in mouse and human cells

    PubMed Central

    Lee, Stella Suyong; Bohrson, Craig; Pike, Alexandra Mims; Wheelan, Sarah Jo; Greider, Carol Widney

    2015-01-01

    Summary Short telomeres induce a DNA damage response, senescence and apoptosis; thus, maintaining telomere length equilibrium is essential for cell viability. Telomerase addition of telomere repeats is tightly regulated in cells. To probe pathways that regulate telomere addition, we developed the ADDIT assay to measure new telomere addition at a single telomere in vivo. Sequence analysis showed telomerase specific addition of repeats onto a new telomere occurred in just 48 hr. Using the ADDIT assay, we found that ATM is required for addition of new repeats onto telomeres in mouse cells. Evaluation of bulk telomeres, in both human and mouse cells, showed that blocking ATM inhibited telomere elongation. Finally, the activation of ATM through the inhibition of PARP1 resulted in increased telomere elongation, supporting the central role of the ATM pathway in regulating telomere addition. Understanding this role of ATM may yield new areas for possible therapeutic intervention in telomere-mediated disease. PMID:26586427

  5. Redundant Function of Plasmacytoid and Conventional Dendritic Cells Is Required To Survive a Natural Virus Infection

    PubMed Central

    Kaminsky, Lauren W.; Sei, Janet J.; Parekh, Nikhil J.; Davies, Michael L.; Reider, Irene E.; Krouse, Tracy E.

    2015-01-01

    ABSTRACT Viruses that spread systemically from a peripheral site of infection cause morbidity and mortality in the human population. Innate myeloid cells, including monocytes, macrophages, monocyte-derived dendritic cells (mo-DC), and dendritic cells (DC), respond early during viral infection to control viral replication, reducing virus spread from the peripheral site. Ectromelia virus (ECTV), an orthopoxvirus that naturally infects the mouse, spreads systemically from the peripheral site of infection and results in death of susceptible mice. While phagocytic cells have a requisite role in the response to ECTV, the requirement for individual myeloid cell populations during acute immune responses to peripheral viral infection is unclear. In this study, a variety of myeloid-specific depletion methods were used to dissect the roles of individual myeloid cell subsets in the survival of ECTV infection. We showed that DC are the primary producers of type I interferons (T1-IFN), requisite cytokines for survival, following ECTV infection. DC, but not macrophages, monocytes, or granulocytes, were required for control of the virus and survival of mice following ECTV infection. Depletion of either plasmacytoid DC (pDC) alone or the lymphoid-resident DC subset (CD8α+ DC) alone did not confer lethal susceptibility to ECTV. However, the function of at least one of the pDC or CD8α+ DC subsets is required for survival of ECTV infection, as mice depleted of both populations were susceptible to ECTV challenge. The presence of at least one of these DC subsets is sufficient for cytokine production that reduces ECTV replication and virus spread, facilitating survival following infection. IMPORTANCE Prior to the eradication of variola virus, the orthopoxvirus that causes smallpox, one-third of infected people succumbed to the disease. Following successful eradication of smallpox, vaccination rates with the smallpox vaccine have significantly dropped. There is now an increasing

  6. Membrane Organization and Cell Fusion During Mating in Fission Yeast Requires Multipass Membrane Protein Prm1

    PubMed Central

    Curto, M.-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M.-Henar

    2014-01-01

    The involvement of Schizosaccharomyces pombe prm1+ in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell–cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1+ and the dni+ genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell–cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell–cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  7. Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells.

    PubMed Central

    Bokoch, G M; Prossnitz, V

    1992-01-01

    The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation. Images PMID:1310693

  8. Oct1 and OCA-B are selectively required for CD4 memory T cell function.

    PubMed

    Shakya, Arvind; Goren, Alon; Shalek, Alex; German, Cody N; Snook, Jeremy; Kuchroo, Vijay K; Yosef, Nir; Chan, Raymond C; Regev, Aviv; Williams, Matthew A; Tantin, Dean

    2015-11-16

    Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4(+) memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4(+) T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2, Ifng, and Zbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4(+) T cell memory. PMID:26481684

  9. Oct1 and OCA-B are selectively required for CD4 memory T cell function

    PubMed Central

    Shakya, Arvind; Goren, Alon; Shalek, Alex; German, Cody N.; Snook, Jeremy; Kuchroo, Vijay K.; Yosef, Nir; Chan, Raymond C.; Regev, Aviv

    2015-01-01

    Epigenetic changes are crucial for the generation of immunological memory. Failure to generate or maintain these changes will result in poor memory responses. Similarly, augmenting or stabilizing the correct epigenetic states offers a potential method of enhancing memory. Yet the transcription factors that regulate these processes are poorly defined. We find that the transcription factor Oct1 and its cofactor OCA-B are selectively required for the in vivo generation of CD4+ memory T cells. More importantly, the memory cells that are formed do not respond properly to antigen reencounter. In vitro, both proteins are required to maintain a poised state at the Il2 target locus in resting but previously stimulated CD4+ T cells. OCA-B is also required for the robust reexpression of multiple other genes including Ifng. ChIPseq identifies ∼50 differentially expressed direct Oct1 and OCA-B targets. We identify an underlying mechanism involving OCA-B recruitment of the histone lysine demethylase Jmjd1a to targets such as Il2, Ifng, and Zbtb32. The findings pinpoint Oct1 and OCA-B as central mediators of CD4+ T cell memory. PMID:26481684

  10. Conserved Promoter Motif Is Required for Cell Cycle Timing of dnaX Transcription in Caulobacter

    PubMed Central

    Keiler, Kenneth C.; Shapiro, Lucy

    2001-01-01

    Cells use highly regulated transcriptional networks to control temporally regulated events. In the bacterium Caulobacter crescentus, many cellular processes are temporally regulated with respect to the cell cycle, and the genes required for these processes are expressed immediately before the products are needed. Genes encoding factors required for DNA replication, including dnaX, dnaA, dnaN, gyrB, and dnaK, are induced at the G1/S-phase transition. By analyzing mutations in the dnaX promoter, we identified a motif between the −10 and −35 regions that is required for proper timing of gene expression. This motif, named RRF (for repression of replication factors), is conserved in the promoters of other coordinately induced replication factors. Because mutations in the RRF motif result in constitutive gene expression throughout the cell cycle, this sequence is likely to be the binding site for a cell cycle-regulated transcriptional repressor. Consistent with this hypothesis, Caulobacter extracts contain an activity that binds specifically to the RRF in vitro. PMID:11466289

  11. Axonal and Schwann Cell BACE1 Is Equally Required for Remyelination of Peripheral Nerves

    PubMed Central

    Hu, Xiangyou; Hu, Jinxuan; Dai, Lu; Trapp, Bruce

    2015-01-01

    Inhibition of β-site APP cleaving enzyme 1 (BACE1) is being pursued as a therapeutic target for treating patients with Alzheimer's disease because BACE1 is the sole β-secretase for generating β-amyloid peptide. Knowledge regarding the other cellular functions of BACE1 is therefore critical for the safe use of BACE1 inhibitors in human patients. BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination in injured sciatic nerves. Since BACE1 is expected to be ubiquitously expressed, we asked whether axonal or Schwann cell BACE1 is required for optimal remyelination. By swapping sciatic nerve segments from BACE1-null mice with the corresponding wild-type nerve segments or vice versa, we tested how a deficiency of BACE1 in Schwann cells or axons affects remyelination. Our results show that BACE1 in axons and Schwann cells is similarly important for remyelination of regenerated axons. Nerve injury induces BACE1 transcription and protein levels are elevated in Schwann cells. Expression of type I neuregulin 1 (Nrg1), rather than type III Nrg1, was induced by Schwann cells, and the abolished Nrg1 cleavage in BACE1-null Schwann cells contributed to decreased remyelination of regenerated axons. Hence, this study is the first to demonstrate the equal importance of axonal and Schwann cell BACE1 for remyelination of injured nerves. PMID:25740511

  12. Antitumor immunity induced by hybrid murine tumor cells: requirements for optimal immunization

    SciTech Connect

    McCune, C.S.; O'Donnell, R.W.; Horan, P.K.; Budd, H.S.; Spennacchio, J.L.; Chuang, C.; Henshaw, E.C.

    1982-09-01

    Hybrid tumor cells have been evaluated for their ability to induce specific antitumor immunity in inbred female C3H/He mice challenged with the syngeneic BA tumor. Hybrid cells were produced by fusion of BA cells with a BALB/c renal adenocarcinoma, which is hypoxanthine phosphoribosyl transferase-deficient and grows well in culture. Corynebacterium parvum was evaluated as an adjuvant for BA and hybrid cells. The BA tumor was shown to be poorly immunogenic, and four weekly injections of BA cells alone or C. parvum alone did not confer significant immunity. When BA cells and C. parvum were mixed, survival time was prolonged and most mice remained tumor-free. Hybrid cell lines derived from the BA tumor were produced in culture in unlimited quantities and were successfully used as immunogens. The addition of C. parvum to hybrids gave a significant incremental increase in survival when compared to the survival resulting from immunization by hybrids without adjuvant. When hybrids without adjuvant were used, several weekly injections were required for effective immunization. Irradiated and unirradiated hybrids were compared, and it was found that irradiation did not diminish hybrid immunogenicity. The potential problems and advantages of this concept of therapy are discussed.

  13. Survival of Igα-Deficient Mature B Cells Requires BAFF-R Function.

    PubMed

    Levit-Zerdoun, Ella; Becker, Martin; Pohlmeyer, Roland; Wilhelm, Isabel; Maity, Palash Chandra; Rajewsky, Klaus; Reth, Michael; Hobeika, Elias

    2016-03-01

    Expression of a functional BCR is essential for the development of mature B cells and has been invoked in the control of their maintenance. To test this maintenance function in a new experimental setting, we used the tamoxifen-inducible mb1-CreER(T2) mouse strain to delete or truncate either the mb-1 gene encoding the BCR signaling subunit Igα or the VDJ segment of the IgH (H chain [HC]). In this system, Cre-mediated deletion of the mb-1 gene is accompanied by expression of a GFP reporter. We found that, although the Igα-deficient mature B cells survive for >20 d in vivo, the HC-deficient or Igα tail-truncated B cell population is short-lived, with the HC-deficient cells displaying signs of an unfolded protein response. We also show that Igα-deficient B cells still respond to the prosurvival factor BAFF in culture and require BAFF-R signaling for their in vivo maintenance. These results suggest that, under certain conditions, the loss of the BCR can be tolerated by mature B cells for some time, whereas HC-deficient B cells, potentially generated by aberrant somatic mutations in the germinal center, are rapidly eliminated. PMID:26843325

  14. Fuel cells for transport: can the promise be fulfilled? Technical requirements and demands from customers

    NASA Astrophysics Data System (ADS)

    Klaiber, Thomas

    The paper discusses the technical requirements and the customer demands for vehicles that have an on-board methanol reformer and fuel cells. The research concentrates on the technical developmental risks which include minimizing volume, reducing weight and, at the same time, improving efficiency and system dynamics. Fuel cell powered vehicles with methanol reformers are not only suitable for a niche market but also these vehicles will compete with conventional vehicles. The greatest hindrance will be the price of the fuel cell. A possible progressive development of the number of fuel cell powered vehicles in conjunction with a reduction in costs will be discussed in the paper. When fuel cell vehicles come to the market it is necessary that an infrastructure for the fuel methanol or hydrogen is installed. Therefore, it will only be possible to introduce fuel cell vehicles into special markets, e.g. California. Such a process will need to be subsidized by additional incentives like tax concessions. Today there are many technical risks and unsolved problems relating to production technologies, infrastructure, and costs. Nevertheless, among the alternative power units, the fuel cell seems to be the only one that might be competitive to the conventional power unit, especially relating to emissions.

  15. Differential requirement for OBF-1 during antibody-secreting cell differentiation

    PubMed Central

    Corcoran, Lynn M.; Hasbold, Jhagvaral; Dietrich, Wendy; Hawkins, Edwin; Kallies, Axel; Nutt, Stephen L.; Tarlinton, David M.; Matthias, Patrick; Hodgkin, Philip D.

    2005-01-01

    Resting B cells can be cultured to induce antibody-secreting cell (ASC) differentiation in vitro. A quantitative analysis of cell behavior during such a culture allows the influences of different stimuli and gene products to be measured. The application of this analytical system revealed that the OBF-1 transcriptional coactivator, whose loss impairs antibody production in vivo, has two effects on ASC development. Although OBF-1 represses early T cell–dependent (TD) differentiation, it is also critical for the completion of the final stages of ASC development. Under these conditions, the loss of OBF-1 blocks the genetic program of ASC differentiation so that Blimp-1/prdm1 induction fails, and bcl-6, Pax5, and AID are not repressed as in control ASC. Retroviral complementation confirmed that OBF-1 was the critical entity. Surprisingly, when cells were cultured in lipopolysaccharide to mimic T cell–independent conditions, OBF-1–null B cells differentiated normally to ASC. In the OBF-1−/− ASC generated under either culture regimen, antibody production was normal or only modestly reduced, revealing that Ig genes are not directly dependent on OBF-1 for their expression. The differential requirement for OBF-1 in TD ASC generation was confirmed in vivo. These studies define a new regulatory role for OBF-1 in determining the cell-autonomous capacity of B cells to undergo terminal differentiation in response to different immunological signals. PMID:15867091

  16. Survival of the Replication Checkpoint Deficient Cells Requires MUS81-RAD52 Function

    PubMed Central

    Murfuni, Ivana; Basile, Giorgia; Subramanyam, Shyamal; Malacaria, Eva; Bignami, Margherita; Spies, Maria; Franchitto, Annapaola; Pichierri, Pietro

    2013-01-01

    In checkpoint-deficient cells, DNA double-strand breaks (DSBs) are produced during replication by the structure-specific endonuclease MUS81. The mechanism underlying MUS81-dependent cleavage, and the effect on chromosome integrity and viability of checkpoint deficient cells is only partly understood, especially in human cells. Here, we show that MUS81-induced DSBs are specifically triggered by CHK1 inhibition in a manner that is unrelated to the loss of RAD51, and does not involve formation of a RAD51 substrate. Indeed, CHK1 deficiency results in the formation of a RAD52-dependent structure that is cleaved by MUS81. Moreover, in CHK1-deficient cells depletion of RAD52, but not of MUS81, rescues chromosome instability observed after replication fork stalling. However, when RAD52 is down-regulated, recovery from replication stress requires MUS81, and loss of both these proteins results in massive cell death that can be suppressed by RAD51 depletion. Our findings reveal a novel RAD52/MUS81-dependent mechanism that promotes cell viability and genome integrity in checkpoint-deficient cells, and disclose the involvement of MUS81 to multiple processes after replication stress. PMID:24204313

  17. Early nucleosome deposition on, and replication of, HSV DNA requires cell factor PCNA

    PubMed Central

    Sanders, Iryna; Boyer, Mark; Fraser, Nigel W.

    2015-01-01

    Herpes Simplex Virus (HSV) is a double stranded DNA virus that can cause lytic infections in epithelial cells of the skin and latent infections in neuronal cells of the peripheral nervous system. After virion attachment to the cell membrane, the capsid enters the cytoplasm and is transported to the nucleus. Following docking at the nuclear pore, the HSV DNA, and contents of the virion, are injected into the nucleus. The viral DNA that enters the nucleus is devoid of histones, but begins to be covered with them soon after entry. The covering of histones, in the form of nucleosomes, reaches a maximum during the early stages of infection and drops off during late infection (after DNA replication). However during latency the genome is saturated with nucleosomes. In this study, we examine the role of cell Proliferating Cell Nuclear Antigen (PCNA) a cellular DNA polymerase accessory protein (processivity factor), and cell DNA polymerases in histone deposition during the early stages of HSV infection. Using SiRNA knockdown, and a cytosine arabinoside (araC) chemical inhibitor, we conclude that PCNA is important for viral replication and histone deposition. However, cell DNA polymerases that bind PCNA do not appear to be required for these processes and PCNA does not appear to bind to the viral DNA polymerase (which has its own viral processivity factor). PMID:25672886

  18. TCF1 Is Required for the T Follicular Helper Cell Response to Viral Infection.

    PubMed

    Wu, Tuoqi; Shin, Hyun Mu; Moseman, E Ashley; Ji, Yun; Huang, Bonnie; Harly, Christelle; Sen, Jyoti M; Berg, Leslie J; Gattinoni, Luca; McGavern, Dorian B; Schwartzberg, Pamela L

    2015-09-29

    T follicular helper (TFH) and T helper 1 (Th1) cells generated after viral infections are critical for the control of infection and the development of immunological memory. However, the mechanisms that govern the differentiation and maintenance of these two distinct lineages during viral infection remain unclear. We found that viral-specific TFH and Th1 cells showed reciprocal expression of the transcriptions factors TCF1 and Blimp1 early after infection, even before the differential expression of the canonical TFH marker CXCR5. Furthermore, TCF1 was intrinsically required for the TFH cell response to viral infection; in the absence of TCF1, the TFH cell response was severely compromised, and the remaining TCF1-deficient TFH cells failed to maintain TFH-associated transcriptional and metabolic signatures, which were distinct from those in Th1 cells. Mechanistically, TCF1 functioned through forming negative feedback loops with IL-2 and Blimp1. Our findings demonstrate an essential role of TCF1 in TFH cell responses to viral infection. PMID:26365183

  19. Proliferation of Double-Strand Break-Resistant Polyploid Cells Requires Drosophila FANCD2.

    PubMed

    Bretscher, Heidi S; Fox, Donald T

    2016-06-01

    Conserved DNA-damage responses (DDRs) sense genome damage and prevent mitosis of broken chromosomes. How cells lacking DDRs cope with broken chromosomes during mitosis is poorly understood. DDRs are frequently inactivated in cells with extra genomes (polyploidy), suggesting that study of polyploidy can reveal how cells with impaired DDRs/genome damage continue dividing. Here, we show that continued division and normal organ development occurs in polyploid, DDR-impaired Drosophila papillar cells. As papillar cells become polyploid, they naturally accumulate broken acentric chromosomes but do not apoptose/arrest the cell cycle. To survive mitosis with acentric chromosomes, papillar cells require Fanconi anemia proteins FANCD2 and FANCI, as well as Blm helicase, but not canonical DDR signaling. FANCD2 acts independently of previous S phases to promote alignment and segregation of acentric DNA produced by double-strand breaks, thus avoiding micronuclei and organ malformation. Because polyploidy and impaired DDRs can promote cancer, our findings provide insight into disease-relevant DNA-damage tolerance mechanisms. PMID:27270041

  20. Memory CD8(+) T Cells Require Increased Concentrations of Acetate Induced by Stress for Optimal Function.

    PubMed

    Balmer, Maria L; Ma, Eric H; Bantug, Glenn R; Grählert, Jasmin; Pfister, Simona; Glatter, Timo; Jauch, Annaïse; Dimeloe, Sarah; Slack, Emma; Dehio, Philippe; Krzyzaniak, Magdalena A; King, Carolyn G; Burgener, Anne-Valérie; Fischer, Marco; Develioglu, Leyla; Belle, Réka; Recher, Mike; Bonilla, Weldy V; Macpherson, Andrew J; Hapfelmeier, Siegfried; Jones, Russell G; Hess, Christoph

    2016-06-21

    How systemic metabolic alterations during acute infections impact immune cell function remains poorly understood. We found that acetate accumulates in the serum within hours of systemic bacterial infections and that these increased acetate concentrations are required for optimal memory CD8(+) T cell function in vitro and in vivo. Mechanistically, upon uptake by memory CD8(+) T cells, stress levels of acetate expanded the cellular acetyl-coenzyme A pool via ATP citrate lyase and promoted acetylation of the enzyme GAPDH. This context-dependent post-translational modification enhanced GAPDH activity, catalyzing glycolysis and thus boosting rapid memory CD8(+) T cell responses. Accordingly, in a murine Listeria monocytogenes model, transfer of acetate-augmented memory CD8(+) T cells exerted superior immune control compared to control cells. Our results demonstrate that increased systemic acetate concentrations are functionally integrated by CD8(+) T cells and translate into increased glycolytic and functional capacity. The immune system thus directly relates systemic metabolism with immune alertness. PMID:27212436

  1. Axonal and Schwann cell BACE1 is equally required for remyelination of peripheral nerves.

    PubMed

    Hu, Xiangyou; Hu, Jinxuan; Dai, Lu; Trapp, Bruce; Yan, Riqiang

    2015-03-01

    Inhibition of β-site APP cleaving enzyme 1 (BACE1) is being pursued as a therapeutic target for treating patients with Alzheimer's disease because BACE1 is the sole β-secretase for generating β-amyloid peptide. Knowledge regarding the other cellular functions of BACE1 is therefore critical for the safe use of BACE1 inhibitors in human patients. BACE1 deficiency in mice causes hypomyelination during development and impairs remyelination in injured sciatic nerves. Since BACE1 is expected to be ubiquitously expressed, we asked whether axonal or Schwann cell BACE1 is required for optimal remyelination. By swapping sciatic nerve segments from BACE1-null mice with the corresponding wild-type nerve segments or vice versa, we tested how a deficiency of BACE1 in Schwann cells or axons affects remyelination. Our results show that BACE1 in axons and Schwann cells is similarly important for remyelination of regenerated axons. Nerve injury induces BACE1 transcription and protein levels are elevated in Schwann cells. Expression of type I neuregulin 1 (Nrg1), rather than type III Nrg1, was induced by Schwann cells, and the abolished Nrg1 cleavage in BACE1-null Schwann cells contributed to decreased remyelination of regenerated axons. Hence, this study is the first to demonstrate the equal importance of axonal and Schwann cell BACE1 for remyelination of injured nerves. PMID:25740511

  2. An essential single domain response regulator required for normal cell division and differentiation in Caulobacter crescentus.

    PubMed Central

    Hecht, G B; Lane, T; Ohta, N; Sommer, J M; Newton, A

    1995-01-01

    Signal transduction pathways mediated by sensor histidine kinases and cognate response regulators control a variety of physiological processes in response to environmental conditions. Here we show that in Caulobacter crescentus these systems also play essential roles in the regulation of polar morphogenesis and cell division. Previous studies have implicated histidine kinase genes pleC and divJ in the regulation of these developmental events. We now report that divK encodes an essential, cell cycle-regulated homolog of the CheY/Spo0F subfamily and present evidence that this protein is a cognate response regulator of the histidine kinase PleC. The purified kinase domain of PleC, like that of DivJ, can serve as an efficient phosphodonor to DivK and as a phospho-DivK phosphatase. Based on these and earlier genetic results we propose that PleC and DivK are members of a signal transduction pathway that couples motility and stalk formation to completion of a late cell division cycle event. Gene disruption experiments and the filamentous phenotype of the conditional divK341 mutant reveal that DivK also functions in an essential signal transduction pathway required for cell division, apparently in response to another histidine kinase. We suggest that phosphotransfer mediated by these two-component signal transduction systems may represent a general mechanism regulating cell differentiation and cell division in response to successive cell cycle checkpoints. Images PMID:7664732

  3. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    SciTech Connect

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-15

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells.

  4. Kif11 dependent cell cycle progression in radial glial cells is required for proper neurogenesis in the zebrafish neural tube.

    PubMed

    Johnson, Kimberly; Moriarty, Chelsea; Tania, Nessy; Ortman, Alissa; DiPietrantonio, Kristina; Edens, Brittany; Eisenman, Jean; Ok, Deborah; Krikorian, Sarah; Barragan, Jessica; Golé, Christophe; Barresi, Michael J F

    2014-03-01

    Radial glia serve as the resident neural stem cells in the embryonic vertebrate nervous system, and their proliferation must be tightly regulated to generate the correct number of neuronal and glial cell progeny in the neural tube. During a forward genetic screen, we recently identified a zebrafish mutant in the kif11 loci that displayed a significant increase in radial glial cell bodies at the ventricular zone of the spinal cord. Kif11, also known as Eg5, is a kinesin-related, plus-end directed motor protein responsible for stabilizing and separating the bipolar mitotic spindle. We show here that Gfap+ radial glial cells express kif11 in the ventricular zone and floor plate. Loss of Kif11 by mutation or pharmacological inhibition with S-trityl-L-cysteine (STLC) results in monoastral spindle formation in radial glial cells, which is characteristic of mitotic arrest. We show that M-phase radial glia accumulate over time at the ventricular zone in kif11 mutants and STLC treated embryos. Mathematical modeling of the radial glial accumulation in kif11 mutants not only confirmed an ~226× delay in mitotic exit (likely a mitotic arrest), but also predicted two modes of increased cell death. These modeling predictions were supported by an increase in the apoptosis marker, anti-activated Caspase-3, which was also found to be inversely proportional to a decrease in cell proliferation. In addition, treatment with STLC at different stages of neural development uncovered two critical periods that most significantly require Kif11 function for stem cell progression through mitosis. We also show that loss of Kif11 function causes specific reductions in oligodendroglia and secondary interneurons and motorneurons, suggesting these later born populations require proper radial glia division. Despite these alterations to cell cycle dynamics, survival, and neurogenesis, we document unchanged cell densities within the neural tube in kif11 mutants, suggesting that a mechanism of

  5. Access of protective antiviral antibody to neuronal tissues requires CD4 T-cell help.

    PubMed

    Iijima, Norifumi; Iwasaki, Akiko

    2016-05-26

    Circulating antibodies can access most tissues to mediate surveillance and elimination of invading pathogens. Immunoprivileged tissues such as the brain and the peripheral nervous system are shielded from plasma proteins by the blood-brain barrier and blood-nerve barrier, respectively. Yet, circulating antibodies must somehow gain access to these tissues to mediate their antimicrobial functions. Here we examine the mechanism by which antibodies gain access to neuronal tissues to control infection. Using a mouse model of genital herpes infection, we demonstrate that both antibodies and CD4 T cells are required to protect the host after immunization at a distal site. We show that memory CD4 T cells migrate to the dorsal root ganglia and spinal cord in response to infection with herpes simplex virus type 2. Once inside these neuronal tissues, CD4 T cells secrete interferon-γ and mediate local increase in vascular permeability, enabling antibody access for viral control. A similar requirement for CD4 T cells for antibody access to the brain is observed after intranasal challenge with vesicular stomatitis virus. Our results reveal a previously unappreciated role of CD4 T cells in mobilizing antibodies to the peripheral sites of infection where they help to limit viral spread. PMID:27225131

  6. Glycosylphosphatidylinositol-Anchored Proteins Are Required for Cell Wall Synthesis and Morphogenesis in Arabidopsis

    PubMed Central

    Gillmor, C. Stewart; Lukowitz, Wolfgang; Brininstool, Ginger; Sedbrook, John C.; Hamann, Thorsten; Poindexter, Patricia; Somerville, Chris

    2005-01-01

    Mutations at five loci named PEANUT1-5 (PNT) were identified in a genetic screen for radially swollen embryo mutants. pnt1 cell walls showed decreased crystalline cellulose, increased pectins, and irregular and ectopic deposition of pectins, xyloglucans, and callose. Furthermore, pnt1 pollen is less viable than the wild type, and pnt1 embryos were delayed in morphogenesis and showed defects in shoot and root meristems. The PNT1 gene encodes the Arabidopsis thaliana homolog of mammalian PIG-M, an endoplasmic reticulum–localized mannosyltransferase that is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor. All five pnt mutants showed strongly reduced accumulation of GPI-anchored proteins, suggesting that they all have defects in GPI anchor synthesis. Although the mutants are seedling lethal, pnt1 cells are able to proliferate for a limited time as undifferentiated callus and do not show the massive deposition of ectopic cell wall material seen in pnt1 embryos. The different phenotype of pnt1 cells in embryos and callus suggest a differential requirement for GPI-anchored proteins in cell wall synthesis in these two tissues and points to the importance of GPI anchoring in coordinated multicellular growth. PMID:15772281

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

    PubMed Central

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

    2016-01-01

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

  8. Endothelial cell FGF signaling is required for injury response but not for vascular homeostasis

    PubMed Central

    Oladipupo, Sunday S.; Smith, Craig; Santeford, Andrea; Park, Changwon; Sene, Abdoulaye; Wiley, Luke A.; Osei-Owusu, Patrick; Hsu, Joann; Zapata, Nicole; Liu, Fang; Nakamura, Rei; Lavine, Kory J.; Blumer, Kendall J.; Choi, Kyunghee; Apte, Rajendra S.; Ornitz, David M.

    2014-01-01

    Endothelial cells (ECs) express fibroblast growth factor receptors (FGFRs) and are exquisitely sensitive to FGF signals. However, whether the EC or another vascular cell type requires FGF signaling during development, homeostasis, and response to injury is not known. Here, we show that Flk1-Cre or Tie2-Cre mediated deletion of FGFR1 and FGFR2 (Fgfr1/2Flk1-Cre or Fgfr1/2Tie2-Cre mice), which results in deletion in endothelial and hematopoietic cells, is compatible with normal embryonic development. As adults, Fgfr1/2Flk1-Cre mice maintain normal blood pressure and vascular reactivity and integrity under homeostatic conditions. However, neovascularization after skin or eye injury was significantly impaired in both Fgfr1/2Flk1-Cre and Fgfr1/2Tie2-Cre mice, independent of either hematopoietic cell loss of FGFR1/2 or vascular endothelial growth factor receptor 2 (Vegfr2) haploinsufficiency. Also, impaired neovascularization was associated with delayed cutaneous wound healing. These findings reveal a key requirement for cell-autonomous EC FGFR signaling in injury-induced angiogenesis, but not for vascular homeostasis, identifying the EC FGFR signaling pathway as a target for diseases associated with aberrant vascular proliferation, such as age-related macular degeneration, and for modulating wound healing without the potential toxicity associated with direct manipulation of systemic FGF or VEGF activity. PMID:25139991

  9. B and T cells are required for mouse mammary tumor virus spread within the mammary gland.

    PubMed

    Golovkina, T V; Dudley, J P; Ross, S R

    1998-09-01

    Mouse mammary tumor virus (MMTV) is an infectious retrovirus transmitted through milk from mother to newborns. MMTV encodes a superantigen (SAg) whose activity is indispensable for the virus life cycle, since a genetically engineered virus with a mutation in the sag gene neither amplified in cells of the immune system of suckling pups nor infected their mammary glands. When wild-type MMTV was injected directly into the mammary glands of uninfected pubescent mice, their lymphoid as well as mammary gland cells became virus infected. To test whether this infection of lymphoid cells was dependent on SAg activity and required for virus spread within the mammary gland, we performed mammary gland injections of wild-type MMTV(C3H) into two strains of transgenic mice that lacked SAg-cognate, V beta 14+ T cells. Neither the MTV-ORF or LEL strains showed infection of their mammary glands. Moreover, no MMTV infection of their peripheral lymphocytes was detected. Similar experiments with mice lacking B cells (mu-chain knockouts) showed no detectable virus spread in the mammary glands or lymphoid tissues. These data suggest that SAg activity and MMTV-infected lymphocytes are required, not only for initial steps of viral infection, but also for virus spread within the mammary gland. Virus spread at late times in infection determines whether MMTV induces mammary tumors. PMID:9725233

  10. Derivation of lung mesenchymal lineages from the fetal mesothelium requires hedgehog signaling for mesothelial cell entry

    PubMed Central

    Dixit, Radhika; Ai, Xingbin; Fine, Alan

    2013-01-01

    Recent studies have shown that mesothelial progenitors contribute to mesenchymal lineages of developing organs. To what extent the overlying mesothelium contributes to lung development remains unknown. To rigorously address this question, we employed Wt1CreERT2/+ mice for high-fidelity lineage tracing after confirming that Cre recombinase was mesothelial specific and faithfully recapitulated endogenous Wilms’ tumor 1 (Wt1) gene expression. We visualized WT1+ mesothelial cell entry into the lung by live imaging and identified their progenies in subpopulations of bronchial smooth muscle cells, vascular smooth muscle cells and desmin+ fibroblasts by lineage tagging. Derivation of these lineages was only observed with Cre recombinase activation during early lung development. Using loss-of-function assays in organ cultures, and targeted mesothelial-restricted hedgehog loss-of-function mice, we demonstrated that mesothelial cell movement into the lung requires the direct action of hedgehog signaling. By contrast, hedgehog signaling was not required for fetal mesothelial heart entry. These findings further support a paradigm wherein the mesothelium is a source of progenitors for mesenchymal lineages during organogenesis and indicate that signals controlling mesothelial cell entry are organ specific. PMID:24130328

  11. Autoreactive marginal zone B cells are spontaneously activated but lymph node B cells require T cell help

    PubMed Central

    Mandik-Nayak, Laura; Racz, Jennifer; Sleckman, Barry P.; Allen, Paul M.

    2006-01-01

    In K/BxN mice, arthritis is induced by autoantibodies against glucose-6-phosphate-isomerase (GPI). To investigate B cell tolerance to GPI in nonautoimmune mice, we increased the GPI-reactive B cell frequency using a low affinity anti-GPI H chain transgene. Surprisingly, anti-GPI B cells were not tolerant to this ubiquitously expressed and circulating autoantigen. Instead, they were found in two functionally distinct compartments: an activated population in the splenic marginal zone (MZ) and an antigenically ignorant one in the recirculating follicular/lymph node (LN) pool. This difference in activation was due to increased autoantigen availability in the MZ. Importantly, the LN anti-GPI B cells remained functionally competent and could be induced to secrete autoantibodies in response to cognate T cell help in vitro and in vivo. Therefore, our study of low affinity autoreactive B cells reveals two distinct but potentially concurrent mechanisms for their activation, of which one is T cell dependent and the other is T cell independent. PMID:16880262

  12. FOXOs support the metabolic requirements of normal and tumor cells by promoting IDH1 expression

    PubMed Central

    Charitou, Paraskevi; Rodriguez-Colman, Maria; Gerrits, Johan; van Triest, Miranda; Groot Koerkamp, Marian; Hornsveld, Marten; Holstege, Frank; Verhoeven-Duif, Nanda M; Burgering, Boudewijn MT

    2015-01-01

    FOXO transcription factors are considered bona fide tumor suppressors; however, recent studies showed FOXOs are also required for tumor survival. Here, we identify FOXOs as transcriptional activators of IDH1. FOXOs promote IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise stimulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation and inhibits TET enzymes and histone demethylases. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion. PMID:25648147

  13. Identification of TRAPPC8 as a Host Factor Required for Human Papillomavirus Cell Entry

    PubMed Central

    Ishii, Yoshiyuki; Nakahara, Tomomi; Kataoka, Michiyo; Kusumoto-Matsuo, Rika; Mori, Seiichiro; Takeuchi, Takamasa; Kukimoto, Iwao

    2013-01-01

    Human papillomavirus (HPV) is a non-enveloped virus composed of a circular DNA genome and two capsid proteins, L1 and L2. Multiple interactions between its capsid proteins and host cellular proteins are required for infectious HPV entry, including cell attachment and internalization, intracellular trafficking and viral genome transfer into the nucleus. Using two variants of HPV type 51, the Ma and Nu strains, we have previously reported that MaL2 is required for efficient pseudovirus (PsV) transduction. However, the cellular factors that confer this L2 dependency have not yet been identified. Here we report that the transport protein particle complex subunit 8 (TRAPPC8) specifically interacts with MaL2. TRAPPC8 knockdown in HeLa cells yielded reduced levels of reporter gene expression when inoculated with HPV51Ma, HPV16, and HPV31 PsVs. TRAPPC8 knockdown in HaCaT cells also showed reduced susceptibility to infection with authentic HPV31 virions, indicating that TRAPPC8 plays a crucial role in native HPV infection. Immunofluorescence microscopy revealed that the central region of TRAPPC8 was exposed on the cell surface and colocalized with inoculated PsVs. The entry of Ma, Nu, and L2-lacking PsVs into cells was equally impaired in TRAPPC8 knockdown HeLa cells, suggesting that TRAPPC8-dependent endocytosis plays an important role in HPV entry that is independent of L2 interaction. Finally, expression of GFP-fused L2 that can also interact with TRAPPC8 induced dispersal of the Golgi stack structure in HeLa cells, a phenotype also observed by TRAPPC8 knockdown. These results suggest that during viral intracellular trafficking, binding of L2 to TRAPPC8 inhibits its function resulting in Golgi destabilization, a process that may assist HPV genome escape from the trans-Golgi network. PMID:24244674

  14. Inflammasome-independent NLRP3 is required for epithelial-mesenchymal transition in colon cancer cells.

    PubMed

    Wang, Hong; Wang, Yajing; Du, Qianming; Lu, Ping; Fan, Huimin; Lu, Jinrong; Hu, Rong

    2016-03-15

    Inflammasome NLRP3 plays a crucial role in the process of colitis and colitis--associated colon cancer. Even though much is known regarding the NLRP3 inflammasome that regulates pro-inflammatory cytokine release in innate immune cells, the role of NLRP3 in non-immune cells is still unclear. In this study, we showed that NLRP3 was highly expressed in mesenchymal-like colon cancer cells (SW620), and was upregulated by tumor necrosis factors-α (TNF-α) and transforming growth factor-β1 (TGF-β1) respectively, during EMT in colon cancer epithelial cells HCT116 and HT29. Knockdown of NLRP3 retained epithelial spindle-like morphology of HCT116 and HT29 cells and reversed the mesenchymal characteristic of SW620 cells, indicated by the decreased expression of vimentin and MMP9 and increased expression of E-cadherin. In addition, knockdown of NLRP3 in colorectal carcinoma cells displayed diminished cell migration and invasion. Interestingly, during the EMT process induced by TNF-α or TGF-β1, the cleaved caspase-1 and ASC speck were not detected, indicating that NLRP3 functions in an inflammasome-independent way. Further studies demonstrated that NLRP3 protein expression was regulated by NF-κB signaling in TNF-α or TGF-β1-induced EMT, as verified by the NF-κB inhibitor Bay 11-7082. Moreover, NLRP3 knockdown reduced the expression of Snail1, indicating that NLRP3 may promote EMT through regulating Snail1. In summary, our results showed that the NLRP3 expression, not the inflammasome activation, was required for EMT in colorectal cancer cells. PMID:26968633

  15. Stable inhibitory activity of regulatory T cells requires the transcription factor Helios.

    PubMed

    Kim, Hye-Jung; Barnitz, R Anthony; Kreslavsky, Taras; Brown, Flavian D; Moffett, Howell; Lemieux, Madeleine E; Kaygusuz, Yasemin; Meissner, Torsten; Holderried, Tobias A W; Chan, Susan; Kastner, Philippe; Haining, W Nicholas; Cantor, Harvey

    2015-10-16

    The maintenance of immune homeostasis requires regulatory T cells (T(regs)). Given their intrinsic self-reactivity, T(regs) must stably maintain a suppressive phenotype to avoid autoimmunity. We report that impaired expression of the transcription factor (TF) Helios by FoxP3(+) CD4 and Qa-1-restricted CD8 T(regs) results in defective regulatory activity and autoimmunity in mice. Helios-deficient T(regs) develop an unstable phenotype during inflammatory responses characterized by reduced FoxP3 expression and increased effector cytokine expression secondary to diminished activation of the STAT5 pathway. CD8 T(regs) also require Helios-dependent STAT5 activation for survival and to prevent terminal T cell differentiation. The definition of Helios as a key transcription factor that stabilizes T(regs) in the face of inflammatory responses provides a genetic explanation for a core property of T(regs). PMID:26472910

  16. Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress

    PubMed Central

    Malcolm, Tim I. M.; Villarese, Patrick; Fairbairn, Camilla J.; Lamant, Laurence; Trinquand, Amélie; Hook, C. Elizabeth; Burke, G. A. Amos; Brugières, Laurence; Hughes, Katherine; Payet, Dominique; Merkel, Olaf; Schiefer, Ana-Iris; Ashankyty, Ibraheem; Mian, Shahid; Wasik, Mariusz; Turner, Martin; Kenner, Lukas; Asnafi, Vahid; Macintyre, Elizabeth; Turner, Suzanne D.

    2016-01-01

    Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM–ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy. PMID:26753883

  17. Appressorium formation in the corn smut fungus Ustilago maydis requires a G2 cell cycle arrest

    PubMed Central

    Castanheira, Sónia; Pérez-Martín, José

    2015-01-01

    Many of the most important plant diseases are caused by fungal pathogens that form specialized cell structures to breach the leaf surface as well as to proliferate inside the plant. To initiate pathogenic development, the fungus responds to a set of inductive cues. Some of them are of extracellular nature (environmental signals) while others respond to intracellular conditions (developmental signals). These signals have to be integrated into a single response that has as a major outcome changes in the morphogenesis of the fungus. The cell cycle regulation is pivotal during these cellular differentiations, and we hypothesized that cell cycle regulation would be likely to provide control points for infection development by fungal pathogens. Although efforts have been done in various fungal systems, there is still limited information available regarding the relationship of these processes with the induction of the virulence programs. Hence, the role of fungal cell cycle regulators –which are wide conserved elements– as true virulence factors, has yet to be defined. Here we discuss the recent finding that the formation of the appressorium, a structure required for plant penetration, in the corn smut fungus Ustilago maydis seems to be incompatible with an active cell cycle and, therefore genetic circuits evolved in this fungus to arrest the cell cycle during the growth of this fungus on plant surface, before the appressorium-mediated penetration into the plant tissue. PMID:25876077

  18. Appressorium formation in the corn smut fungus Ustilago maydis requires a G2 cell cycle arrest.

    PubMed

    Castanheira, Sónia; Pérez-Martín, José

    2015-01-01

    Many of the most important plant diseases are caused by fungal pathogens that form specialized cell structures to breach the leaf surface as well as to proliferate inside the plant. To initiate pathogenic development, the fungus responds to a set of inductive cues. Some of them are of extracellular nature (environmental signals) while others respond to intracellular conditions (developmental signals). These signals have to be integrated into a single response that has as a major outcome changes in the morphogenesis of the fungus. The cell cycle regulation is pivotal during these cellular differentiations, and we hypothesized that cell cycle regulation would be likely to provide control points for infection development by fungal pathogens. Although efforts have been done in various fungal systems, there is still limited information available regarding the relationship of these processes with the induction of the virulence programs. Hence, the role of fungal cell cycle regulators -which are wide conserved elements- as true virulence factors, has yet to be defined. Here we discuss the recent finding that the formation of the appressorium, a structure required for plant penetration, in the corn smut fungus Ustilago maydis seems to be incompatible with an active cell cycle and, therefore genetic circuits evolved in this fungus to arrest the cell cycle during the growth of this fungus on plant surface, before the appressorium-mediated penetration into the plant tissue. PMID:25876077

  19. Polycomb repressive complex 2 component Suz12 is required for hematopoietic stem cell function and lymphopoiesis.

    PubMed

    Lee, Stanley C W; Miller, Sarah; Hyland, Craig; Kauppi, Maria; Lebois, Marion; Di Rago, Ladina; Metcalf, Donald; Kinkel, Sarah A; Josefsson, Emma C; Blewitt, Marnie E; Majewski, Ian J; Alexander, Warren S

    2015-07-01

    Polycomb repressive complex 2 (PRC2) is a chromatin modifier that regulates stem cells in embryonic and adult tissues. Loss-of-function studies of PRC2 components have been complicated by early embryonic dependence on PRC2 activity and the partial functional redundancy of enhancer of zeste homolog 1 (Ezh1) and enhancer of zeste homolog 2 (Ezh2), which encode the enzymatic component of PRC2. Here, we investigated the role of PRC2 in hematopoiesis by conditional deletion of suppressor of zeste 12 protein homolog (Suz12), a core component of PRC2. Complete loss of Suz12 resulted in failure of hematopoiesis, both in the embryo and the adult, with a loss of maintenance of hematopoietic stem cells (HSCs). In contrast, partial loss of PRC2 enhanced HSC self-renewal. Although Suz12 was required for lymphoid development, deletion in individual blood cell lineages revealed that it was dispensable for the development of granulocytic, monocytic, and megakaryocytic cells. Collectively, these data reveal the multifaceted role of PRC2 in hematopoiesis, with divergent dose-dependent effects in HSC and distinct roles in maturing blood cells. Because PRC2 is a potential target for cancer therapy, the significant consequences of modest changes in PRC2 activity, as well as the cell and developmental stage-specific effects, will need to be carefully considered in any therapeutic context. PMID:26036803

  20. tmie Is required for gentamicin uptake by the hair cells of mice.

    PubMed

    Park, Seojin; Lee, Jeong-Han; Cho, Hyun-Ju; Lee, Kyu-yup; Kim, Myoung Ok; Yun, Byung-Wook; Ryoo, ZaeYoung

    2013-04-01

    The circling (cir/cir) mouse is a spontaneous model of deafness due to deletion of a 40-kb genomic region that includes the transmembrane inner ear (tmie) gene. In addition to being deaf, cir/cir mice exhibit abnormal behaviors including circling and hyperactivity. Here we investigated differences between 3-d-old (that is, before hair-cell degeneration) cir/cir and phenotypically normal (+/cir) mice and the reason underlying the degeneration of the inner ear structure of cir/cir mice. To this end, we used gentamicin, gentamicin-Texas red conjugate, and FM1-43 to investigate mechanotransducer channel activity in the hair cells of cir/cir mice; these compounds are presumed to enter hair cells through the mechanotransducer channel. Although the structure of the inner ear of +/cir mice was equivalent to that of cir/cir mice, the hair cells of cir/cir mice (unlike +/cir) did not take up gentamicin, gentamicin-Texas red conjugate, or FM1-43. These findings suggest that hair cells in cir/cir mice demonstrate abnormal maturation and mechanotransduction. In addition, our current results indicate that tmie is required for maturation and maintenance of hair cells. PMID:23582420

  1. Anaplastic large cell lymphoma arises in thymocytes and requires transient TCR expression for thymic egress.

    PubMed

    Malcolm, Tim I M; Villarese, Patrick; Fairbairn, Camilla J; Lamant, Laurence; Trinquand, Amélie; Hook, C Elizabeth; Burke, G A Amos; Brugières, Laurence; Hughes, Katherine; Payet, Dominique; Merkel, Olaf; Schiefer, Ana-Iris; Ashankyty, Ibraheem; Mian, Shahid; Wasik, Mariusz; Turner, Martin; Kenner, Lukas; Asnafi, Vahid; Macintyre, Elizabeth; Turner, Suzanne D

    2016-01-01

    Anaplastic large cell lymphoma (ALCL) is a peripheral T-cell lymphoma presenting mostly in children and young adults. The natural progression of this disease is largely unknown as is the identity of its true cell of origin. Here we present a model of peripheral ALCL pathogenesis where the malignancy is initiated in early thymocytes, before T-cell receptor (TCR) β-rearrangement, which is bypassed in CD4/NPM-ALK transgenic mice following Notch1 expression. However, we find that a TCR is required for thymic egress and development of peripheral murine tumours, yet this TCR must be downregulated for T-cell lymphomagenesis. In keeping with this, clonal TCR rearrangements in human ALCL are predominantly in-frame, but often aberrant, with clonal TCRα but no comparable clonal TCRβ rearrangement, yielding events that would not normally be permissive for survival during thymic development. Children affected by ALCL may thus harbour thymic lymphoma-initiating cells capable of seeding relapse after chemotherapy. PMID:26753883

  2. LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding

    PubMed Central

    Layer, Justin H.; Alford, Catherine E.; McDonald, W. Hayes

    2015-01-01

    LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R320LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens. PMID:26598604

  3. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels

    PubMed Central

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G.; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J.; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M.; Kaczmarek, Leonard K.; Flavell, Richard A.

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling. PMID:26815481

  4. Requirement of full TCR repertoire for regulatory T cells to maintain intestinal homeostasis

    PubMed Central

    Nishio, Junko; Baba, Minato; Atarashi, Koji; Tanoue, Takeshi; Negishi, Hideo; Yanai, Hideyuki; Habu, Sonoko; Hori, Shohei; Honda, Kenya; Taniguchi, Tadatsugu

    2015-01-01

    The regulation of intestinal homeostasis by the immune system involves the dynamic interplay between gut commensal microbiota and resident immune cells. It is well known that a large and diverse lymphocyte antigen receptor repertoire enables the immune system to recognize and respond to a wide range of invading pathogens. There is also an emerging appreciation for a critical role the T-cell receptor (TCR) repertoire serves in the maintenance of peripheral tolerance by regulatory T cells (Tregs). Nevertheless, how the diversity of the TCR repertoire in Tregs affects intestinal homeostasis remains unknown. To address this question, we studied mice whose T cells express a restricted TCR repertoire. We observed the development of spontaneous colitis, accompanied by the induction of T-helper type 17 cells in the colon that is driven by gut commensal microbiota. We provide further evidence that a restricted TCR repertoire causes a loss of tolerogenicity to microbiota, accompanied by a paucity of peripherally derived, Helios− Tregs and hyperactivation of migratory dendritic cells. These results thus reveal a new facet of the TCR repertoire in which Tregs require a diverse TCR repitoire for intestinal homeostasis, suggesting an additional driving force in the evolutional significance of the TCR repertoire. PMID:26420876

  5. T Cell Receptor Mediated Calcium Entry Requires Alternatively Spliced Cav1.1 Channels.

    PubMed

    Matza, Didi; Badou, Abdallah; Klemic, Kathryn G; Stein, Judith; Govindarajulu, Usha; Nadler, Monica J; Kinet, Jean-Pierre; Peled, Amnon; Shapira, Oz M; Kaczmarek, Leonard K; Flavell, Richard A

    2016-01-01

    The process of calcium entry in T cells is a multichannel and multi-step process. We have studied the requirement for L-type calcium channels (Cav1.1) α1S subunits during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Overexpression studies using cloned T cell Cav1.1 in 293HEK cells (that lack TCR) suggest that the gating of these channels was altered. Knockdown of Cav1.1 channels in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling. PMID:26815481

  6. Requirement of full TCR repertoire for regulatory T cells to maintain intestinal homeostasis.

    PubMed

    Nishio, Junko; Baba, Minato; Atarashi, Koji; Tanoue, Takeshi; Negishi, Hideo; Yanai, Hideyuki; Habu, Sonoko; Hori, Shohei; Honda, Kenya; Taniguchi, Tadatsugu

    2015-10-13

    The regulation of intestinal homeostasis by the immune system involves the dynamic interplay between gut commensal microbiota and resident immune cells. It is well known that a large and diverse lymphocyte antigen receptor repertoire enables the immune system to recognize and respond to a wide range of invading pathogens. There is also an emerging appreciation for a critical role the T-cell receptor (TCR) repertoire serves in the maintenance of peripheral tolerance by regulatory T cells (Tregs). Nevertheless, how the diversity of the TCR repertoire in Tregs affects intestinal homeostasis remains unknown. To address this question, we studied mice whose T cells express a restricted TCR repertoire. We observed the development of spontaneous colitis, accompanied by the induction of T-helper type 17 cells in the colon that is driven by gut commensal microbiota. We provide further evidence that a restricted TCR repertoire causes a loss of tolerogenicity to microbiota, accompanied by a paucity of peripherally derived, Helios(-) Tregs and hyperactivation of migratory dendritic cells. These results thus reveal a new facet of the TCR repertoire in which Tregs require a diverse TCR repitoire for intestinal homeostasis, suggesting an additional driving force in the evolutional significance of the TCR repertoire. PMID:26420876

  7. Sensor Needs and Requirements for Fuel Cells and CIDI/SIDI Engines

    SciTech Connect

    Glass, R.S.

    2000-03-01

    To reduce U.S. dependence on imported oil, improve urban air quality, and decrease greenhouse gas emissions, the Department of Energy (DOE) is developing advanced vehicle technologies and fuels. Enabling technologies for fuel cell power systems and direct-injection engines are being developed by DOE through the Partnership for a New Generation of Vehicles (PNGV), a government-industry collaboration to produce vehicles having up to three times the fuel economy of conventional mid-size automobiles. Sensors have been identified as a research and development need for both fuel cell and direct-injection systems, because current sensor technologies do not adequately meet requirements. Sensors are needed for emission control, for passenger safety and comfort, to increase system lifetime, and for system performance enhancement through feedback and control. These proceedings document the results of a workshop to define sensor requirements for proton exchange membrane (PEM) fuel cell systems and direct-injection engines for automotive applications. The recommendations from this workshop will be incorporated into the multi-year R&D plan of the DOE Office of Advanced Automotive Technologies. The objectives of the workshop were to: define the requirements for sensors; establish R&D priorities; identify the technical targets and technical barriers; and facilitate collaborations among participants. The recommendations from this workshop will be incorporated into the multi-year R&D plan of the DOE Office of Advanced Automotive Technologies.

  8. Notch2 is required for maintaining sustentacular cell function in the adult mouse main olfactory epithelium

    PubMed Central

    Rodriguez, Steve; Sickles, Heather M.; DeLeonardis, Chris; Alcaraz, Ana; Gridley, Thomas; Lin, David M.

    2008-01-01

    Notch receptors are expressed in neurons and glia in the adult nervous system, but why this expression persists is not well-understood. Here we examine the role of the Notch pathway in the postnatal mouse main olfactory system, and show evidence consistent with a model where Notch2 is required for maintaining sustentacular cell function. In the absence of Notch2, the laminar nature of these glial-like cells is disrupted. Hes1, Hey1, and Six1, which are downstream effectors of the Notch pathway, are down-regulated, and cytochrome P450 and Glutathione S-transferase (GST) expression by sustentacular cells is reduced. Functional levels of GST activity are also reduced. These disruptions are associated with increased olfactory sensory neuron degeneration. Surprisingly, expression of Notch3 is also down-regulated. This suggests the existence of a feedback loop where expression of Notch3 is initially independent of Notch2, but requires Notch2 for maintained expression. While the Notch pathway has previously been shown to be important for promoting gliogenesis during development, this is the first demonstration that the persistent expression of Notch receptors is required for maintaining glial function in adult. PMID:18155189

  9. Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients

    SciTech Connect

    Orme, I.M.; Collins, F.M.

    1983-07-01

    The results of this study demonstrate that spleen cells taken from mice at the height of the primary immune response to intravenous infection with Mycobacterium tuberculosis possess the capacity to transfer adoptive protection to M. tuberculosis-infected recipients, but only if these recipients are first rendered T cell-deficient, either by thymectomy and gamma irradiation, or by sublethal irradiation. A similar requirement was necessary to demonstrate the adoptive protection of the lungs after exposure to an acute aerosol-delivered M. tuberculosis infection. In both infectious models successful adoptive immunotherapy was shown to be mediated by T lymphocytes, which were acquired in the donor animals in response to the immunizing infection. It is proposed that the results of this study may serve as a basic model for the subsequent analysis of the nature of the T cell-mediated immune response to both systemic and aerogenic infections with M. tuberculosis.

  10. Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes

    PubMed Central

    Inoue, Shinya; Kondo, Shigeru; Parichy, David M.; Watanabe, Masakatsu

    2014-01-01

    Summary Skin pigment pattern formation in zebrafish requires pigment-cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N-glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries. PMID:24734316

  11. Tubulin glycylases are required for primary cilia, control of cell proliferation and tumor development in colon

    PubMed Central

    Rocha, Cecilia; Papon, Laura; Cacheux, Wulfran; Marques Sousa, Patricia; Lascano, Valeria; Tort, Olivia; Giordano, Tiziana; Vacher, Sophie; Lemmers, Benedicte; Mariani, Pascale; Meseure, Didier; Medema, Jan Paul; Bièche, Ivan; Hahne, Michael; Janke, Carsten

    2014-01-01

    TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development. PMID:25180231

  12. Tubulin glycylases are required for primary cilia, control of cell proliferation and tumor development in colon.

    PubMed

    Rocha, Cecilia; Papon, Laura; Cacheux, Wulfran; Marques Sousa, Patricia; Lascano, Valeria; Tort, Olivia; Giordano, Tiziana; Vacher, Sophie; Lemmers, Benedicte; Mariani, Pascale; Meseure, Didier; Medema, Jan Paul; Bièche, Ivan; Hahne, Michael; Janke, Carsten

    2014-10-01

    TTLL3 and TTLL8 are tubulin glycine ligases catalyzing posttranslational glycylation of microtubules. We show here for the first time that these enzymes are required for robust formation of primary cilia. We further discover the existence of primary cilia in colon and demonstrate that TTLL3 is the only glycylase in this organ. As a consequence, colon epithelium shows a reduced number of primary cilia accompanied by an increased rate of cell division in TTLL3-knockout mice. Strikingly, higher proliferation is compensated by faster tissue turnover in normal colon. In a mouse model for tumorigenesis, lack of TTLL3 strongly promotes tumor development. We further demonstrate that decreased levels of TTLL3 expression are linked to the development of human colorectal carcinomas. Thus, we have uncovered a novel role for tubulin glycylation in primary cilia maintenance, which controls cell proliferation of colon epithelial cells and plays an essential role in colon cancer development. PMID:25180231

  13. Epithelial cell migration requires the interaction between the vimentin and keratin intermediate filaments

    PubMed Central

    Velez-delValle, Cristina; Marsch-Moreno, Meytha; Castro-Muñozledo, Federico; Galván-Mendoza, Ivan J.; Kuri-Harcuch, Walid

    2016-01-01

    Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells. PMID:27072292

  14. Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells

    SciTech Connect

    Morita, Akinori; Tanimoto, Keiji; Murakami, Tomoki; Morinaga, Takeshi; Hosoi, Yoshio

    2014-01-24

    Highlights: • Oxidative ATM activation can occur in the absence of nuclear DNA damage response. • The oxidized Hep G2 cells were subjected to subcellular fractionation. • The obtained results suggest that the ATM activation occurs in mitochondria. • ATM failed to respond to oxidative stress in mitochondria-depleted Hep G2 cells. • Mitochondria are required for the oxidative activation of ATM. - Abstract: Ataxia–telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized ATM can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of ATM, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that ATM can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated ATM was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and active ATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria

  15. Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.

    PubMed

    Oztan, Asli; Silvis, Mark; Weisz, Ora A; Bradbury, Neil A; Hsu, Shu-Chan; Goldenring, James R; Yeaman, Charles; Apodaca, Gerard

    2007-10-01

    The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A. PMID:17686995

  16. Human mitochondrial transcription factor A is required for the segregation of mitochondrial DNA in cultured cells.

    PubMed

    Kasashima, Katsumi; Sumitani, Megumi; Endo, Hitoshi

    2011-01-15

    The segregation and transmission of the mitochondrial genome in humans are complicated processes but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. However, the molecular mechanism of the segregation of mitochondrial DNA (mtDNA) is largely unclear. In this study, we demonstrated that human mitochondrial transcription factor A (TFAM) is required for the segregation of mtDNA in cultured cells. RNAi-mediated knockdown of TFAM in HeLa cells resulted in the enlarged mtDNA, as indicated by the assembly of fluorescent signals stained with PicoGreen. Fluorescent in situ hybridization confirmed the enlarged mtDNA and further showed the existence of increased numbers of mitochondria lacking mtDNA signals in TFAM knockdown cells. By complementation analysis, the C-terminal tail of TFAM, which enhances its affinity with DNA, was found to be required for the appropriate distribution of mtDNA. Furthermore, we found that TFAM knockdown induced asymmetric segregation of mtDNA between dividing daughter cells. These results suggest an essential role for human TFAM in symmetric segregation of mtDNA. PMID:20955698

  17. Clustering of neuronal sodium channels requires contact with myelinating Schwann cells.

    PubMed

    Ching, W; Zanazzi, G; Levinson, S R; Salzer, J L

    1999-01-01

    Efficient and rapid conduction of action potentials by saltatory conduction requires the clustering of voltage-gated sodium channels at nodes of Ranvier. This clustering results from interactions between neurons and myelinating glia, although it has not been established whether this glial signal is contact-dependent or soluble. To investigate the nature of this signal, we examined sodium channel clustering in co-cultures of embryonic rat dorsal root ganglion neurons and Schwann cells. Cultures maintained under conditions promoting or preventing myelination were immunostained with antibodies against the alpha subunit of the sodium channel and against ankyrin(G), a cytoskeletal protein associated with these channels. Consistent with previous in vivo studies (Vabnick et al., 1996), sodium channels and ankyrin G cluster at the onset of myelination. These clusters form adjacent to the ends of the myelinating Schwann cells and appear to fuse to form mature nodes. In contrast, sodium channels and ankyrin G do not cluster in neurons grown alone or in co-cultures where myelination is precluded by growing cells in defined media. Conditioned media from myelinating co-cultures also failed to induce sodium channel or ankyrin G clusters in cultures of neurons alone. Finally, no clusters develop in the amyelinated portions of suspended fascicles of dorsal root ganglia explants despite being in close proximity to myelinated segments in other areas of the dish. These results indicate that clustering of sodium channels requires contact with myelinating Schwann cells. PMID:10739572

  18. The intracellular bacterium Rhodococcus equi requires Mac-1 to bind to mammalian cells.

    PubMed Central

    Hondalus, M K; Diamond, M S; Rosenthal, L A; Springer, T A; Mosser, D M

    1993-01-01

    Rhodococcus equi is a facultative intracellular bacterium of macrophages that causes disease in immunocompromised individuals, particularly those with AIDS. In this report, we demonstrate that R. equi binding to mammalian cells requires complement and is mediated primarily by the leukocyte complement receptor, Mac-1. Bacteria bind to macrophages poorly unless exogenous complement is added to the incubation medium. The addition of fresh nonimmune serum, which contains no detectable antibodies to R. equi, greatly enhances bacterial binding to macrophages, whereas heat inactivation of this serum or immunological depletion of C3 from the serum reduces binding to levels only slightly higher than those of binding under serum-free conditions. Human serum depleted of C2 or C4 is fully opsonic, indicating that complement activation and fixation occur by the alternative pathway. The serum-dependent binding of rhodococci to macrophages is mediated primarily by the macrophage complement receptor type 3, Mac-1 (CD11b/CD18). Bacteria do not bind to fibroblastoid or epithelial cells that lack this receptor. Most of the bacterial binding to macrophages is inhibited by a monoclonal antibody to Mac-1 but is unaffected by a monoclonal antibody to complement receptor type 1. Furthermore, opsonized, but not unopsonized, bacteria bind to purified Mac-1 immobilized on plastic. In addition, in the presence of opsonic complement, rhodococci bind efficiently to fibroblastoid cells transfected with cloned Mac-1 but relatively poorly to cells transfected with the complement receptor type 1. Hence, R. equi fixes complement by activating the alternative complement pathway, and this fixation is a requirement for bacterial adhesion and invasion. Furthermore, complement fixation defines rhodococcal host cell tropism, since R. equi binds specifically and exclusively to cells expressing Mac-1. Images PMID:8514396

  19. STAT2 Is Required for TLR-Induced Murine Dendritic Cell Activation and Cross-Presentation.

    PubMed

    Xu, Jun; Lee, Michael H; Chakhtoura, Marita; Green, Benjamin L; Kotredes, Kevin P; Chain, Robert W; Sriram, Uma; Gamero, Ana M; Gallucci, Stefania

    2016-07-01

    TLR-stimulated cross-presentation by conventional dendritic cells (cDCs) is important in host defense and antitumor immunity. We recently reported that cDCs lacking the type I IFN signaling molecule STAT2 are impaired in cross-presenting tumor Ags to CD8(+) T cells. To investigate how STAT2 affects cross-presentation, we determined its requirements for dendritic cell activation. In this study, we report that STAT2 is essential for the activation of murine female cDCs upon TLR3, -4, -7, and -9 stimulation. In response to various TLR ligands, Stat2(-/-) cDCs displayed reduced expression of costimulatory molecules and type I IFN-stimulated genes. The cDC responses to exogenous IFN-α that we evaluated required STAT2 activation, indicating that the canonical STAT1-STAT2 heterodimers are the primary signaling transducers of type I IFNs in cDCs. Interestingly, LPS-induced production of IL-12 was STAT2 and type I IFN receptor (IFNAR) dependent, whereas LPS-induced production of TNF-α and IL-6 was STAT2 and IFNAR independent, suggesting a specific role of the IFNAR-STAT2 axis in the stimulation of proinflammatory cytokines by LPS in cDCs. In contrast, R848- and CpG-induced cytokine production was less influenced by the IFNAR-STAT2 axis. Short kinetics and IFNAR blockade studies showed that STAT2 main function is to transduce signals triggered by autocrine type I IFNs. Importantly, Stat2(-/-) cDCs were deficient in cross-presenting to CD8(+) T cells in vitro upon IFN-α, CpG, and LPS stimulation, and also in cross-priming and licensing cytotoxic T cell killers in vivo. We conclude that STAT2 plays a critical role in TLR-induced dendritic cell activation and cross-presentation, and thus is vital in host defense. PMID:27233962

  20. Diaphanous-Related Formin 2 and Profilin I Are Required for Gastrulation Cell Movements

    PubMed Central

    Lai, Shih-Lei; Chan, Tun-Hao; Lin, Meng-Ju; Huang, Wei-Pang; Lou, Show-Wan; Lee, Shyh-Jye

    2008-01-01

    Intensive cellular movements occur during gastrulation. These cellular movements rely heavily on dynamic actin assembly. Rho with its associated proteins, including the Rho-activated formin, Diaphanous, are key regulators of actin assembly in cellular protrusion and migration. However, the function of Diaphanous in gastrulation cell movements remains unclear. To study the role of Diaphanous in gastrulation, we isolated a partial zebrafish diaphanous-related formin 2 (zdia2) clone with its N-terminal regulatory domains. The GTPase binding domain of zDia2 is highly conserved compared to its mammalian homologues. Using a yeast two-hybrid assay, we showed that zDia2 interacts with constitutively-active RhoA and Cdc42. The zdia2 mRNAs were ubiquitously expressed during early embryonic development in zebrafish as determined by RT-PCR and whole-mount in situ hybridization analyses. Knockdown of zdia2 by antisense morpholino oligonucleotides (MOs) blocked epiboly formation and convergent extension in a dose-dependent manner, whereas ectopic expression of a human mdia gene partially rescued these defects. Time-lapse recording further showed that bleb-like cellular processes of blastoderm marginal deep marginal cells and pseudopod-/filopod-like processes of prechordal plate cells and lateral cells were abolished in the zdia2 morphants. Furthermore, zDia2 acts cell-autonomously since transplanted zdia2-knockdown cells exhibited low protrusive activity with aberrant migration in wild type host embryos. Lastly, co-injection of antisense MOs of zdia2 and zebrafish profilin I (zpfn 1), but not zebrafish profilin II, resulted in a synergistic inhibition of gastrulation cell movements. These results suggest that zDia2 in conjunction with zPfn 1 are required for gastrulation cell movements in zebrafish. PMID:18941507

  1. Curing of yeast [PSI+] prion by guanidine inactivation of Hsp104 does not require cell division.

    PubMed

    Wu, Yue-Xuan; Greene, Lois E; Masison, Daniel C; Eisenberg, Evan

    2005-09-01

    Propagation of the yeast prion [PSI+], a self-replicating aggregated form of Sup35p, requires Hsp104. One model to explain this phenomenon proposes that, in the absence of Hsp104, Sup35p aggregates enlarge but fail to replicate thus becoming diluted out as the yeast divide. To test this model, we used live imaging of Sup35p-GFP to follow the changes that occur in [PSI+] cells after the addition of guanidine to inactivate Hsp104. After guanidine addition there was initially an increase in aggregation of Sup35p-GFP; but then, before the yeast divided, the aggregates began to dissolve, and after approximately 6 h the Sup35-GFP looked identical to the Sup35-GFP in [psi+] cells. Although plating studies showed that the yeast were still [PSI+], this reduction in aggregation suggested that curing of [PSI+] by inactivation of Hsp104 might be independent of cell division. This was tested by measuring the rate of curing of [PSI+] cells in both dividing and nondividing cells. Cell division was inhibited by adding either alpha factor or farnesol. Remarkably, with both of these methods, we found that the rate of curing was not significantly affected by cell division. Thus, cell division is not a determining factor for curing [PSI+] by inactivating Hsp104 with guanidine. Rather, curing apparently occurs because Sup35-GFP polymers slowly depolymerize in the absence of Hsp104 activity. Hsp104 then counteracts this curing possibly by catalyzing formation of new polymers. PMID:16123122

  2. Analysis of dynamic requirements for fuel cell systems for vehicle applications

    NASA Astrophysics Data System (ADS)

    Pischinger, Stefan; Schönfelder, Carsten; Ogrzewalla, Jürgen

    Conventional vehicles with internal combustion engines, as well as battery powered electric vehicles, achieve one of the most important customer requirements; achieving extremely short response times to load changes. Also, fast acceleration times from a cold start to full power in the range of seconds are practicable. New fuel cell-based propulsion systems, as well as auxiliary power units, have to fulfill the same demands to become competitive. This includes heating-up the system to operating temperature as well as the control strategy for start-up. An additional device to supply starting air is necessary, if the compressor motor can only be operated with fuel cell voltage. Since the system components (for example, the air supply or the fuel supply) are not mechanically coupled, as is the case with conventional internal combustion engines, these components have to be controlled by different sensors and actuators. This can be an advantage in optimizing the system, but it also can represent an additional challenge. This paper describes the fuel cell system requirements regarding transient operation and their dependence on system structure. In particular, the requirements for peripheral components such as air supply, fuel supply and the balance of heat in a fuel cell system are examined. Furthermore, the paper outlines the necessity of an electric storage device and its resultant capacity, which will enable faster load changes. Acceleration and deceleration of the vehicle are accomplished through the use of the electric storage device, while the fuel cell system only has to deliver the mean power consumption without higher load peaks. On the basis of system simulation, different concepts are evaluated for use as a propulsion system or APU and, then, critical components are identified. The effects of advanced control strategies regarding the dynamic behavior of the system are demonstrated. Technically, a fuel cell system could be a viable propulsion system alternative

  3. Analysis of dynamic requirements for fuel cell systems for vehicle applications

    NASA Astrophysics Data System (ADS)

    Pischinger, Stefan; Schönfelder, Carsten; Ogrzewalla, Jürgen

    Conventional vehicles with internal combustion engines, as well as battery powered electric vehicles, achieve one of the most important customer requirements; achieving extremely short response times to load changes. Also, fast acceleration times from a cold start to full power in the range of seconds are practicable. New fuel cell-based propulsion systems, as well as auxiliary power units, have to fulfill the same demands to become competitive. This includes heating-up the system to operating temperature as well as the control strategy for start-up. An additional device to supply starting air is necessary, if the compressor motor can only be operated with fuel cell voltage. Since the system components (for example, the air supply or the fuel supply) are not mechanically coupled, as is the case with conventional internal combustion engines, these components have to be controlled by different sensors and actuators. This can be an advantage in optimizing the system, but it also can represent an additional challenge. This paper describes the fuel cell system requirements regarding transient operation and their dependence on system structure. In particular, the requirements for peripheral components such as air supply, fuel supply and the balance of heat in a fuel cell system are examined. Furthermore, the paper outlines the necessity of an electric storage device and its resultant capacity, which will enable faster load changes. Acceleration and deceleration of the vehicle are accomplished through the use of the electric storage device, while the fuel cell system only has to deliver the mean power consumption without higher load peaks. On the basis of system simulation, different concepts are evaluated for use as a propulsion system or APU and, then, critical components are identified. The effects of advanced control strategies regarding the dynamic behavior of the system are demonstrated. Technically, a fuel cell system could be a viable propulsion system alternative

  4. Cripto is required for mesoderm and endoderm cell allocation during mouse gastrulation

    PubMed Central

    Jin, Jiu-Zhen; Ding, Jixiang

    2013-01-01

    During mouse gastrulation, cells in the primitive streak undergo epithelial-mesenchymal transformation and the resulting mesenchymal cells migrate out laterally to form mesoderm and definitive endoderm across the entire embryonic cylinder. The mechanisms underlying mesoderm and endoderm specification, migration, and allocation is poorly understood. In this study, we focused on the function of mouse Cripto, a member of the EGF-CFC gene family that is highly expressed in the primitive streak and migrating mesoderm cells on embryonic day 6.5. Conditional inactivation of Cripto during gastrulation leads to varied defects in mesoderm and endoderm development. Mutant embryos display accumulation of mesenchymal cells around the shortened primitive streak indicating a functional requirement of Cripto during the formation of mesoderm layer in gastrulation. In addition, some mutant embryos showed poor formation and abnormal allocation of definitive endoderm cells on embryonic day 7.5. Consistently, many mutant embryos that survived to embryonic day 8.5 displayed defects in ventral closure of the gut endoderm causing cardia bifida. Detailed analyses revealed that both the Fgf8-Fgfr1 pathway and p38 MAP kinase activation are partially affected by the loss of Cripto function. These results demonstrate a critical role for Cripto during mouse gastrulation, especially in mesoderm and endoderm formation and allocation. PMID:23747598

  5. Group 3 innate lymphoid cells continuously require the transcription factor GATA-3 after commitment.

    PubMed

    Zhong, Chao; Cui, Kairong; Wilhelm, Christoph; Hu, Gangqing; Mao, Kairui; Belkaid, Yasmine; Zhao, Keji; Zhu, Jinfang

    2016-02-01

    The transcription factor GATA-3 is indispensable for the development of all innate lymphoid cells (ILCs) that express the interleukin 7 receptor α-chain (IL-7Rα). However, the function of low GATA-3 expression in committed group 3 ILCs (ILC3 cells) has not been identified. We found that GATA-3 regulated the homeostasis of ILC3 cells by controlling IL-7Rα expression. In addition, GATA-3 served a critical function in the development of the NKp46(+) ILC3 subset by regulating the balance between the transcription factors T-bet and RORγt. Among NKp46(+) ILC3 cells, although GATA-3 positively regulated genes specific to the NKp46(+) ILC3 subset, it negatively regulated genes specific to lymphoid tissue-inducer (LTi) or LTi-like ILC3 cells. Furthermore, GATA-3 was required for IL-22 production in both ILC3 subsets. Thus, despite its low expression, GATA-3 was critical for the homeostasis, development and function of ILC3 subsets. PMID:26595886

  6. PKN3 is required for malignant prostate cell growth downstream of activated PI 3-kinase

    PubMed Central

    Leenders, Frauke; Möpert, Kristin; Schmiedeknecht, Anett; Santel, Ansgar; Czauderna, Frank; Aleku, Manuela; Penschuck, Silke; Dames, Sibylle; Sternberger, Maria; Röhl, Thomas; Wellmann, Axel; Arnold, Wolfgang; Giese, Klaus; Kaufmann, Jörg; Klippel, Anke

    2004-01-01

    Chronic activation of the phosphoinositide 3-kinase (PI3K)/PTEN signal transduction pathway contributes to metastatic cell growth, but up to now effectors mediating this response are poorly defined. By simulating chronic activation of PI3K signaling experimentally, combined with three-dimensional (3D) culture conditions and gene expression profiling, we aimed to identify novel effectors that contribute to malignant cell growth. Using this approach we identified and validated PKN3, a barely characterized protein kinase C-related molecule, as a novel effector mediating malignant cell growth downstream of activated PI3K. PKN3 is required for invasive prostate cell growth as assessed by 3D cell culture assays and in an orthotopic mouse tumor model by inducible expression of short hairpin RNA (shRNA). We demonstrate that PKN3 is regulated by PI3K at both the expression level and the catalytic activity level. Therefore, PKN3 might represent a preferred target for therapeutic intervention in cancers that lack tumor suppressor PTEN function or depend on chronic activation of PI3K. PMID:15282551

  7. Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation.

    PubMed

    Wu, Fang; Gehring, Heinz

    2009-02-01

    Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5'-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC(50) up to 25 microM) of glioma cells (LN229) than alpha-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CH(X))(4)-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N(1)-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO. PMID:18922879

  8. 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

  9. Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification

    PubMed Central

    Boube, Muriel; Faucher, Christian; Joulia, Laurent; Cribbs, David L.; Bourbon, Henri-Marc

    2000-01-01

    The origins of specificity in gene expression are a central concern in understanding developmental control. Mediator protein complexes regulate transcriptional initiation, acting as modular adaptors linking specific transcription factors to core RNA polymerase II. Here, we identified the Drosophila homologs of 23 human mediator genes and mutations of two, dTRAP240 and of dTRAP80 (the putative fly homolog of yeast SRB4). Clonal analysis indicates a general role for dTRAP80 necessary for cell viability. The dTRAP240 gene is also essential, but cells lacking its function are viable and proliferate normally. Clones reveal localized developmental activities including a sex comb cell identity function. This contrasts with the ubiquitous nuclear accumulation of dTRAP240 protein in imaginal discs. Synergistic genetic interactions support shared developmental cell and segment identity functions of dTRAP240 and dTRAP80, potentially within a common complex. Further, they identify the homeotic Sex combs reduced product, required for the same cell/tissue identities, as a functional partner of these mediator proteins. PMID:11090137

  10. BMP9-Induced Survival Effect in Liver Tumor Cells Requires p38MAPK Activation

    PubMed Central

    García-Álvaro, María; Addante, Annalisa; Roncero, Cesáreo; Fernández, Margarita; Fabregat, Isabel; Sánchez, Aránzazu; Herrera, Blanca

    2015-01-01

    The study of bone morphogenetic proteins (BMPs) role in tumorigenic processes, and specifically in the liver, has gathered importance in the last few years. Previous studies have shown that BMP9 is overexpressed in about 40% of hepatocellular carcinoma (HCC) patients. In vitro data have also shown evidence that BMP9 has a pro-tumorigenic action, not only by inducing epithelial to mesenchymal transition (EMT) and migration, but also by promoting proliferation and survival in liver cancer cells. However, the precise mechanisms driving these effects have not yet been established. In the present work, we deepened our studies into the intracellular mechanisms implicated in the BMP9 proliferative and pro-survival effect on liver tumor cells. In HepG2 cells, BMP9 induces both Smad and non-Smad signaling cascades, specifically PI3K/AKT and p38MAPK. However, only the p38MAPK pathway contributes to the BMP9 growth-promoting effect on these cells. Using genetic and pharmacological approaches, we demonstrate that p38MAPK activation, although dispensable for the BMP9 proliferative activity, is required for the BMP9 protective effect on serum withdrawal-induced apoptosis. These findings contribute to a better understanding of the signaling pathways involved in the BMP9 pro-tumorigenic role in liver tumor cells. PMID:26343646

  11. Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering, Tubulogenesis and Motility

    PubMed Central

    Li, Ran; Knight, Jennifer F.; Park, Morag; Pendergast, Ann Marie

    2015-01-01

    Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling. PMID:25946048

  12. Donor CD47 controls T cell alloresponses and is required for tolerance induction following hepatocyte allotransplantation

    PubMed Central

    Zhang, Mingyou; Wang, Hui; Tan, Shulian; Navarro-Alvarez, Nalu; Zheng, Yang; Yang, Yong-Guang

    2016-01-01

    CD47-deficient hepatocyte transplantation induces rapid innate immune cell activation and subsequent associated graft loss in syngeneic recipients. However, the role of donor CD47 in regulation of T-cell alloresponses is poorly understood. We addressed this question by assessing OVA-specific immune responses in mice following hepatocyte transplantation from CD47-competent or -deficient OVA-transgenic donors. Compared to sham-operated controls, intrasplenic transplantation of CD47-deficient OVA+ hepatocytes significantly accelerated rejection of OVA+ skin grafted 7 days after hepatocyte transplantation. In contrast, mice receiving CD47-competent OVA+ hepatocytes showed prolonged and even indefinite survival of OVA+ skin allografts. T cells from mice receiving CD47-deficient, but not CD47-competent, OVA+ hepatocytes showed significantly enhanced responses to OVA+ stimulators compared to sham-operated controls. In contrast to the production of tolerogenic cytokines (IL-4 and IL-10) in the recipients of CD47-competent hepatocytes, mice receiving CD47-deficient hepatocytes showed elevated production of IFN-γ and IL-1α. Moreover, significant expansion of myeloid-derived suppressor cells was detected in the recipients of CD47-competent hepatocytes, which was required for tolerance induction in these mice. Thus, donor CD47 plays an important role in the control of T-cell alloresponses and tolerance induction following hepatocyte transplantation. Our data also suggest that intrasplenic hepatocyte transplantation may provide a means to induce allograft tolerance. PMID:27230788

  13. Brg1 directly regulates Olig2 transcription and is required for oligodendrocyte progenitor cell specification.

    PubMed

    Matsumoto, Steven; Banine, Fatima; Feistel, Kerstin; Foster, Scott; Xing, Rubing; Struve, Jaime; Sherman, Larry S

    2016-05-15

    The Olig2 basic-helix-loop-helix transcription factor promotes oligodendrocyte specification in early neural progenitor cells (NPCs), including radial glial cells, in part by recruiting SWI/SNF chromatin remodeling complexes to the enhancers of genes involved in oligodendrocyte differentiation. How Olig2 expression is regulated during oligodendrogliogenesis is not clear. Here, we find that the Brg1 subunit of SWI/SNF complexes interacts with a proximal Olig2 promoter and represses Olig2 transcription in the mouse cortex at E14, when oligodendrocyte progenitors (OPCs) are not yet found in this location. Brg1 does not interact with the Olig2 promoter in the E14 ganglionic eminence, where NPCs differentiate into Olig2-positive OPCs. Consistent with these findings, Brg1-null NPCs demonstrate precocious expression of Olig2 in the cortex. However, these cells fail to differentiate into OPCs. We further find that Brg1 is necessary for neuroepithelial-to-radial glial cell transition, but not neuronal differentiation despite a reduction in expression of the pro-neural transcription factor Pax6. Collectively, these and earlier findings support a model whereby Brg1 promotes neurogenic radial glial progenitor cell specification but is dispensable for neuronal differentiation. Concurrently, Brg1 represses Olig2 expression and the specification of OPCs, but is required for OPC differentiation and oligodendrocyte maturation. PMID:27067865

  14. Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation.

    PubMed

    Brennan, Todd V; Lin, Liwen; Brandstadter, Joshua D; Rendell, Victoria R; Dredge, Keith; Huang, Xiaopei; Yang, Yiping

    2016-01-01

    Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell-based antitumor therapies. PMID:26649979

  15. Glycosaminoglycans are required for translocation of amphipathic cell-penetrating peptides across membranes.

    PubMed

    Pae, Janely; Liivamägi, Laura; Lubenets, Dmitri; Arukuusk, Piret; Langel, Ülo; Pooga, Margus

    2016-08-01

    Cell-penetrating peptides (CPPs) are considered as one of the most promising tools to mediate the cellular delivery of various biologically active compounds that are otherwise cell impermeable. CPPs can internalize into cells via two different pathways - endocytosis and direct translocation across the plasma membrane. In both cases, the initial step of internalization requires interactions between CPPs and different plasma membrane components. Despite the extensive research, it is not yet fully understood, which of these cell surface molecules mediate the direct translocation of CPPs across the plasma- and endosomal membrane. In the present study we used giant plasma membrane vesicles (GPMVs) as a model membrane system to elucidate the specific molecular mechanisms behind the internalization and the role of cell surface glycosaminoglycans (GAGs) in the translocation of four well-known CPPs, classified as cationic (nona-arginine, Tat peptide) and amphipathic (transportan and TP10). We demonstrate here that GAGs facilitate the translocation of amphipathic CPPs, but not the internalization of cationic CPPs; and that the uptake is not mediated by a specific GAG class, but rather the overall amount of these polysaccharides is crucial for the internalization of amphipathic peptides. PMID:27117133

  16. [Regulatory requirements regarding cell-based medicinal products for human and veterinary use - a comparison].

    PubMed

    Kuhlmann-Gottke, Johanna; Duchow, Karin

    2015-11-01

    At present, there is no separate regulatory framework for cell-based medicinal products (CBMP) for veterinary use at the European or German level. Current European and national regulations exclusively apply to the corresponding medicinal products for human use. An increasing number of requests for the regulatory classification of CBMP for veterinary use, such as allogeneic stem cell preparations and dendritic cell-based autologous tumour vaccines, and a rise in scientific advice for companies developing these products, illustrate the need for adequate legislation. Currently, advice is given and decisions are made on a case-by-case basis regarding the regulatory classification and authorisation requirements.Since some of the CBMP - in particular in the area of stem-cell products - are developed in parallel for human and veterinary use, there is an urgent need to create specific legal definitions, regulations, and guidelines for these complex innovative products in the veterinary sector as well. Otherwise, there is a risk that that the current legal grey area regarding veterinary medicinal products will impede therapeutic innovations in the long run. A harmonised EU-wide approach is desirable. Currently the European legislation on veterinary medicinal products is under revision. In this context, veterinary therapeutics based on allogeneic cells and tissues will be defined and regulated. Certainly, the legal framework does not have to be as comprehensive as for human CBMP; a leaner solution is conceivable, similar to the special provisions for advanced-therapy medicinal products laid down in the German Medicines Act. PMID:26369765

  17. Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates.

    PubMed

    Mundell, Nathan A; Labosky, Patricia A

    2011-02-01

    Neural crest (NC) progenitors generate a wide array of cell types, yet molecules controlling NC multipotency and self-renewal and factors mediating cell-intrinsic distinctions between multipotent versus fate-restricted progenitors are poorly understood. Our earlier work demonstrated that Foxd3 is required for maintenance of NC progenitors in the embryo. Here, we show that Foxd3 mediates a fate restriction choice for multipotent NC progenitors with loss of Foxd3 biasing NC toward a mesenchymal fate. Neural derivatives of NC were lost in Foxd3 mutant mouse embryos, whereas abnormally fated NC-derived vascular smooth muscle cells were ectopically located in the aorta. Cranial NC defects were associated with precocious differentiation towards osteoblast and chondrocyte cell fates, and individual mutant NC from different anteroposterior regions underwent fate changes, losing neural and increasing myofibroblast potential. Our results demonstrate that neural potential can be separated from NC multipotency by the action of a single gene, and establish novel parallels between NC and other progenitor populations that depend on this functionally conserved stem cell protein to regulate self-renewal and multipotency. PMID:21228004

  18. Neural crest stem cell multipotency requires Foxd3 to maintain neural potential and repress mesenchymal fates

    PubMed Central

    Mundell, Nathan A.; Labosky, Patricia A.

    2011-01-01

    Neural crest (NC) progenitors generate a wide array of cell types, yet molecules controlling NC multipotency and self-renewal and factors mediating cell-intrinsic distinctions between multipotent versus fate-restricted progenitors are poorly understood. Our earlier work demonstrated that Foxd3 is required for maintenance of NC progenitors in the embryo. Here, we show that Foxd3 mediates a fate restriction choice for multipotent NC progenitors with loss of Foxd3 biasing NC toward a mesenchymal fate. Neural derivatives of NC were lost in Foxd3 mutant mouse embryos, whereas abnormally fated NC-derived vascular smooth muscle cells were ectopically located in the aorta. Cranial NC defects were associated with precocious differentiation towards osteoblast and chondrocyte cell fates, and individual mutant NC from different anteroposterior regions underwent fate changes, losing neural and increasing myofibroblast potential. Our results demonstrate that neural potential can be separated from NC multipotency by the action of a single gene, and establish novel parallels between NC and other progenitor populations that depend on this functionally conserved stem cell protein to regulate self-renewal and multipotency. PMID:21228004

  19. Pancreatic β cells require NeuroD to achieve and maintain functional maturity

    PubMed Central

    Gu, Chunyan; Stein, Gretchen H.; Pan, Ning; Goebbels, Sandra; Hörnberg, Hanna; Nave, Klaus-Armin; Herrera, Pedro; White, Peter; Kaestner, Klaus H.; Sussel, Lori; Lee, Jacqueline E.

    2010-01-01

    Summary NeuroD, an insulin transactivator, is critical for development of the endocrine pancreas, and NeuroD mutations cause MODY6 in humans. To investigate the role of NeuroD in differentiated β cells, we generated mice in which neuroD is deleted in insulin-expressing cells. These mice exhibit severe glucose intolerance. Islets lacking NeuroD respond poorly to glucose and display a glucose metabolic profile similar to immature β cells, featuring increased expression of glycolytic genes and LDH-A, elevated basal insulin secretion and O2 consumption, and overexpression of NPY. Moreover, the mutant islets appear to have defective KATP channel-mediated insulin secretion. Unexpectedly, virtually all insulin in the mutant mice is derived from ins2, whereas ins1 expression is almost extinguished. Overall, these results indicate that NeuroD is required for β cell maturation and demonstrate the importance of NeuroD in the acquisition and maintenance of fully functional glucose responsive β cells. PMID:20374962

  20. MT1-MMP is required for myeloid cell fusion via regulation of Rac1 signaling

    PubMed Central

    Gonzalo, Pilar; Guadamillas, Marta C.; Hernández-Riquer, Mª Victoria; Pollán, Ángela; Grande-García, Araceli; Bartolomé, Rubén A.; Vasanji, Amit; Ambrogio, Chiara; Chiarle, Roberto; Teixidó, Joaquín; Risteli, Juha; Apte, Suneel S.; del Pozo, Miguel A.; Arroyo, Alicia G.

    2009-01-01

    SUMMARY Cell fusion is essential for fertilization, myotube formation, and inflammation. Macrophages fuse in various circumstances but the molecular signals involved in the distinct steps of their fusion are not fully characterized. Using null mice and derived cells, we show that the protease MT1-MMP is necessary for macrophage fusion during osteoclast and giant cell formation in vitro and in vivo. Specifically, MT1-MMP is required for lamellipodia formation and for proper cell morphology and motility of bone marrow myeloid progenitors prior to membrane fusion. These functions of MT1-MMP do not depend on MT1-MMP catalytic activity or downstream pro-MMP-2 activation. Instead, MT1-MMP-null cells show a decreased Rac1 activity and reduced membrane targeting of Rac1 and the adaptor protein p130Cas. Retroviral rescue experiments and protein binding assays delineate a signaling pathway in which MT1-MMP, via its cytosolic tail, contributes to macrophage migration and fusion by regulating Rac1 activity through an association with p130Cas. PMID:20152179

  1. Commensal Microbiota are Required for Systemic Inflammation Triggered by Necrotic Dendritic Cells

    PubMed Central

    Young, Jennifer A.; He, Tina H.; Reizis, Boris; Winoto, Astar

    2013-01-01

    Summary The relationship between dendritic cells (DCs) and commensal microflora in shaping systemic immune responses is not well understood. Here we report that mice deficient for Fas-associated death domain in DCs developed systemic inflammation associated with elevated pro-inflammatory cytokines and increased myeloid and B cells. These mice exhibited reduced DCs in gut-associated lymphoid tissues due to RIP3-dependent necroptosis, while DC functions remained intact. Induction of systemic inflammation required DC necroptosis and commensal microbiota signals that activated MyD88-dependent pathways in other cell types. Systemic inflammation was abrogated with administration of broad-spectrum antibiotics or complete, but not DC-specific, deletion of MyD88. Thus, we have identified a previously unappreciated role for commensal microbiota in priming immune cells for inflammatory responses against necrotic cells. These studies demonstrate the impact intestinal microflora have on the immune system and their role in eliciting proper immune responses to harmful stimuli. PMID:23727238

  2. Mammalian Cardiac Regeneration After Fetal Myocardial Infarction Requires Cardiac Progenitor Cell Recruitment

    PubMed Central

    Allukian, Myron; Xu, Junwang; Morris, Michael; Caskey, Robert; Dorsett-Martin, Wanda; Plappert, Theodore; Griswold, Michael; Gorman, Joseph H.; Gorman, Robert C.; Liechty, Kenneth W.

    2013-01-01

    Background In contrast to the adult, fetal sheep consistently regenerate functional myocardium after myocardial infarction. We hypothesize that this regeneration is due to the recruitment of cardiac progenitor cells to the infarct by stromal-derived factor-1α (SDF-1α) and that its competitive inhibition will block the regenerative fetal response. Methods A 20% apical infarct was created in adult and fetal sheep by selective permanent coronary artery ligation. Lentiviral overexpression of mutant SDF-1α competitively inhibited SDF-1α in fetal infarcts. Echocardiography was performed to assess left ventricular function and infarct size. Cardiac progenitor cell recruitment and proliferation was assessed in fetal infarcts at 1 month by immunohistochemistry for nkx2.5 and 5-bromo-2-deoxyuridine. Results Competitive inhibition of SDF-1α converted the regenerative fetal response into a reparative response, similar to the adult. SDF-inhibited fetal infarcts demonstrated significant infarct expansion by echocardiography (p < 0.001) and a significant decrease in the number of nkx2.5+ cells repopulating the infarct (p < 0.001). Conclusions The fetal regenerative response to myocardial infarction requires the recruitment of cardiac progenitor cells and is dependent on SDF1α. This novel model of mammalian cardiac regeneration after myocardial infarction provides a powerful tool to better understand cardiac progenitor cell biology and to develop strategies to cardiac regeneration in the adult. PMID:23816072

  3. Human papilloma virus DNAs immortalize normal human mammary epithelial cells and reduce their growth factor requirements

    SciTech Connect

    Band, V.; Zajchowski, D.; Kulesa, V.; Sager, R. )

    1990-01-01

    Human papilloma virus (HPV) types 16 and 18 are most commonly associated with cervical carcinoma in patients and induce immortalization of human keratinocytes in culture. HPV has not been associated with breast cancer. This report describes the immortalization of normal human mammary epithelial cells (76N) by plasmid pHPV18 or pHPV16, each containing the linearized viral genome. Transfectants were grown continuously for more than 60 passages, whereas 76N cells senesce after 18-20 passages. The transfectants also differ from 76N cells in cloning in a completely defined medium called D2 and growing a minimally supplemented defined medium (D3) containing epidermal growth factor. All transfectant tested contain integrated HPV DNA, express HPV RNA, and produce HPV E7 protein. HPV transfectants do not form tumors in a nude mouse assay. It is concluded that products of the HPV genome induce immortalization of human breast epithelial cells and reduce their growth factor requirements. This result raises the possibility that HPV might be involved in breast cancer. Furthermore, other tissue-specific primary epithelial cells that are presently difficult to grown and investigate may also be immortalized by HPV.

  4. Rapid CD4+ T-cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9.

    PubMed

    Atif, Shaikh M; Lee, Seung-Joo; Li, Lin-Xi; Uematsu, Satoshi; Akira, Shizuo; Gorjestani, Sara; Lin, Xin; Schweighoffer, Edina; Tybulewicz, Victor L J; McSorley, Stephen J

    2015-02-01

    Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4(+) T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses. PMID:25430631

  5. Rapid CD4+ T-cell responses to bacterial flagellin require dendritic cell expression of Syk and CARD9

    PubMed Central

    Atif, Shaikh M; Lee, Seung-Joo; Li, Lin-Xi; Uematsu, Satoshi; Akira, Shizuo; Gorjestani, Sara; Lin, Xin; Schweighoffer, Edina; Tybulewicz, Victor L J; McSorley, Stephen J

    2015-01-01

    Toll-like receptors (TLRs) can recognize microbial patterns and utilize adaptor molecules, such as-MyD88 or (TRIF TIR-domain-containing adapter-inducing interferon-β), to initiate downstream signaling that ultimately affects the initiation of adaptive immunity. In addition to this inflammatory role, TLR5 expression on dendritic cells can favor antigen presentation of flagellin peptides and thus increase the sensitivity of flagellin-specific T-cell responses in vitro and in vivo. Here, we examined the role of alternative signaling pathways that might regulate flagellin antigen presentation in addition to MyD88. These studies suggest a requirement for spleen tyrosine kinase, a noncanonical TLR-signaling adaptor molecule, and its downstream molecule CARD9 in regulating the sensitivity of flagellin-specific CD4+ T-cell responses in vitro and in vivo. Thus, a previously unappreciated signaling pathway plays an important role in regulating the dominance of flagellin-specific T-cell responses. PMID:25430631

  6. Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

    SciTech Connect

    Arulanandam, Rozanne; Geletu, Mulu; Feracci, Helene; Raptis, Leda

    2010-03-10

    Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

  7. UBIAD1-mediated vitamin K2 synthesis is required for vascular endothelial cell survival and development

    PubMed Central

    Hegarty, Jeffrey M.; Yang, Hongbo; Chi, Neil C.

    2013-01-01

    Multi-organ animals, such as vertebrates, require the development of a closed vascular system to ensure the delivery of nutrients to, and the transport of waste from, their organs. As a result, an organized vascular network that is optimal for tissue perfusion is created through not only the generation of new blood vessels but also the remodeling and maintenance of endothelial cells via apoptotic and cell survival pathways. Here, we show that UBIAD1, a vitamin K2/menaquinone-4 biosynthetic enzyme, functions cell-autonomously to regulate endothelial cell survival and maintain vascular homeostasis. From a recent vascular transgene-assisted zebrafish forward genetic screen, we have identified a ubiad1 mutant, reddish/reh, which exhibits cardiac edema as well as cranial hemorrhages and vascular degeneration owing to defects in endothelial cell survival. These findings are further bolstered by the expression of UBIAD1 in human umbilical vein endothelial cells and human heart tissue, as well as the rescue of the reh cardiac and vascular phenotypes with either zebrafish or human UBIAD1. Furthermore, we have discovered that vitamin K2, which is synthesized by UBIAD1, can also rescue the reh vascular phenotype but not the reh cardiac phenotype. Additionally, warfarin-treated zebrafish, which have decreased active vitamin K, display similar vascular degeneration as reh mutants, but exhibit normal cardiac function. Overall, these findings reveal an essential role for UBIAD1-generated vitamin K2 to maintain endothelial cell survival and overall vascular homeostasis; however, an alternative UBIAD1/vitamin K-independent pathway may regulate cardiac function. PMID:23533172

  8. Ferroportin and Exocytoplasmic Ferroxidase Activity Are Required for Brain Microvascular Endothelial Cell Iron Efflux*

    PubMed Central

    McCarthy, Ryan C.; Kosman, Daniel J.

    2013-01-01

    The mechanism(s) of iron flux across the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier remains unknown. Although both hephaestin (Hp) and the ferrous iron permease ferroportin (Fpn) have been identified in BMVEC, their roles in iron efflux have not been examined. Using a human BMVEC line (hBMVEC), we have demonstrated that these proteins are required for iron efflux from these cells. Expression of both Hp and Fpn protein was confirmed in hBMVEC by immunoblot and indirect immunofluorescence; we show that hBMVEC express soluble ceruloplasmin (Cp) transcript as well. Depletion of endogenous Hp and Cp via copper chelation leads to the reduction of hBMVEC Fpn protein levels as well as a complete inhibition of 59Fe efflux. Both hBMVEC Fpn protein and 59Fe efflux activity are restored upon incubation with 6.6 nm soluble plasma Cp. These results are independent of the source of cell iron, whether delivered as transferrin- or non-transferrin-bound 59Fe. Our results demonstrate that iron efflux from hBMVEC Fpn requires the action of an exocytoplasmic ferroxidase, which can be either endogenous Hp or extracellular Cp. PMID:23640881

  9. Hmga2 is required for neural crest cell specification in Xenopus laevis.

    PubMed

    Macrì, Simone; Simula, Luca; Pellarin, Ilenia; Pegoraro, Silvia; Onorati, Marco; Sgarra, Riccardo; Manfioletti, Guidalberto; Vignali, Robert

    2016-03-01

    HMGA proteins are small nuclear proteins that bind DNA by conserved AT-hook motifs, modify chromatin architecture and assist in gene expression. Two HMGAs (HMGA1 and HMGA2), encoded by distinct genes, exist in mammals and are highly expressed during embryogenesis or reactivated in tumour progression. We here addressed the in vivo role of Xenopus hmga2 in the neural crest cells (NCCs). We show that hmga2 is required for normal NCC specification and development. hmga2 knockdown leads to severe disruption of major skeletal derivatives of anterior NCCs. We show that, within the NCC genetic network, hmga2 acts downstream of msx1, and is required for msx1, pax3 and snail2 activities, thus participating at different levels of the network. Because of hmga2 early effects in NCC specification, the subsequent epithelial-mesenchymal transition (EMT) and migration of NCCs towards the branchial pouches are also compromised. Strictly paralleling results on embryos, interfering with Hmga2 in a breast cancer cell model for EMT leads to molecular effects largely consistent with those observed on NCCs. These data indicate that Hmga2 is recruited in key molecular events that are shared by both NCCs and tumour cells. PMID:26806704

  10. 21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All...

  11. 21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All...

  12. 21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All...

  13. 21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All...

  14. 21 CFR 1271.85 - What donor testing is required for different types of cells and tissues?

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... of cells and tissues? 1271.85 Section 1271.85 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... FOOD AND DRUG ADMINISTRATION HUMAN CELLS, TISSUES, AND CELLULAR AND TISSUE-BASED PRODUCTS Donor Eligibility § 1271.85 What donor testing is required for different types of cells and tissues? (a) All...

  15. Signalling Through Retinoic Acid Receptors is Required for Reprogramming of Both Mouse Embryonic Fibroblast Cells and Epiblast Stem Cells to Induced Pluripotent Stem Cells

    PubMed Central

    Yang, Jian; Wang, Wei; Ooi, Jolene; Campos, Lia S.

    2015-01-01

    Abstract We previously demonstrated that coexpressing retinoic acid (RA) receptor gamma and liver receptor homolog‐1 (LRH1 or NR5A2) with OCT4, MYC, KLF4, and SOX2 (4F) rapidly reprograms mouse embryonic fibroblast cells (MEFs) into induced pluripotent stem cells (iPSCs). Here, we further explore the role of RA in reprogramming and report that the six factors (6F) efficiently and directly reprogram MEFs into integration‐free iPSCs in defined medium (N2B27) in the absence of feeder cells. Through genetic and chemical approaches, we find that RA signalling is essential, in a highly dose‐sensitive manner, for MEF reprogramming. The removal of exogenous RA from N2B27, the inhibition of endogenous RA synthesis or the expression of a dominant‐negative form of RARA severely impedes reprogramming. By contrast, supplementing N2B27 with various retinoids substantially boosts reprogramming. In addition, when coexpressed with LRH1, RA receptors (RARs) can promote reprogramming in the absence of both exogenous and endogenously synthesized RA. Remarkably, the reprogramming of epiblast stem cells into embryonic stem cell‐like cells also requires low levels of RA, which can modulate Wnt signalling through physical interactions of RARs with β‐catenin. These results highlight the important functions of RA signalling in reprogramming somatic cells and primed stem cells to naïve pluripotency. Stem Cells 2015;33:1390–1404 PMID:25546009

  16. Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

    PubMed Central

    Bennett, A M; Hausdorff, S F; O'Reilly, A M; Freeman, R M; Neel, B G

    1996-01-01

    Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable. PMID:8622663

  17. Supraphysiological Levels of Quercetin Glycosides are Required to Alter Mineralization in Saos2 Cells

    PubMed Central

    Nash, Leslie A.; Peters, Sandra J.; Sullivan, Philip J.; Ward, Wendy E.

    2016-01-01

    Flavonoid intake is positively correlated to bone mineral density (BMD) in women. Flavonoids such as quercetin exhibit strong anti-oxidant and anti-inflammatory activity that may be beneficial for bone health. Quercetin, previously shown to positively influence osteoblasts, is metabolized into glycosides including rutin and hyperoside. We compared the effects of these glycosides on mineralization in human osteoblast (Saos2) cells. Administration of rutin (≥25 µM) and hyperoside (≥5 µM) resulted in higher mineral content, determined using the alizarin red assay. This was accompanied by higher alkaline phosphatase activity with no cell toxicity. The expression of osteopontin, sclerostin, TNFα and IL6, known stimuli for decreasing osteoblast activity, were reduced with the addition of rutin or hyperoside. In summary, rutin and hyperoside require supraphysiological levels, when administered individually, to positively influence osteoblast activity. This information may be useful in developing nutraceuticals to support bone health. PMID:27136576

  18. A cell-free CENP-A assembly system defines the chromatin requirements for centromere maintenance

    PubMed Central

    Westhorpe, Frederick G.; Fuller, Colin J.

    2015-01-01

    Centromeres are defined by the presence of CENP-A nucleosomes in chromatin and are essential for accurate chromosome segregation. Centromeric chromatin epigenetically seeds new CENP-A nucleosome formation, thereby maintaining functional centromeres as cells divide. The features within centromeric chromatin that direct new CENP-A assembly remain unclear. Here, we developed a cell-free CENP-A assembly system that enabled the study of chromatin-bound CENP-A and soluble CENP-A separately. We show that two distinct domains of CENP-A within existing CENP-A nucleosomes are required for new CENP-A assembly and that CENP-A nucleosomes recruit the CENP-A assembly factors CENP-C and M18BP1 independently. Furthermore, we demonstrate that the mechanism of CENP-C recruitment to centromeres is dependent on the density of underlying CENP-A nucleosomes. PMID:26076692

  19. Supraphysiological Levels of Quercetin Glycosides are Required to Alter Mineralization in Saos2 Cells.

    PubMed

    Nash, Leslie A; Peters, Sandra J; Sullivan, Philip J; Ward, Wendy E

    2016-01-01

    Flavonoid intake is positively correlated to bone mineral density (BMD) in women. Flavonoids such as quercetin exhibit strong anti-oxidant and anti-inflammatory activity that may be beneficial for bone health. Quercetin, previously shown to positively influence osteoblasts, is metabolized into glycosides including rutin and hyperoside. We compared the effects of these glycosides on mineralization in human osteoblast (Saos2) cells. Administration of rutin (≥25 µM) and hyperoside (≥5 µM) resulted in higher mineral content, determined using the alizarin red assay. This was accompanied by higher alkaline phosphatase activity with no cell toxicity. The expression of osteopontin, sclerostin, TNFα and IL6, known stimuli for decreasing osteoblast activity, were reduced with the addition of rutin or hyperoside. In summary, rutin and hyperoside require supraphysiological levels, when administered individually, to positively influence osteoblast activity. This information may be useful in developing nutraceuticals to support bone health. PMID:27136576

  20. JunB is required for endothelial cell morphogenesis by regulating core-binding factor β

    PubMed Central

    Licht, Alexander H.; Pein, Oliver T.; Florin, Lore; Hartenstein, Bettina; Reuter, Hendrik; Arnold, Bernd; Lichter, Peter; Angel, Peter; Schorpp-Kistner, Marina

    2006-01-01

    The molecular mechanism triggering the organization of endothelial cells (ECs) in multicellular tubules is mechanistically still poorly understood. We demonstrate that cell-autonomous endothelial functions of the AP-1 subunit JunB are required for proper endothelial morphogenesis both in vivo in mouse embryos with endothelial-specific ablation of JunB and in in vitro angiogenesis models. By cDNA microarray analysis, we identified core-binding factor β (CBFβ), which together with the Runx proteins forms the heterodimeric core-binding transcription complex CBF, as a novel JunB target gene. In line with our findings, expression of the CBF target MMP-13 was impaired in JunB-deficient ECs. Reintroduction of CBFβ into JunB-deficient ECs rescued the tube formation defect and MMP-13 expression, indicating an important role for CBFβ in EC morphogenesis. PMID:17158955

  1. Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii.

    PubMed

    Shimakata, T; Tsubokura, K; Kusaka, T

    1986-06-01

    When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids. PMID:3717946

  2. Meis1 Is Required for the Maintenance of Postnatal Thymic Epithelial Cells

    PubMed Central

    Hirayama, Takehiro; Asano, Yusuke; Iida, Hajime; Watanabe, Takeshi; Nakamura, Takuro; Goitsuka, Ryo

    2014-01-01

    Most epithelial tissues retain stem/progenitor cells to maintain homeostasis of the adult tissues; however, the existence of a thymic epithelial cell (TEC) progenitor capable of maintaining homeostasis of the postnatal thymus remains unclear. Here, we show that a cell population expressing high levels of Meis1, a homeodomain transcription factor, is enriched in TECs with an immature cellular phenotype. These TECs selectively express genes involved in embryonic thymic organogenesis and epithelial stem cell maintenance, and also have the potential to proliferate and differentiate into mature TEC populations. Furthermore, postnatal inactivation of Meis1 in TECs caused disorganization of the thymic architecture, which ultimately leads to premature disappearance of the thymus. There was an age-associated reduction in the proportion of the TEC population expressing high levels of Meis1, which may also be related to thymic involution. These findings indicate that Meis1 is potentially involved in the maintenance of postnatal TECs with progenitor activity that is required for homeostasis of the postnatal thymus. PMID:24594519

  3. The APC tumor suppressor is required for epithelial cell polarization and three-dimensional morphogenesis.

    PubMed

    Lesko, Alyssa C; Goss, Kathleen H; Yang, Frank F; Schwertner, Adam; Hulur, Imge; Onel, Kenan; Prosperi, Jenifer R

    2015-03-01

    The Adenomatous Polyposis Coli (APC) tumor suppressor has been previously implicated in the control of apical-basal polarity; yet, the consequence of APC loss-of-function in epithelial polarization and morphogenesis has not been characterized. To test the hypothesis that APC is required for the establishment of normal epithelial polarity and morphogenesis programs, we generated APC-knockdown epithelial cell lines. APC depletion resulted in loss of polarity and multi-layering on permeable supports, and enlarged, filled spheroids with disrupted polarity in 3D culture. Importantly, these effects of APC knockdown were independent of Wnt/β-catenin signaling, but were rescued with either full-length or a carboxy (c)-terminal segment of APC. Moreover, we identified a gene expression signature associated with APC knockdown that points to several candidates known to regulate cell-cell and cell-matrix communication. Analysis of epithelial tissues from mice and humans carrying heterozygous APC mutations further supports the importance of APC as a regulator of epithelial behavior and tissue architecture. These data also suggest that the initiation of epithelial-derived tumors as a result of APC mutation or gene silencing may be driven by loss of polarity and dysmorphogenesis. PMID:25578398

  4. PHGDH Expression Is Required for Mitochondrial Redox Homeostasis, Breast Cancer Stem Cell Maintenance, and Lung Metastasis.

    PubMed

    Samanta, Debangshu; Park, Youngrok; Andrabi, Shaida A; Shelton, Laura M; Gilkes, Daniele M; Semenza, Gregg L

    2016-08-01

    Intratumoral hypoxia stimulates enrichment of breast cancer stem cells (BCSC), which are critical for metastasis and patient mortality. Here we report a metabolic adaptation that is required for hypoxia-induced BCSC enrichment and metastasis. Hypoxia-inducible factors coordinately regulate expression of genes encoding phosphoglycerate dehydrogenase (PHGDH) and five downstream enzymes in the serine synthesis pathway and mitochondrial one-carbon (folate) cycle. RNAi-mediated silencing of PHGDH expression in both estrogen receptor-positive and negative breast cancer cells led to decreased NADPH levels, disturbed mitochondrial redox homeostasis, and increased apoptosis, which abrogated BCSC enrichment under hypoxic conditions. PHGDH-deficient cells exhibited increased oxidant levels and apoptosis, as well as loss of BCSC enrichment, in response to treatment with carboplatin or doxorubicin. PHGDH-deficient cells were relatively weakly tumorigenic and tumors that did form were deficient in BCSCs, abolishing metastatic capacity. Our findings highlight a role for PHGDH in the formation of secondary (recurrent or metastatic) tumors, with potential implications for therapeutic targeting of advanced cancers. Cancer Res; 76(15); 4430-42. ©2016 AACR. PMID:27280394

  5. ERRs Mediate a Metabolic Switch Required for Somatic Cell Reprogramming to Pluripotency

    PubMed Central

    Kida, Yasuyuki S.; Kawamura, Teruhisa; Wei, Zong; Sogo, Takahiro; Jacinto, Sandra; Shigeno-Kamitsuji, Asako; Yoshihara, Eiji; Liddle, Christopher; Ecker, Joseph R.; Yu, Ruth T.; Atkins, Annette R.; Downes, Michael; Evans, Ronald M.

    2015-01-01

    Summary Cell metabolism is adaptive to extrinsic demands, however the intrinsic metabolic demands that drive the induced pluripotent stem cell (iPSC) program remain unclear. While glycolysis increases throughout the reprogramming process, we show that the estrogen related nuclear receptors (ERRα and γ) and their partnered co-factors PGC-1α and β, are transiently induced at an early stage resulting in a burst of oxidative phosphorylation (OXPHOS) activity. Up-regulation of ERRα or γ is required for both the OXPHOS burst in human and mouse cells, respectively, as well as iPSC generation itself. Failure to induce this metabolic switch collapses the reprogramming process. Furthermore, we identify a rare pool of Sca1−/CD34− sortable cells that is highly enriched in bona fide reprogramming progenitors. Transcriptional profiling confirmed that these progenitors are ERRγ and PGC-1β positive and have undergone extensive metabolic reprogramming. These studies characterize a previously unrecognized, ERR-dependent metabolic gate prior to establishment of induced pluripotency. PMID:25865501

  6. The chromatin remodeler Mi-2beta is required for CD4 expression and T cell development.

    PubMed

    Williams, Christine J; Naito, Taku; Arco, Pablo Gómez-Del; Seavitt, John R; Cashman, Susan M; De Souza, Beverly; Qi, Xiaoqing; Keables, Piper; Von Andrian, Ulrich H; Georgopoulos, Katia

    2004-06-01

    Changes in chromatin structure underlie the activation or silencing of genes during development. The chromatin remodeler Mi-2beta is highly expressed in thymocytes and is presumed to be a transcriptional repressor because of its presence in the nucleosome remodeling deacetylase (NuRD) complex. Using conditional inactivation, we show that Mi-2beta is required at several steps during T cell development: for differentiation of beta selected immature thymocytes, for developmental expression of CD4, and for cell divisions in mature T cells. We further show that Mi-2beta plays a direct role in promoting CD4 gene expression. Mi-2beta associates with the CD4 enhancer as well as the E box binding protein HEB and the histone acetyltransferase (HAT) p300, enabling their recruitment to the CD4 enhancer and causing histone H3-hyperacetylation to this regulatory region. These findings provide important insights into the regulation of CD4 expression during T cell development and define a role for Mi-2beta in gene activation. PMID:15189737

  7. Heparan sulfate mimetic PG545-mediated antilymphoma effects require TLR9-dependent NK cell activation

    PubMed Central

    Brennan, Todd V.; Lin, Liwen; Brandstadter, Joshua D.; Rendell, Victoria R.; Dredge, Keith; Huang, Xiaopei; Yang, Yiping

    2015-01-01

    Heparan sulfate (HS) is an essential component of the extracellular matrix (ECM), which serves as a barrier to tumor invasion and metastasis. Heparanase promotes tumor growth by cleaving HS chains of proteoglycan and releasing HS-bound angiogenic growth factors and facilitates tumor invasion and metastasis by degrading the ECM. HS mimetics, such as PG545, have been developed as antitumor agents and are designed to suppress angiogenesis and metastasis by inhibiting heparanase and competing for the HS-binding domain of angiogenic growth factors. However, how PG545 exerts its antitumor effect remains incompletely defined. Here, using murine models of lymphoma, we determined that the antitumor effects of PG545 are critically dependent on NK cell activation and that NK cell activation by PG545 requires TLR9. We demonstrate that PG545 does not activate TLR9 directly but instead enhances TLR9 activation through the elevation of the TLR9 ligand CpG in DCs. Specifically, PG545 treatment resulted in CpG accumulation in the lysosomal compartment of DCs, leading to enhanced production of IL-12, which is essential for PG545-mediated NK cell activation. Overall, these results reveal that PG545 activates NK cells and that this activation is critical for the antitumor effect of PG545. Moreover, our findings may have important implications for improving NK cell–based antitumor therapies. PMID:26649979

  8. Requirements for efficient cell-type proportioning: regulatory timescales, stochasticity and lateral inhibition.

    PubMed

    Pfeuty, B; Kaneko, K

    2016-01-01

    The proper functioning of multicellular organisms requires the robust establishment of precise proportions between distinct cell types. This developmental differentiation process typically involves intracellular regulatory and stochastic mechanisms to generate cell-fate diversity as well as intercellular signaling mechanisms to coordinate cell-fate decisions at tissue level. We thus surmise that key insights about the developmental regulation of cell-type proportion can be captured by the modeling study of clustering dynamics in population of inhibitory-coupled noisy bistable systems. This general class of dynamical system is shown to exhibit a very stable two-cluster state, but also metastability, collective oscillations or noise-induced state hopping, which can prevent from timely and reliably reaching a robust and well-proportioned clustered state. To circumvent these obstacles or to avoid fine-tuning, we highlight a general strategy based on dual-time positive feedback loops, such as mediated through transcriptional versus epigenetic mechanisms, which improves proportion regulation by coordinating early and flexible lineage priming with late and firm commitment. This result sheds new light on the respective and cooperative roles of multiple regulatory feedback, stochasticity and lateral inhibition in developmental dynamics. PMID:27172110

  9. Requirements for efficient cell-type proportioning: regulatory timescales, stochasticity and lateral inhibition

    NASA Astrophysics Data System (ADS)

    Pfeuty, B.; Kaneko, K.

    2016-04-01

    The proper functioning of multicellular organisms requires the robust establishment of precise proportions between distinct cell types. This developmental differentiation process typically involves intracellular regulatory and stochastic mechanisms to generate cell-fate diversity as well as intercellular signaling mechanisms to coordinate cell-fate decisions at tissue level. We thus surmise that key insights about the developmental regulation of cell-type proportion can be captured by the modeling study of clustering dynamics in population of inhibitory-coupled noisy bistable systems. This general class of dynamical system is shown to exhibit a very stable two-cluster state, but also metastability, collective oscillations or noise-induced state hopping, which can prevent from timely and reliably reaching a robust and well-proportioned clustered state. To circumvent these obstacles or to avoid fine-tuning, we highlight a general strategy based on dual-time positive feedback loops, such as mediated through transcriptional versus epigenetic mechanisms, which improves proportion regulation by coordinating early and flexible lineage priming with late and firm commitment. This result sheds new light on the respective and cooperative roles of multiple regulatory feedback, stochasticity and lateral inhibition in developmental dynamics.

  10. ERRs Mediate a Metabolic Switch Required for Somatic Cell Reprogramming to Pluripotency.

    PubMed

    Kida, Yasuyuki S; Kawamura, Teruhisa; Wei, Zong; Sogo, Takahiro; Jacinto, Sandra; Shigeno, Asako; Kushige, Hiroko; Yoshihara, Eiji; Liddle, Christopher; Ecker, Joseph R; Yu, Ruth T; Atkins, Annette R; Downes, Michael; Evans, Ronald M

    2015-05-01

    Cell metabolism is adaptive to extrinsic demands; however, the intrinsic metabolic demands that drive the induced pluripotent stem cell (iPSC) program remain unclear. Although glycolysis increases throughout the reprogramming process, we show that the estrogen-related nuclear receptors (ERRα and ERRγ) and their partnered co-factors PGC-1α and PGC-1β are transiently induced at an early stage, resulting in a burst of oxidative phosphorylation (OXPHOS) activity. Upregulation of ERRα or ERRγ is required for the OXPHOS burst in both human and mouse cells, respectively, as well as iPSC generation itself. Failure to induce this metabolic switch collapses the reprogramming process. Furthermore, we identify a rare pool of Sca1(-)/CD34(-) sortable cells that is highly enriched in bona fide reprogramming progenitors. Transcriptional profiling confirmed that these progenitors are ERRγ and PGC-1β positive and have undergone extensive metabolic reprogramming. These studies characterize a previously unrecognized, ERR-dependent metabolic gate prior to establishment of induced pluripotency. PMID:25865501

  11. Mortalin antibody-conjugated quantum dot transfer from human mesenchymal stromal cells to breast cancer cells requires cell–cell interaction

    SciTech Connect

    Pietilä, Mika; Lehenkari, Petri; Kuvaja, Paula; Kaakinen, Mika; Kaul, Sunil C.; Wadhwa, Renu; Uemura, Toshimasa

    2013-11-01

    The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48 h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell–cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.

  12. Signal Strength and Metabolic Requirements Control Cytokine-Induced Th17 Differentiation of Uncommitted Human T Cells.

    PubMed

    Kastirr, Ilko; Crosti, Mariacristina; Maglie, Stefano; Paroni, Moira; Steckel, Bodo; Moro, Monica; Pagani, Massimilliano; Abrignani, Sergio; Geginat, Jens

    2015-10-15

    IL-17 production defines Th17 cells, which orchestrate immune responses and autoimmune diseases. Human Th17 cells can be efficiently generated with appropriate cytokines from precommitted precursors, but the requirements of uncommitted T cells are still ill defined. In standard human Th17 cultures, IL-17 production was restricted to CCR6(+)CD45RA(+) T cells, which expressed CD95 and produced IL-17 ex vivo, identifying them as Th17 memory stem cells. Uncommitted naive CD4(+) T cells upregulated CCR6, RORC2, and IL-23R expression with Th17-promoting cytokines but in addition required sustained TCR stimulation, late mammalian target of rapamycin (mTOR) activity, and HIF-1α to produce IL-17. However, in standard high-density cultures, nutrients like glucose and amino acids became progressively limiting, and mTOR activity was consequently not sustained, despite ongoing TCR stimulation and T cell proliferation. Sustained, nutrient-dependent mTOR activity also induced spontaneous IL-22 and IFN-γ production, but these cytokines had also unique metabolic requirements. Thus, glucose promoted IL-12-independent Th1 differentiation, whereas aromatic amino acid-derived AHR ligands were selectively required for IL-22 production. The identification of Th17 memory stem cells and the stimulation requirements for induced human Th17/22 differentiation have important implications for T cell biology and for therapies targeting the mTOR pathway. PMID:26378072

  13. Extreme cellular adaptations and cell differentiation required by a cyanobacterium for carbonate excavation

    PubMed Central

    Guida, Brandon Scott; Garcia-Pichel, Ferran

    2016-01-01

    Some cyanobacteria, known as euendoliths, excavate and grow into calcium carbonates, with their activity leading to significant marine and terrestrial carbonate erosion and to deleterious effects on coral reef and bivalve ecology. Despite their environmental relevance, the mechanisms by which they can bore have remained elusive and paradoxical, in that, as oxygenic phototrophs, cyanobacteria tend to alkalinize their surroundings, which will encourage carbonate precipitation, not dissolution. Therefore, cyanobacteria must rely on unique adaptations to bore. Studies with the filamentous euendolith, Mastigocoleus testarum, indicated that excavation requires both cellular energy and transcellular calcium transport, mediated by P-type ATPases, but the cellular basis for this phenomenon remains obscure. We present evidence that excavation in M. testarum involves two unique cellular adaptations. Long-range calcium transport is based on active pumping at multiple cells along boring filaments, orchestrated by the preferential localization of calcium ATPases at one cell pole, in a ring pattern, facing the cross-walls, and by repeating this placement and polarity, a pattern that breaks at branching and apical cells. In addition, M. testarum differentiates specialized cells we call calcicytes, that which accumulate calcium at concentrations more than 500-fold those found in other cyanobacteria, concomitantly and drastically lowering photosynthetic pigments and enduring severe cytoplasmatic alkalinization. Calcicytes occur commonly, but not exclusively, in apical parts of the filaments distal to the excavation front. We suggest that calcicytes allow for fast calcium flow at low, nontoxic concentrations through undifferentiated cells by providing buffering storage for excess calcium before final excretion to the outside medium. PMID:27140633

  14. Bacillus anthracis tagO Is Required for Vegetative Growth and Secondary Cell Wall Polysaccharide Synthesis

    PubMed Central

    Lunderberg, J. Mark; Liszewski Zilla, Megan; Missiakas, Dominique

    2015-01-01

    ABSTRACT Bacillus anthracis elaborates a linear secondary cell wall polysaccharide (SCWP) that retains surface (S)-layer and associated proteins via their S-layer homology (SLH) domains. The SCWP is comprised of trisaccharide repeats [→4)-β-ManNAc-(1→4)-β-GlcNAc-(1→6)-α-GlcNAc-(1→] and tethered via acid-labile phosphodiester bonds to peptidoglycan. Earlier work identified UDP-GlcNAc 2-epimerases GneY (BAS5048) and GneZ (BAS5117), which act as catalysts of ManNAc synthesis, as well as a polysaccharide deacetylase (BAS5051), as factors contributing to SCWP synthesis. Here, we show that tagO (BAS5050), which encodes a UDP-N-acetylglucosamine:undecaprenyl-P N-acetylglucosaminyl 1-P transferase, the enzyme that initiates the synthesis of murein linkage units, is required for B. anthracis SCWP synthesis and S-layer assembly. Similar to gneY-gneZ mutants, B. anthracis strains lacking tagO cannot maintain cell shape or support vegetative growth. In contrast, mutations in BAS5051 do not affect B. anthracis cell shape, vegetative growth, SCWP synthesis, or S-layer assembly. These data suggest that TagO-mediated murein linkage unit assembly supports SCWP synthesis and attachment to the peptidoglycan via acid-labile phosphodiester bonds. Further, B. anthracis variants unable to synthesize SCWP trisaccharide repeats cannot sustain cell shape and vegetative growth. IMPORTANCE Bacillus anthracis elaborates an SCWP to support vegetative growth and envelope assembly. Here, we show that some, but not all, SCWP synthesis is dependent on tagO-derived murein linkage units and subsequent attachment of SCWP to peptidoglycan. The data implicate secondary polymer modifications of peptidoglycan and subcellular distributions as a key feature of the cell cycle in Gram-positive bacteria and establish foundations for work on the molecular functions of the SCWP and on inhibitors with antibiotic attributes. PMID:26324447

  15. Extreme cellular adaptations and cell differentiation required by a cyanobacterium for carbonate excavation.

    PubMed

    Guida, Brandon Scott; Garcia-Pichel, Ferran

    2016-05-17

    Some cyanobacteria, known as euendoliths, excavate and grow into calcium carbonates, with their activity leading to significant marine and terrestrial carbonate erosion and to deleterious effects on coral reef and bivalve ecology. Despite their environmental relevance, the mechanisms by which they can bore have remained elusive and paradoxical, in that, as oxygenic phototrophs, cyanobacteria tend to alkalinize their surroundings, which will encourage carbonate precipitation, not dissolution. Therefore, cyanobacteria must rely on unique adaptations to bore. Studies with the filamentous euendolith, Mastigocoleus testarum, indicated that excavation requires both cellular energy and transcellular calcium transport, mediated by P-type ATPases, but the cellular basis for this phenomenon remains obscure. We present evidence that excavation in M. testarum involves two unique cellular adaptations. Long-range calcium transport is based on active pumping at multiple cells along boring filaments, orchestrated by the preferential localization of calcium ATPases at one cell pole, in a ring pattern, facing the cross-walls, and by repeating this placement and polarity, a pattern that breaks at branching and apical cells. In addition, M. testarum differentiates specialized cells we call calcicytes, that which accumulate calcium at concentrations more than 500-fold those found in other cyanobacteria, concomitantly and drastically lowering photosynthetic pigments and enduring severe cytoplasmatic alkalinization. Calcicytes occur commonly, but not exclusively, in apical parts of the filaments distal to the excavation front. We suggest that calcicytes allow for fast calcium flow at low, nontoxic concentrations through undifferentiated cells by providing buffering storage for excess calcium before final excretion to the outside medium. PMID:27140633

  16. α6-integrin is required for the adhesion and vasculogenic potential of hemangioma stem cells

    PubMed Central

    Smadja, David M.; Guerin, Coralie L.; Boscolo, Elisa; Bieche, Ivan; Mulliken, John B.; Bischoff, Joyce

    2013-01-01

    Background Infantile hemangioma (IH) is the most common tumor of infancy. Hemangioma stem cells (HemSC) are a mesenchymal subpopulation isolated from IH CD133+ cells. HemSC can differentiate into endothelial and pericyte/smooth muscle cells and form vascular networks when injected in immune-deficient mice. α6-Integrin subunit has been implicated in the tumorgenicity of glioblastoma stem cells and the homing properties of hematopoietic, endothelial and mesenchymal progenitor cells. Therefore, we investigated the possible function(s) of α6-integrin in HemSC. Methods/Results We documented α6-integrin expression in IH tumor specimens and HemSC by RT-qPCR and flow cytometry. We examined the effect of blocking or silencing α6-integrin on the adhesive and proliferative properties of HemSCin vitro and the vasculogenic and homing properties of HemSCin vivo. Targeting α6-integrin in cultured HemSC inhibited adhesion to laminin but had no effect on proliferation. Vessel-forming ability in Matrigel implants and hepatic homing after intravenous delivery were significantly decreased in α6-integrin siRNA transfected HemSC. Conclusion α6-Integrin is required for HemSC adherence to laminin, vessel formation in vivo and for homing to the liver. Thus, we uncovered an important role for α6 integrin in the vasculogenic properties of HemSC. Our results suggest that α6-integrin expression on HemSC could be a new target for anti-hemangioma therapy. PMID:24022922

  17. Immune Cell Inhibition by SLAMF7 Is Mediated by a Mechanism Requiring Src Kinases, CD45, and SHIP-1 That Is Defective in Multiple Myeloma Cells

    PubMed Central

    Guo, Huaijian; Cruz-Munoz, Mario-Ernesto; Wu, Ning; Robbins, Michael

    2014-01-01

    Signaling lymphocytic activation molecule F7 (SLAMF7) is a receptor present on immune cells, including natural killer (NK) cells. It is also expressed on multiple myeloma (MM) cells. This led to development of an anti-SLAMF7 antibody, elotuzumab, showing efficacy against MM. SLAMF7 mediates activating or inhibitory effects in NK cells, depending on whether cells express or do not express the adaptor EAT-2. Since MM cells lack EAT-2, we elucidated the inhibitory effectors of SLAMF7 in EAT-2-negative NK cells and tested whether these effectors were triggered in MM cells. SLAMF7-mediated inhibition in NK cells lacking EAT-2 was mediated by SH2 domain-containing inositol phosphatase 1 (SHIP-1), which was recruited via tyrosine 261 of SLAMF7. Coupling of SLAMF7 to SHIP-1 required Src kinases, which phosphorylated SLAMF7. Although MM cells lack EAT-2, elotuzumab did not induce inhibitory signals in these cells. This was at least partly due to a lack of CD45, a phosphatase required for Src kinase activation. A defect in SLAMF7 function was also observed in CD45-deficient NK cells. Hence, SLAMF7-triggered inhibition is mediated by a mechanism involving Src kinases, CD45, and SHIP-1 that is defective in MM cells. This defect might explain why elotuzumab eliminates MM cells by an indirect mechanism involving the activation of NK cells. PMID:25312647

  18. IgE-mediated enhancement of CD4+ T cell responses requires antigen presentation by CD8α− conventional dendritic cells

    PubMed Central

    Ding, Zhoujie; Dahlin, Joakim S.; Xu, Hui; Heyman, Birgitta

    2016-01-01

    IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4+ T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)+ B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c+ cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c+ cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α− conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4+ T cell proliferation ex vivo than were CD8α+ cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α− cDCs which induce proliferation of CD4+ T cells. PMID:27306570

  19. IgE-mediated enhancement of CD4(+) T cell responses requires antigen presentation by CD8α(-) conventional dendritic cells.

    PubMed

    Ding, Zhoujie; Dahlin, Joakim S; Xu, Hui; Heyman, Birgitta

    2016-01-01

    IgE, forming an immune complex with small proteins, can enhance the specific antibody and CD4(+) T cell responses in vivo. The effects require the presence of CD23 (Fcε-receptor II)(+) B cells, which capture IgE-complexed antigens (Ag) in the circulation and transport them to splenic B cell follicles. In addition, also CD11c(+) cells, which do not express CD23, are required for IgE-mediated enhancement of T cell responses. This suggests that some type of dendritic cell obtains IgE-Ag complexes from B cells and presents antigenic peptides to T cells. To elucidate the nature of this dendritic cell, mice were immunized with ovalbumin (OVA)-specific IgE and OVA, and different populations of CD11c(+) cells, obtained from the spleens four hours after immunization, were tested for their ability to present OVA. CD8α(-) conventional dendritic cells (cDCs) were much more efficient in inducing specific CD4(+) T cell proliferation ex vivo than were CD8α(+) cDCs or plasmacytoid dendritic cells. Thus, IgE-Ag complexes administered intravenously are rapidly transported to the spleen by recirculating B cells where they are delivered to CD8α(-) cDCs which induce proliferation of CD4(+) T cells. PMID:27306570

  20. Pulse power requirements for large aperture optical switches based on plasma electrode Pockels cells

    SciTech Connect

    Rhodes, M.A.; Taylor, J.

    1992-06-01

    We discuss very large-aperture optical switches (greater than 30 [times] 30 cm) as an enabling technology for inertial confinement fusion drivers based on multipass laser amplifiers. Large-scale laser fusion drivers such as the Nova laser have been based on single-pass amplifier designs in part because of the unavailability of a suitable large-aperture switch. We are developing an optical switch based on a Pockels cell employing plasma-electrodes. A plasma-electrode Pockels cell (PEPC) is a longitudinal-mode Pockels cell in which a plasma discharge is formed on each side of an electro-optic crystal (typically KDP or deuterated KDP, often designated KD*P). The plasmas formed on either side of the crystal act as transparent electrodes for a switching-pulse and are intended to allow uniform charging of the entire crystal. The switching-pulse is a nominally rectangular high-voltage pulse equal to the half-wave voltage V[sub x] ( 8 kV for KD*P or 17 kV for KDP) and is applied across the crystal via the plasma-electrodes. When the crystal is charged to V[sub x], the polarization of an incoming, linearly polarized, laser beam is rotated by 90[degree]. When used in conjunction with an appropriate, passive polarizer, an optical switch is thus realized. A switch with a clear aperture of 37 [times] 37 cm is now in construction for the Beamlet laser which will serve as a test bed for this switch as well as other technologies required for an advanced NOVA laser design. In this paper, we discuss the unique power electronics requirements of PEPC optical switches.

  1. Pulse power requirements for large aperture optical switches based on plasma electrode Pockels cells

    SciTech Connect

    Rhodes, M.A.; Taylor, J.

    1992-06-01

    We discuss very large-aperture optical switches (greater than 30 {times} 30 cm) as an enabling technology for inertial confinement fusion drivers based on multipass laser amplifiers. Large-scale laser fusion drivers such as the Nova laser have been based on single-pass amplifier designs in part because of the unavailability of a suitable large-aperture switch. We are developing an optical switch based on a Pockels cell employing plasma-electrodes. A plasma-electrode Pockels cell (PEPC) is a longitudinal-mode Pockels cell in which a plasma discharge is formed on each side of an electro-optic crystal (typically KDP or deuterated KDP, often designated KD*P). The plasmas formed on either side of the crystal act as transparent electrodes for a switching-pulse and are intended to allow uniform charging of the entire crystal. The switching-pulse is a nominally rectangular high-voltage pulse equal to the half-wave voltage V{sub x} ( 8 kV for KD*P or 17 kV for KDP) and is applied across the crystal via the plasma-electrodes. When the crystal is charged to V{sub x}, the polarization of an incoming, linearly polarized, laser beam is rotated by 90{degree}. When used in conjunction with an appropriate, passive polarizer, an optical switch is thus realized. A switch with a clear aperture of 37 {times} 37 cm is now in construction for the Beamlet laser which will serve as a test bed for this switch as well as other technologies required for an advanced NOVA laser design. In this paper, we discuss the unique power electronics requirements of PEPC optical switches.

  2. Stemness factor Sall4 is required for DNA damage response in embryonic stem cells

    PubMed Central

    Xiong, Jianhua; Todorova, Dilyana; Su, Ning-Yuan; Kim, Jinchul; Lee, Pei-Jen; Shen, Zhouxin; Briggs, Steven P.

    2015-01-01

    Mouse embryonic stem cells (ESCs) are genetically more stable than somatic cells, thereby preventing the passage of genomic abnormalities to their derivatives including germ cells. The underlying mechanisms, however, remain largely unclear. In this paper, we show that the stemness factor Sall4 is required for activating the critical Ataxia Telangiectasia Mutated (ATM)–dependent cellular responses to DNA double-stranded breaks (DSBs) in mouse ESCs and confer their resistance to DSB-induced cytotoxicity. Sall4 is rapidly mobilized to the sites of DSBs after DNA damage. Furthermore, Sall4 interacts with Rad50 and stabilizes the Mre11–Rad50–Nbs1 complex for the efficient recruitment and activation of ATM. Sall4 also interacts with Baf60a, a member of the SWI/SNF (switch/sucrose nonfermentable) ATP-dependent chromatin-remodeling complex, which is responsible for recruiting Sall4 to the site of DNA DSB damage. Our findings provide novel mechanisms to coordinate stemness of ESCs with DNA damage response, ensuring genomic stability during the expansion of ESCs. PMID:25733712

  3. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae

    PubMed Central

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  4. SKAP, an outer kinetochore protein, is required for mouse germ cell development

    PubMed Central

    Grey, Corinne; Espeut, Julien; Ametsitsi, Rachel; Kumar, Rajeev; Luksza, Malgorzata; Brun, Christine; Verlhac, Marie-Hélene; Suja, José Angél; de Massy, Bernard

    2016-01-01

    In sexually reproducing organisms, accurate gametogenesis is crucial for the transmission of genetic material from one generation to the next. This requires the faithful segregation of chromosomes during mitotic and meiotic divisions. One of the main players in this process is the kinetochore, a large multi-protein complex that forms at the interface of centromeres and microtubules. Here, we analyzed the expression profile and function of small kinetochore-associated protein (SKAP) in the mouse. We found that two distinct SKAP isoforms are specifically expressed in the germline: a smaller isoform, which is detected in spermatogonia and spermatocytes and localized in the outer mitotic and meiotic kinetochores from metaphase to telophase, and a larger isoform, which is expressed in the cytoplasm of elongating spermatids. We generated SKAP-deficient mice and found that testis size and sperm production were severely reduced in mutant males. This phenotype was partially caused by defects during spermatogonia proliferation before entry into meiosis. We conclude that mouse SKAP, while being dispensable for somatic cell divisions, has an important role in the successful outcome of male gametogenesis. In germ cells, analogous to what has been suggested in studies using immortalized cells, SKAP most likely stabilizes the interaction between kinetochores and microtubules, where it might be needed as an extra safeguard to ensure the correct segregation of mitotic and meiotic chromosomes. PMID:26667018

  5. A Bruno-like gene is required for stem cell maintenance in planarians.

    PubMed

    Guo, Tingxia; Peters, Antoine H F M; Newmark, Phillip A

    2006-08-01

    The regenerative abilities of freshwater planarians are based on neoblasts, stem cells maintained throughout the animal's life. We show that a member of the Bruno-like family of RNA binding proteins is critical for regulating neoblasts in the planarian Schmidtea mediterranea. Smed-bruno-like (bruli) mRNA and protein are expressed in neoblasts and the central nervous system. Following bruli RNAi, which eliminates detectable Bruli protein, planarians initiate the proliferative response to amputation and form small blastemas but then undergo tissue regression and lysis. We characterize the neoblast population by using antibodies recognizing SMEDWI-1 and Histone H4 (monomethyl-K20) and cell-cycle markers to label subsets of neoblasts and their progeny. bruli knockdown results in a dramatic reduction/elimination of neoblasts. Our analyses indicate that neoblasts lacking Bruli can respond to wound stimuli and generate progeny that can form blastemas and differentiate; yet, they are unable to self-renew. These results suggest that Bruli is required for stem cell maintenance. PMID:16890156

  6. Regulatory requirements for clinical trial and marketing authorisation application for cell-based medicinal products.

    PubMed

    Salmikangas, P; Flory, E; Reinhardt, J; Hinz, T; Maciulaitis, R

    2010-01-01

    The new era of regenerative medicine has led to rapid development of new innovative therapies especially for diseases and tissue/organ defects for which traditional therapies and medicinal products have not provided satisfactory outcome. Although the clinical use and developments of cell-based medicinal products (CBMPs) could be witnessed already for a decade, robust scientific and regulatory provisions for these products have only recently been enacted. The new Regulation for Advanced Therapies (EC) 1394/2007 together with the revised Annex I, Part IV of Directive 2001/83/EC provides the new legal framework for CBMPs. The wide variety of cell-based products and the foreseen limitations (small sample sizes, short shelf life) vs. particular risks (microbiological purity, variability, immunogenicity, tumourigenicity) associated with CBMPs have called for a flexible, case-by-case regulatory approach for these products. Consequently, a risk-based approach has been developed to allow definition of the amount of scientific data needed for a Marketing Authorisation Application (MAA) of each CBMP. The article provides further insight into the initial risk evaluation, as well as to the quality, non-clinical, and clinical requirements of CBMPs. Special somatic cell therapies designed for active immunotherapy are also addressed. PMID:19940964

  7. Vgll2a is required for neural crest cell survival during zebrafish craniofacial development

    PubMed Central

    Johnson, Christopher W.; Hernandez-Lagunas, Laura; Feng, Weiguo; Melvin, Vida Senkus; Williams, Trevor; Artinger, Kristin Bruk

    2011-01-01

    Invertebrate and vertebrate vestigial (vg) and vestigial-like (vgl) genes are involved in embryonic patterning and cell fate determination. These genes encode cofactors that interact with members of the TEAD/Scalloped family of transcription factors and modulate their activity. We have previously shown that, in mice, Vgll2 is differentially expressed in the developing facial prominences. In this study, we show that the zebrafish ortholog vgll2a is expressed in the pharyngeal endoderm and ectoderm surrounding the neural crest derived mesenchyme of the pharyngeal arches. Moreover, both the FGF and retinoic acid (RA) signaling pathways, which are critical components of the hierarchy controlling craniofacial patterning, regulate this domain of vgll2a expression. Consistent with these observations, vgll2a is required within the pharyngeal endoderm for NCC survival and pharyngeal cartilage development. Specifically, knockdown of Vgll2a in zebrafish embryos using Morpholino injection results in increased cell death within the pharyngeal arches, aberrant endodermal pouch morphogenesis, and hypoplastic cranial cartilages. Overall, our data reveal a novel non-cell autonomous role for Vgll2a in development of the NCC-derived vertebrate craniofacial skeleton. PMID:21741961

  8. Autophagy-associated alpha-arrestin signaling is required for conidiogenous cell development in Magnaporthe oryzae.

    PubMed

    Dong, Bo; Xu, Xiaojin; Chen, Guoqing; Zhang, Dandan; Tang, Mingzhi; Xu, Fei; Liu, Xiaohong; Wang, Hua; Zhou, Bo

    2016-01-01

    Conidiation patterning is evolutionarily complex and mechanism concerning conidiogenous cell differentiation remains largely unknown. Magnaporthe oryzae conidiates in a sympodial way and uses its conidia to infect host and disseminate blast disease. Arrestins are multifunctional proteins that modulate receptor down-regulation and scaffold components of intracellular trafficking routes. We here report an alpha-arrestin that regulates patterns of conidiation and contributes to pathogenicity in M. oryzae. We show that disruption of ARRDC1 generates mutants which produce conidia in an acropetal array and ARRDC1 significantly affects expression profile of CCA1, a virulence-related transcription factor required for conidiogenous cell differentiation. Although germ tubes normally develop appressoria, penetration peg formation is dramatically impaired and Δarrdc1 mutants are mostly nonpathogenic. Fluorescent analysis indicates that EGFP-ARRDC1 puncta are well colocalized with DsRed2-Atg8, and this distribution profile could not be altered in Δatg9 mutants, suggesting ARRDC1 enters into autophagic flux before autophagosome maturation. We propose that M. oryzae employs ARRDC1 to regulate specific receptors in response to conidiation-related signals for conidiogenous cell differentiation and utilize autophagosomes for desensitization of conidiogenous receptor, which transmits extracellular signal to the downstream elements of transcription factors. Our investigation extends novel significance of autophagy-associated alpha-arrestin signaling to fungal parasites. PMID:27498554

  9. Fancd2 is required for nuclear retention of Foxo3a in hematopoietic stem cell maintenance.

    PubMed

    Li, Xiaoli; Li, Jie; Wilson, Andrew; Sipple, Jared; Schick, Jonathan; Pang, Qishen

    2015-01-30

    Functional maintenance of hematopoietic stem cells (HSCs) is constantly challenged by stresses like DNA damage and oxidative stress. Here we show that the Fanconi anemia protein Fancd2 and stress transcriptional factor Foxo3a cooperate to prevent HSC exhaustion in mice. Deletion of both Fancd2 and Foxo3a led to an initial expansion followed by a progressive decline of bone marrow stem and progenitor cells. Limiting dilution transplantation and competitive repopulating experiments demonstrated a dramatic reduction of competitive repopulating units and progressive decline in hematopoietic repopulating ability of double-knockout (dKO) HSCs. Analysis of the transcriptome of dKO HSCs revealed perturbation of multiple pathways implicated in HSC exhaustion. Fancd2 deficiency strongly promoted cytoplasmic localization of Foxo3a in HSCs, and re-expression of Fancd2 completely restored nuclear Foxo3a localization. By co-expressing a constitutively active CA-FOXO3a and WT or a nonubiquitinated Fancd2 in dKO bone marrow stem/progenitor cells, we demonstrated that Fancd2 was required for nuclear retention of CA-FOXO3a and for maintaining hematopoietic repopulation of the HSCs. Collectively, these results implicate a functional interaction between the Fanconi anemia DNA repair and FOXO3a pathways in HSC maintenance. PMID:25505262

  10. Human Cells Require Non-stop Ribosome Rescue Activity in Mitochondria

    PubMed Central

    Feaga, Heather A.; Quickel, Michael D.; Hankey-Giblin, Pamela A.; Keiler, Kenneth C.

    2016-01-01

    Bacteria use trans-translation and the alternative rescue factors ArfA (P36675) and ArfB (Q9A8Y3) to hydrolyze peptidyl-tRNA on ribosomes that stall near the 3' end of an mRNA during protein synthesis. The eukaryotic protein ICT1 (Q14197) is homologous to ArfB. In vitro ribosome rescue assays of human ICT1 and Caulobacter crescentus ArfB showed that these proteins have the same activity and substrate specificity. Both ArfB and ICT1 hydrolyze peptidyl-tRNA on nonstop ribosomes or ribosomes stalled with ≤6 nucleotides extending past the A site, but are unable to hydrolyze peptidyl-tRNA when the mRNA extends ≥14 nucleotides past the A site. ICT1 provided sufficient ribosome rescue activity to support viability in C. crescentus cells that lacked both trans-translation and ArfB. Likewise, expression of ArfB protected human cells from death when ICT1 was silenced with siRNA. These data indicate that ArfB and ICT1 are functionally interchangeable, and demonstrate that ICT1 is a ribosome rescue factor. Because ICT1 is essential in human cells, these results suggest that ribosome rescue activity in mitochondria is required in humans. PMID:27029019

  11. Cathepsin L is required for endothelial progenitor cell-induced neovascularization

    SciTech Connect

    Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra; Sasaki, Ken-ichiro; Bruhl, Thomas; Hofmann, Wolf K.; Peters, Christoph; Reinheckel, Thomas; Pennacchio, Len A.; Abolmaali, Nasreddin D.; Chavakis, Emmanouil; Zeiher, Andreas M.; Dimmeler, Stefanie

    2004-01-15

    Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sites of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.

  12. Differential requirement for Caspase-1 autoproteolysis in pathogen-induced cell death and cytokine processing

    PubMed Central

    Broz, Petr; von Moltke, Jakob; Jones, Jonathan W.; Vance, Russell E.; Monack, Denise M.

    2010-01-01

    Summary Activation of the cysteine protease Caspase-1 is a key event in the innate immune response to infections. Synthesized as a pro-protein, Caspase-1 undergoes autoproteolysis within multi-protein complexes called inflammasomes. Activated Caspase-1 is required for proteolytic processing and release of the cytokines interleukin-1β and interleukin-18, and can also cause rapid macrophage cell death. We show that macrophage cell death and cytokine maturation in response to infection with diverse bacterial pathogens can be separated genetically and that two distinct inflammasome complexes mediate these events. Inflammasomes containing the signaling adaptor Asc form a single large ‘focus’ in which Caspase-1 undergoes autoproteolysis and processes IL-1β/IL-18. In contrast, Asc-independent inflammasomes activate Caspase-1 without autoproteolysis and do not form any large structures in the cytosol. Caspase-1 mutants unable to undergo autoproteolysis promoted rapid cell death, but processed IL-1β/18 inefficiently. Our results suggest the formation of spatially and functionally distinct inflammasomes complexes in response to bacterial pathogens. PMID:21147462

  13. High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein.

    PubMed

    Munro, P; Kojima, H; Dupont, J L; Bossu, J L; Poulain, B; Boquet, P

    2001-11-30

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis. Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT. We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP. Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC. In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT. Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin. PMID:11716521

  14. FOXP3+ regulatory T cell development and function require histone/protein deacetylase 3

    PubMed Central

    Wang, Liqing; Liu, Yujie; Han, Rongxiang; Beier, Ulf H.; Bhatti, Tricia R.; Akimova, Tatiana; Greene, Mark I.; Hiebert, Scott W.; Hancock, Wayne W.

    2015-01-01

    Treg dysfunction is associated with a variety of inflammatory diseases. Treg populations are defined by expression of the oligomeric transcription factor FOXP3 and inability to produce IL-2, a cytokine required for T cell maintenance and survival. FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs). Here, we determined that HDAC3 mediates both the development and function of the two main Treg subsets, thymus-derived Tregs and induced Tregs (iTregs). We determined that HDAC3 and FOXP3 physically interact and that HDAC3 expression markedly reduces Il2 promoter activity. In murine models, conditional deletion of Hdac3 during thymic Treg development restored Treg production of IL-2 and blocked the suppressive function of Tregs. HDAC3-deficient mice died from autoimmunity by 4–6 weeks of age; however, injection of WT FOXP3+ Tregs prolonged survival. Adoptive transfer of Hdac3-deficient Tregs, unlike WT Tregs, did not control T cell proliferation in naive mice and did not prevent allograft rejection or colitis. HDAC3 also regulated the development of iTregs, as HDAC3-deficient conventional T cells were not converted into iTregs under polarizing conditions and produced large amounts of IL-2, IL-6, and IL-17. We conclude that HDAC3 is essential for the normal development and suppressive functions of thymic and peripheral FOXP3+ Tregs. PMID:25642770

  15. The requirement of membrane lymphotoxin for the presence of dendritic cells in lymphoid tissues.

    PubMed

    Wu, Q; Wang, Y; Wang, J; Hedgeman, E O; Browning, J L; Fu, Y X

    1999-09-01

    Although several cytokines, including tumor necrosis factor (TNF), can promote the growth of dendritic cells (DCs) in vitro, the cytokines that naturally regulate DC development and function in vivo have not been well defined. Here, we report that membrane lymphotoxin (LT), instead of TNF, regulates the migration of DCs in the spleen. LTalpha(-/-) mice, lacking membrane LTalpha/beta and LTalpha(3), show markedly reduced numbers of DCs in the spleen. Unlike wild-type mice and TNF(-/-) mice that have densely clustered DCs in the T cell zone and around the marginal zone, splenic DCs in LTalpha(-/-) mice are randomly distributed. The reduced number of DCs in lymphoid tissues of LTalpha(-/-) mice is associated with an increased number of DCs in nonlymphoid tissues. The number of splenic DCs in LTalpha(-/-) mice is restored when additional LT-expressing cells are provided. Blocking membrane LTalpha/beta in wild-type mice markedly diminishes the accumulation of DCs in lymphoid tissues. These data suggest that membrane LT is an essential ligand for the presence of DCs in the spleen. Mice deficient in TNF receptor, which is the receptor for both soluble LTalpha(3) and TNF-alpha(3) trimers, have normal numbers of DCs. However, LTbetaR(-/-) mice show reduced numbers of DCs, similar to the mice lacking membrane LT alpha/beta. Taken together, these results support the notion that the signaling via LTbetaR by membrane LTalpha/beta is required for the presence of DCs in lymphoid tissues. PMID:10477548

  16. Osmotic Regulation Is Required for Cancer Cell Survival under Solid Stress.

    PubMed

    McGrail, Daniel J; McAndrews, Kathleen M; Brandenburg, Chandler P; Ravikumar, Nithin; Kieu, Quang Minh N; Dawson, Michelle R

    2015-10-01

    For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress. PMID:26445434

  17. CD8+ T cell migration to the skin requires CD4+ help in a murine model of contact hypersensitivity.

    PubMed

    Fyhrquist, Nanna; Wolff, Henrik; Lauerma, Antti; Alenius, Harri

    2012-01-01

    The relative roles of CD4+ and CD8+ T cells in contact hypersensitivity responses have not been fully solved, and remain an important question. Using an adoptive transfer model, we investigated the role of the respective T cell subset. Magnetic bead separated CD4+ and CD8+ T cells from oxazolone sensitized C57BL/6 mice were transferred into RAG-/- mice, followed by hapten challenge and analysis of inflammatory parameters at 24 hours post exposure. The CD4+ T cell recipient mice developed partial contact hypersensitivity responses to oxazolone. CD8+ T cells caused significant amplification of the response in recipients of both CD4+ and CD8+ T cells including ear swelling, type 1 inflammatory mediators, and cell killing. Unexpectedly, CD8+ T cells were not sufficient to mediate contact hypersensitivity, although abundantly present in the lymph nodes in the CD8+ T cell reconstituted mice. There were no signs of inflammation at the site of hapten exposure, indicating impaired recruitment of CD8+ T cells in the absence of CD4+ T cells. These data show that CD4+ T cells mediate contact hypersensitivity to oxazolone, but CD8+ T cells contribute with the most potent effector mechanisms. Moreover, our results suggest that CD4+ T cell function is required for the mobilization of CD8+ effector T cells to the site of hapten exposure. The results shed new light on the relative importance of CD4+ and CD8+ T cells during the effector phase of contact hypersensitivity. PMID:22916101

  18. Effect of Spermidine Analogues on Cell Growth of Escherichia coli Polyamine Requiring Mutant MA261

    PubMed Central

    Yoshida, Taketo; Sakamoto, Akihiko; Terui, Yusuke; Takao, Koichi; Sugita, Yoshiaki; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei; Kashiwagi, Keiko

    2016-01-01

    The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon. PMID:27434546

  19. Requirement for Ssbp2 in Hematopoietic Stem Cell Maintenance and Stress Response

    PubMed Central

    Li, June; Kurasawa, Yasuhiro; Wang, Yang; Clise-Dwyer, Karen; Klumpp, Sherry A.; Liang, Hong; Tailor, Ramesh C.; Raymond, Aaron C.; Estrov, Zeev; Brandt, Stephen J.; Davis, Richard E.; Zweidler–McKay, Patrick; Amin, Hesham M.; Nagarajan, Lalitha

    2014-01-01

    Transcriptional mechanisms governing hematopoietic stem cell (HSC) quiescence, self-renewal, and differentiation are not fully understood. Sequence-specific single-stranded DNA-binding protein 2 (SSBP2) is a candidate acute myelogenous leukemia (AML) suppressor gene located at chromosome 5q14. SSBP2 binds the transcriptional adaptor protein Lim-domain binding protein 1 (LDB1) and enhances LDB1 stability to regulate gene expression. Notably, Ldb1 is essential for HSC specification during early development and maintenance in adults. We previously reported shortened lifespan and greater susceptibility to B cell lymphomas and carcinomas in Ssbp2 −/− mice. However, whether Ssbp2 plays a regulatory role in normal HSC function and leukemogenesis is unknown. Here, we provide several lines of evidence to demonstrate a requirement for Ssbp2 in the function and transcriptional program of hematopoietic stem and progenitor cells (HSPCs) in vivo. We found that hematopoietic tissues were hypoplastic in Ssbp2−/− mice and the frequency of lymphoid-primed multipotent progenitor cells in bone marrow was reduced. Other significant features of these mice were delayed recovery from 5-fluorouracil treatment and diminished multilineage reconstitution in lethally irradiated bone marrow recipients. Dramatic reduction of Notch1 transcripts and increased expression of transcripts encoding the transcription factor E2a and its downstream target Cdkn1a also distinguished Ssbp2−/− HSPCs from wild-type HSPCs. Finally, a tendency towards coordinated expression of SSBP2 and the AML suppressor NOTCH1 in a subset of The Cancer Genome Atlas AML cases suggested a role for SSBP2 in AML pathogenesis. Collectively, our results uncovered a critical regulatory function for SSBP2 in HSPC gene expression and function. PMID:25238756

  20. Heat induction of heat shock protein 25 requires cellular glutamine in intestinal epithelial cells.

    PubMed

    Phanvijhitsiri, Kittiporn; Musch, Mark W; Ropeleski, Mark J; Chang, Eugene B

    2006-08-01

    Glutamine is considered a nonessential amino acid; however, it becomes conditionally essential during critical illness when consumption exceeds production. Glutamine may modulate the heat shock/stress response, an important adaptive cellular response for survival. Glutamine increases heat induction of heat shock protein (Hsp) 25 in both intestinal epithelial cells (IEC-18) and mesenchymal NIH/3T3 cells, an effect that is neither glucose nor serum dependent. Neither arginine, histidine, proline, leucine, asparagine, nor tyrosine acts as physiological substitutes for glutamine for heat induction of Hsp25. The lack of effect of these amino acids was not caused by deficient transport, although some amino acids, including glutamate (a major direct metabolite of glutamine), were transported poorly by IEC-18 cells. Glutamate uptake could be augmented in a concentration- and time-dependent manner by increasing either media concentration and/or duration of exposure. Under these conditions, glutamate promoted heat induction of Hsp25, albeit not as efficiently as glutamine. Further evidence for the role of glutamine conversion to glutamate was obtained with the glutaminase inhibitor 6-diazo-5-oxo-l-norleucine (DON), which inhibited the effect of glutamine on heat-induced Hsp25. DON inhibited phosphate-dependent glutaminase by 75% after 3 h, decreasing cell glutamate. Increased glutamine/glutamate conversion to glutathione was not involved, since the glutathione synthesis inhibitor, buthionine sulfoximine, did not block glutamine's effect on heat induction of Hsp25. A large drop in ATP levels did not appear to account for the diminished Hsp25 induction during glutamine deficiency. In summary, glutamine is an important amino acid, and its requirement for heat-induced Hsp25 supports a role for glutamine supplementation to optimize cellular responses to pathophysiological stress. PMID:16554407

  1. ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells

    PubMed Central

    Lu, Qing; Schnitzler, Gavin R.; Ueda, Kazutaka; Iyer, Lakshmanan K.; Diomede, Olga I.; Andrade, Tiffany; Karas, Richard H.

    2016-01-01

    Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs), which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen) in vascular injury require the estrogen receptor alpha (ERα). ERα transduces the effects of estrogen via a classical DNA binding, “genomic” signaling pathway and via a more recently-described “rapid” signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα) that is specifically defective in rapid signaling, but is competent to regulate transcription through the “genomic” pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen. PMID:27035664

  2. Controlled synthesis of the DSF cell-cell signal is required for biofilm formation and virulence in Xanthomonas campestris.

    PubMed

    Torres, Pablo S; Malamud, Florencia; Rigano, Luciano A; Russo, Daniela M; Marano, María Rosa; Castagnaro, Atilio P; Zorreguieta, Angeles; Bouarab, Kamal; Dow, John Maxwell; Vojnov, Adrián A

    2007-08-01

    Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell-cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence. PMID:17635553

  3. The acquisition of mechano‐electrical transducer current adaptation in auditory hair cells requires myosin VI

    PubMed Central

    Marcotti, Walter; Corns, Laura F.; Goodyear, Richard J.; Rzadzinska, Agnieszka K.; Avraham, Karen B.; Steel, Karen P.; Richardson, Guy P.

    2016-01-01

    Key points The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano‐electrical transducer (MET) channels.The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli.In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles.We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop.We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. Abstract Mutations in Myo6, the gene encoding the (F‐actin) minus end‐directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano‐electrical transduction. We report that Ca2+‐dependent adaptation of the mechano‐electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links

  4. T cell-dependent IgM memory B cells generated during bacterial infection are required for IgG responses to antigen challenge.

    PubMed

    Yates, Jennifer L; Racine, Rachael; McBride, Kevin M; Winslow, Gary M

    2013-08-01

    Immunological memory has long considered to be harbored in B cells that express high-affinity class-switched IgG. IgM-positive memory B cells can also be generated following immunization, although their physiological role has been unclear. In this study, we show that bacterial infection elicited a relatively large population of IgM memory B cells that were uniquely identified by their surface expression of CD11c, CD73, and programmed death-ligand 2. The cells lacked expression of cell surface markers typically expressed by germinal center B cells, were CD138 negative, and did not secrete Ab ex vivo. The population was also largely quiescent and accumulated somatic mutations. The IgM memory B cells were located in the region of the splenic marginal zone and were not detected in blood or other secondary lymphoid organs. Generation of the memory cells was CD4 T cell dependent and required IL-21R signaling. In vivo depletion of the IgM memory B cells abrogated the IgG recall responses to specific Ag challenge, demonstrating that the cell population was required for humoral memory, and underwent class-switch recombination following Ag encounter. Our findings demonstrate that T cell-dependent IgM memory B cells can be elicited at high frequency and can play an important role in maintaining long-term immunity during bacterial infection. PMID:23804710

  5. The transcription factor E4BP4 is not required for extramedullary pathways of NK cell development.

    PubMed

    Crotta, Stefania; Gkioka, Annita; Male, Victoria; Duarte, João H; Davidson, Sophia; Nisoli, Ilaria; Brady, Hugh J M; Wack, Andreas

    2014-03-15

    NK cells contribute to antitumor and antiviral immunosurveillance. Their development in the bone marrow (BM) requires the transcription factor E4BP4/NFIL3, but requirements in other organs are less well defined. In this study, we show that CD3(-)NK1.1(+)NKp46(+)CD122(+) NK cells of immature phenotype and expressing low eomesodermin levels are found in thymus, spleen, and liver of E4BP4-deficient mice, whereas numbers of mature, eomesodermin(high) conventional NK cells are drastically reduced. E4BP4-deficient CD44(+)CD25(-) double-negative 1 thymocytes efficiently develop in vitro into NK cells with kinetics, phenotype, and functionality similar to wild-type controls, whereas no NK cells develop from E4BP4-deficient BM precursors. In E4BP4/Rag-1 double-deficient (DKO) mice, NK cells resembling those in Rag-1-deficient controls are found in similar numbers in the thymus and liver. However, NK precursors are reduced in DKO BM, and no NK cells develop from DKO BM progenitors in vitro. DKO thymocyte precursors readily develop into NK cells, but DKO BM transfers into nude recipients and NK cells in E4BP4/Rag-1/IL-7 triple-KO mice indicated thymus-independent NK cell development. In the presence of T cells or E4BP4-sufficient NK cells, DKO NK cells have a selective disadvantage, and thymic and hepatic DKO NK cells show reduced survival when adoptively transferred into lymphopenic hosts. This correlates with higher apoptosis rates and lower responsiveness to IL-15 in vitro. In conclusion, we demonstrate E4BP4-independent development of NK cells of immature phenotype, reduced fitness, short t1/2, and potential extramedullary origin. Our data identify E4BP4-independent NK cell developmental pathways and a role for E4BP4 in NK cell homeostasis. PMID:24534532

  6. PrimPol Is Required for Replicative Tolerance of G Quadruplexes in Vertebrate Cells

    PubMed Central

    Schiavone, Davide; Jozwiakowski, Stanislaw K.; Romanello, Marina; Guilbaud, Guillaume; Guilliam, Thomas A.; Bailey, Laura J.; Sale, Julian E.; Doherty, Aidan J.

    2016-01-01

    Summary G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments. PMID:26626482

  7. Pigment cell movement is not required for generation of Turing patterns in zebrafish skin

    PubMed Central

    Bullara, D.; De Decker, Y.

    2015-01-01

    The zebrafish is a model organism for pattern formation in vertebrates. Understanding what drives the formation of its coloured skin motifs could reveal pivotal to comprehend the mechanisms behind morphogenesis. The motifs look and behave like reaction–diffusion Turing patterns, but the nature of the underlying physico-chemical processes is very different, and the origin of the patterns is still unclear. Here we propose a minimal model for such pattern formation based on a regulatory mechanism deduced from experimental observations. This model is able to produce patterns with intrinsic wavelength, closely resembling the experimental ones. We mathematically prove that their origin is a Turing bifurcation occurring despite the absence of cell motion, through an effect that we call differential growth. This mechanism is qualitatively different from the reaction–diffusion originally proposed by Turing, although they both generate the short-range activation and the long-range inhibition required to form Turing patterns. PMID:25959141

  8. Pigment cell movement is not required for generation of Turing patterns in zebrafish skin

    NASA Astrophysics Data System (ADS)

    Bullara, D.; de Decker, Y.

    2015-05-01

    The zebrafish is a model organism for pattern formation in vertebrates. Understanding what drives the formation of its coloured skin motifs could reveal pivotal to comprehend the mechanisms behind morphogenesis. The motifs look and behave like reaction-diffusion Turing patterns, but the nature of the underlying physico-chemical processes is very different, and the origin of the patterns is still unclear. Here we propose a minimal model for such pattern formation based on a regulatory mechanism deduced from experimental observations. This model is able to produce patterns with intrinsic wavelength, closely resembling the experimental ones. We mathematically prove that their origin is a Turing bifurcation occurring despite the absence of cell motion, through an effect that we call differential growth. This mechanism is qualitatively different from the reaction-diffusion originally proposed by Turing, although they both generate the short-range activation and the long-range inhibition required to form Turing patterns.

  9. Nfil3/E4bp4 is required for the development and maturation of NK cells in vivo.

    PubMed

    Kamizono, Shintaro; Duncan, Gordon S; Seidel, Markus G; Morimoto, Akira; Hamada, Koichi; Grosveld, Gerard; Akashi, Koichi; Lind, Evan F; Haight, Jillian P; Ohashi, Pamela S; Look, A Thomas; Mak, Tak W

    2009-12-21

    Nuclear factor interleukin-3 (Nfil3; also known as E4-binding protein 4) is a basic region leucine zipper transcription factor that has antiapoptotic activity in vitro under conditions of growth factor withdrawal. To study the role of Nfil3 in vivo, we generated gene-targeted Nfil3-deficient (Nfil3(-/-)) mice. Nfil3(-/-) mice were born at normal Mendelian frequency and were grossly normal and fertile. Although numbers of T cells, B cells, and natural killer (NK) T cells were normal in Nfil3(-/-) mice, a specific disruption in NK cell development resulted in severely reduced numbers of mature NK cells in the periphery. This defect was NK cell intrinsic in nature, leading to a failure to reject MHC class I-deficient cells in vivo and reductions in both interferon gamma production and cytolytic activity in vitro. Our results confirm the specific and essential requirement of Nfil3 for the development of cells of the NK lineage. PMID:19995955

  10. Protection against henipaviruses in swine requires both, cell-mediated and humoral immune response.

    PubMed

    Pickering, Brad S; Hardham, John M; Smith, Greg; Weingartl, Eva T; Dominowski, Paul J; Foss, Dennis L; Mwangi, Duncan; Broder, Christopher C; Roth, James A; Weingartl, Hana M

    2016-09-14

    Hendra virus (HeV) and Nipah virus (NiV) are members of the genus Henipavirus, within the family Paramyxoviridae. Nipah virus has caused outbreaks of human disease in Bangladesh, Malaysia, Singapore, India and Philippines, in addition to a large outbreak in swine in Malaysia in 1998/1999. Recently, NiV was suspected to be a causative agent of an outbreak in horses in 2014 in the Philippines, while HeV has caused multiple human and equine outbreaks in Australia since 1994. A swine vaccine able to prevent shedding of infectious virus is of veterinary and human health importance, and correlates of protection against henipavirus infection in swine need to be better understood. In the present study, three groups of animals were employed. Pigs vaccinated with adjuvanted recombinant soluble HeV G protein (sGHEV) and challenged with HeV, developed antibody levels considered to be protective prior to the challenge (titers of 320). However, activation of the cell-mediated immune response was not detected, and the animals were only partially protected against challenge with 5×10(5) PFU of HeV per animal. In the second group, cross-neutralizing antibody levels against NiV in the sGHEV vaccinated animals did not reach protective levels, and with no activation of cellular immune memory, these animals were not protected against NiV. Only pigs orally infected with 5×10(4) PFU of NiV per animal were protected against nasal challenge with 5×10(5) PFU of NiV per animal. This group of pigs developed protective antibody levels, as well as cell-mediated immune memory. Peripheral blood mononuclear cells restimulated with UV-inactivated NiV upregulated IFN-gamma, IL-10 and the CD25 activation marker on CD4(+)CD8(+) T memory helper cells and to lesser extent on CD4(-)CD8(+) T cells. In conclusion, both humoral and cellular immune responses were required for protection of swine against henipaviruses. PMID:27544586

  11. Gata2 cis-element is required for hematopoietic stem cell generation in the mammalian embryo.

    PubMed

    Gao, Xin; Johnson, Kirby D; Chang, Yuan-I; Boyer, Meghan E; Dewey, Colin N; Zhang, Jing; Bresnick, Emery H

    2013-12-16

    The generation of hematopoietic stem cells (HSCs) from hemogenic endothelium within the aorta, gonad, mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. We described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13-14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia/leukemia. Here, we demonstrate that the +9.5 element enhances Gata2 expression and is required to generate long-term repopulating HSCs in the AGM. Deletion of the +9.5 element abrogated the capacity of hemogenic endothelium to generate HSC-containing clusters in the aorta. Genomic analyses indicated that the +9.5 element regulated a rich ensemble of genes that control hemogenic endothelium and HSCs, as well as genes not implicated in hematopoiesis. These results reveal a mechanism that controls stem cell emergence from hemogenic endothelium to establish the adult hematopoietic system. PMID:24297994

  12. Gata2 cis-element is required for hematopoietic stem cell generation in the mammalian embryo

    PubMed Central

    Gao, Xin; Johnson, Kirby D.; Chang, Yuan-I; Boyer, Meghan E.; Dewey, Colin N.; Zhang, Jing

    2013-01-01

    The generation of hematopoietic stem cells (HSCs) from hemogenic endothelium within the aorta, gonad, mesonephros (AGM) region of the mammalian embryo is crucial for development of the adult hematopoietic system. We described a deletion of a Gata2 cis-element (+9.5) that depletes fetal liver HSCs, is lethal at E13–14 of embryogenesis, and is mutated in an immunodeficiency that progresses to myelodysplasia/leukemia. Here, we demonstrate that the +9.5 element enhances Gata2 expression and is required to generate long-term repopulating HSCs in the AGM. Deletion of the +9.5 element abrogated the capacity of hemogenic endothelium to generate HSC-containing clusters in the aorta. Genomic analyses indicated that the +9.5 element regulated a rich ensemble of genes that control hemogenic endothelium and HSCs, as well as genes not implicated in hematopoiesis. These results reveal a mechanism that controls stem cell emergence from hemogenic endothelium to establish the adult hematopoietic system. PMID:24297994

  13. Gorab is required for dermal condensate cells to respond to hedgehog signals during hair follicle morphogenesis

    PubMed Central

    Liu, Ying; Snedecor, Elizabeth R.; Choi, Yeon Ja; Yang, Ning; Zhang, Xu; Xu, Yuhuan; Han, Yunlin; Jones, Evan C.; Shroyer, Kenneth R.; Clark, Richard A.; Zhang, Lianfeng; Qin, Chuan; Chen, Jiang

    2015-01-01

    GORAB is a golgin that localizes predominantly at the Golgi apparatus and physically interacts with small GTPases. GORAB is ubiquitously expressed in mammalian tissues, including the skin. However, the biological function of this golgin in skin is unknown. Here, we report that disrupting the expression of the Gorab gene in mice results in hair follicle morphogenesis defects that were characterized by impaired follicular keratinocyte differentiation. This hair follicle phenotype was associated with markedly suppressed hedgehog (Hh) signaling pathway in dermal condensates in vivo. Gorab-deficient dermal mesenchymal cells also displayed significantly reduced capability to respond to Hh pathway activation in vitro. Furthermore, we found that the formation of primary cilium, a cellular organelle that is essential for the Hh pathway, was impaired in mutant dermal papilla cells, suggesting that Gorab may be required for the Hh pathway through facilitating the formation of primary cilia. Thus, data obtained from this study provided insight onto the biological functions of Gorab during embryonic morphogenesis of skin in which Hh signaling and primary cilia exert important functions. PMID:26967474

  14. Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells.

    PubMed

    Satoh, Rumi; Kakugawa, Kiyokazu; Yasuda, Takuwa; Yoshida, Hisahiro; Sibilia, Maria; Katsura, Yoshimoto; Levi, Ben; Abramson, Jakub; Koseki, Yoko; Koseki, Haruhiko; van Ewijk, Willem; Hollander, Georg A; Kawamoto, Hiroshi

    2016-01-01

    Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions. PMID:26789017

  15. Membrane repair of human skeletal muscle cells requires Annexin-A5.

    PubMed

    Carmeille, Romain; Bouvet, Flora; Tan, Sisareuth; Croissant, Coralie; Gounou, Céline; Mamchaoui, Kamel; Mouly, Vincent; Brisson, Alain R; Bouter, Anthony

    2016-09-01

    Defect in membrane repair contributes to the development of limb girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. In healthy skeletal muscle, unraveling membrane repair mechanisms requires to establish an exhaustive list of the components of the resealing machinery. Here we show that human myotubes rendered deficient for Annexin-A5 (AnxA5) suffer from a severe defect in membrane resealing. This defect is rescued by the addition of recombinant AnxA5 while an AnxA5 mutant, which is unable to form 2D protein arrays, has no effect. Using correlative light and electron microscopy, we show that AnxA5 binds to the edges of the torn membrane, as early as a few seconds after sarcolemma injury, where it probably self-assembles into 2D arrays. In addition, we observed that membrane resealing is associated with the presence of a cluster of lipid vesicles at the wounded site. AnxA5 is present at the surface of these vesicles and may thus participate in plugging the cell membrane disruption. Finally, we show that AnxA5 behaves similarly in myotubes from a muscle cell line established from a patient suffering from LGMD2B, a myopathy due to dysferlin mutations, which indicates that trafficking of AnxA5 during sarcolemma damage is independent of the presence of dysferlin. PMID:27286750

  16. Bone Marrow Stromal Cells Stimulate an Angiogenic Program that Requires Endothelial MT1-MMP

    PubMed Central

    Kachgal, Suraj; Carrion, Bita; Janson, Isaac A.; Putnam, Andrew J.

    2012-01-01

    Bone marrow-derived stromal/stem cells (BMSCs) have recently been characterized as mediators of tissue regeneration after injury. In addition to preventing fibrosis at the wound site, BMSCs elicit an angiogenic response within the fibrin matrix. The mechanistic interactions between BMSCs and invading endothelial cells (ECs) during this process are not fully understood. Using a three-dimensional, fibrin-based angiogenesis model, we sought to investigate the proteolytic mechanisms by which BMSCs promote vessel morphogenesis. We find that BMSC-mediated vessel formation depends on the proteolytic ability of membrane type 1-matrix metalloproteinase (MT1-MMP). Knockdown of the protease results in a small network of vessels with enlarged lumens. Contrastingly, vessel morphogenesis is unaffected by the knockdown of MMP-2 and MMP-9. Furthermore, we find that BMSC-mediated vessel morphogenesis in vivo follows mechanisms similar to what we observe in vitro. Subcutaneous, cellular fibrin implants in C.B-17/SCID mice form aberrant vasculature when MMPs are inhibited with a broad spectrum chemical inhibitor, and a very minimal amount of vessels when MT1-MMP proteolytic activity is interrupted in ECs. Other studies have debated the necessity of MT1-MMP in the context of vessel invasion in fibrin, but this study clearly demonstrates its requirement in BMSC-mediated angiogenesis. PMID:22262018

  17. Endothelial cell survival during angiogenesis requires the pro-survival protein MCL1.

    PubMed

    Watson, E C; Whitehead, L; Adams, R H; Dewson, G; Coultas, L

    2016-08-01

    Angiogenesis is essential to match the size of blood vessel networks to the metabolic demands of growing tissues. While many genes and pathways necessary for regulating angiogenesis have been identified, those responsible for endothelial cell (EC) survival during angiogenesis remain largely unknown. We have investigated the in vivo role of myeloid cell leukemia 1 (MCL1), a pro-survival member of the BCL2 family, in EC survival during angiogenesis. EC-specific deletion of Mcl1 resulted in a dose-dependent increase in EC apoptosis in the angiogenic vasculature and a corresponding decline in vessel density. Our results suggest this apoptosis was independent of the BH3-only protein BIM. Despite the known link between apoptosis and blood vessel regression, this was not the cause of reduced vessel density observed in the absence of endothelial MCL1. Rather, the reduction in vessel density was linked to ectopic apoptosis in regions of the angiogenic vasculature where EC proliferation and new vessel growth occurs. We have therefore identified MCL1 as an essential survival factor for ECs that is required for blood vessel production during angiogenesis. PMID:26943318

  18. Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells

    PubMed Central

    Satoh, Rumi; Kakugawa, Kiyokazu; Yasuda, Takuwa; Yoshida, Hisahiro; Sibilia, Maria; Katsura, Yoshimoto; Levi, Ben; Abramson, Jakub; Koseki, Yoko; Koseki, Haruhiko; van Ewijk, Willem; Hollander, Georg A.; Kawamoto, Hiroshi

    2016-01-01

    Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions. PMID:26789017

  19. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1.

    PubMed

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-03-12

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP₃ receptors (IP₃Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP₃R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP₃R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP₃R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment. PMID:25637353

  20. Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

    PubMed Central

    Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

    2015-01-01

    Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP3 receptors (IP3Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP3R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP3R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP3R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment. PMID:25637353

  1. Differential Requirements of TCR Signaling in Homeostatic Maintenance and Function of Dendritic Epidermal T Cells.

    PubMed

    Zhang, Baojun; Wu, Jianxuan; Jiao, Yiqun; Bock, Cheryl; Dai, Meifang; Chen, Benny; Chao, Nelson; Zhang, Weiguo; Zhuang, Yuan

    2015-11-01

    Dendritic epidermal T cells (DETCs) are generated exclusively in the fetal thymus and maintained in the skin epithelium throughout postnatal life of the mouse. DETCs have restricted antigenic specificity as a result of their exclusive usage of a canonical TCR. Although the importance of the TCR in DETC development has been well established, the exact role of TCR signaling in DETC homeostasis and function remains incompletely defined. In this study, we investigated TCR signaling in fully matured DETCs by lineage-restricted deletion of the Lat gene, an essential signaling molecule downstream of the TCR. We found that Lat deletion impaired TCR-dependent cytokine gene activation and the ability of DETCs to undergo proliferative expansion. However, linker for activation of T cells-deficient DETCs were able to maintain long-term population homeostasis, although with a reduced proliferation rate. Mice with Lat deletion in DETCs exhibited delayed wound healing accompanied by impaired clonal expansion within the wound area. Our study revealed differential requirements for TCR signaling in homeostatic maintenance of DETCs and in their effector function during wound healing. PMID:26408667

  2. Gorab Is Required for Dermal Condensate Cells to Respond to Hedgehog Signals during Hair Follicle Morphogenesis.

    PubMed

    Liu, Ying; Snedecor, Elizabeth R; Choi, Yeon Ja; Yang, Ning; Zhang, Xu; Xu, Yuhuan; Han, Yunlin; Jones, Evan C; Shroyer, Kenneth R; Clark, Richard A; Zhang, Lianfeng; Qin, Chuan; Chen, Jiang

    2016-02-01

    GORAB is a golgin that localizes predominantly at the Golgi apparatus and physically interacts with small guanosine triphosphatases. GORAB is ubiquitously expressed in mammalian tissues, including the skin. However, the biological function of this golgin in skin is unknown. Here, we report that disrupting the expression of the Gorab gene in mice results in hair follicle morphogenesis defects that were characterized by impaired follicular keratinocyte differentiation. This hair follicle phenotype was associated with markedly suppressed hedgehog (Hh) signaling pathway in dermal condensates in vivo. Gorab-deficient dermal mesenchymal cells also displayed a significantly reduced capability to respond to Hh pathway activation in vitro. Furthermore, we found that the formation of the primary cilium, a cellular organelle that is essential for the Hh pathway, was impaired in mutant dermal condensate cells, suggesting that Gorab may be required for the Hh pathway through facilitating the formation of primary cilia. Thus, data obtained from this study provided insight into the biological functions of Gorab during embryonic morphogenesis of the skin in which Hh signaling and primary cilia exert important functions. PMID:26967474

  3. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  4. CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    PubMed Central

    Xu, Ying; Wang, Tao

    2016-01-01

    ABSTRACT Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila melanogaster, the main light-sensing rhodopsin (Rh1; encoded by ninaE) and the downstream ion channel, transient receptor potential like (TRPL), are endocytosed in response to light, but the mechanism is unclear. By using an RNA-Sequencing (RNA-Seq) approach, we discovered a protein we named CULD, a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein, that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1-positive or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1, but not of TRPL, suppressed retinal degeneration in culd-null mutant flies. Our data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity. PMID:26598556

  5. Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

    PubMed

    Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

    2016-08-23

    Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs. PMID:27558729

  6. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR)

    PubMed Central

    Trussoni, Christy E.; Tabibian, James H.; Splinter, Patrick L.; O’Hara, Steven P.

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes. PMID:25915403

  7. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    PubMed

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes. PMID:25915403

  8. Differential requirements for H/ACA ribonucleoprotein components in cell proliferation and response to DNA damage.

    PubMed

    Lin, Ping; Mobasher, Maral E; Hakakian, Yasaman; Kakarla, Veena; Naseem, Anum F; Ziai, Heliya; Alawi, Faizan

    2015-12-01

    H/ACA ribonucleoproteins (RNPs) are comprised of four conserved proteins, dyskerin, NHP2, NOP10, and GAR1, and a function-specifying, noncoding H/ACA RNA. H/ACA RNPs contribute to telomerase assembly and stabilization, and posttranscriptional processing of nascent ribosomal RNA and spliceosomal RNA. However, very little is known about the coordinated action of the four proteins in other biologic processes. As described herein, we observed a differential requirement for the proteins in cell proliferation and identified a possible reliance for these factors in regulation of specific DNA damage biomarkers. In particular, GAR1 expression was upregulated following exposure to all forms of genotoxic stress tested. In contrast, levels of the other proteins were either reduced or unaffected. Only GAR1 showed an altered subcellular localization with a shift from the nucleolus to the nucleoplasm after ultraviolet-C irradiation and doxorubicin treatments. Transient siRNA-mediated depletion of GAR1 and dyskerin arrested cell proliferation, whereas loss of either NHP2 or NOP10 had no effect. Finally, loss of dyskerin, GAR1, NHP2, and NOP10, respectively, limited the accumulation of DNA damage biomarkers. However, the individual responses were dependent upon the specific type of damage incurred. In general, loss of GAR1 had the most suppressive effect on the biomarkers tested. Since the specific responses to genotoxic stress, the contribution of each protein to cell proliferation, and the activation of DNA damage biomarkers were not equivalent, this suggests the possibility that at least some of the proteins, most notably GAR1, may potentially function independently of their respective roles within H/ACA RNP complexes. PMID:26265134

  9. Rat dendritic cells function as accessory cells and control the production of a soluble factor required for mitogenic responses of T lymphocytes.

    PubMed Central

    Klinkert, W E; LaBadie, J H; O'Brien, J P; Beyer, C F; Bowers, W E

    1980-01-01

    Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred at a ratio of only one dendritic cell per 200 periodate-treated lymphocytes. This evidence thus indicates strongly that in rats, dendritic cells--not macrophages--function as accessory cells. Further, the number of dendritic cells in a preparation governed the magnitude of the mitogenic response and was limiting in the case of unfractionated lymph node cells. In addition, when oxidized with periodate or with neuraminidase plus galactose oxidase, the dendritic cell served as a very potent indirect stimulator of untreated responder lymphocytes. Both functions of the dendritic cell appeared to lack species specificity, since mouse dendritic cells were very active when tested with rat responder lymphocytes. A soluble factor (accessory cell-replacing factor), produced by cultures of lymph node or spleen cells subjected to oxidative mitogenesis, enabled otherwise unresponsive mitogen-treated lymphocytes to respond. Dendritic cells were required for the production of this factor but may not be solely responsible for its production. Images PMID:6968911

  10. Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton

    PubMed Central

    Cui, Cheng; Chatterjee, Bishwanath; Lozito, Thomas P.; Zhang, Zhen; Francis, Richard J.; Yagi, Hisato; Swanhart, Lisa M.; Sanker, Subramaniam; Francis, Deanne; Yu, Qing; San Agustin, Jovenal T.; Puligilla, Chandrakala; Chatterjee, Tania; Tansey, Terry; Liu, Xiaoqin; Kelley, Matthew W.; Spiliotis, Elias T.; Kwiatkowski, Adam V.; Tuan, Rocky; Pazour, Gregory J.; Hukriede, Neil A.; Lo, Cecilia W.

    2013-01-01

    Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to

  11. Alloproliferation of purified CD4+ T cells to adult human heart endothelial cells, and study of second-signal requirements.

    PubMed Central

    McDouall, R M; Page, C S; Hafizi, S; Yacoub, M H; Rose, M L

    1996-01-01

    Human endothelial cells have been shown to be capable of causing direct allostimulation of T cells. However, the majority of immunological studies of human endothelial cells have been performed on cells of fetal origin. Here we use endothelial cells isolated from the adult human heart, both large vessel (coronary artery, pulmonary artery and aorta) and also microvascular. We have examined the ability of all these endothelial cells to cause direct allostimulation of T cells, and show that purified CD4+ T cells can proliferate in response to adult human heart endothelial cells, the response being dependent on pretreatment of the endothelial cells with interferon-gamma (IFN-gamma) and inhibited by anti-HLA-DR monoclonal antibody. The proliferative responses of CD8+ T cells to adult but not fetal endothelial cells was inconsistent and weak. Proliferative responses were not blocked by CTLA4-Ig, which inhibits T-cell responses to "classical' antigen-presenting cells (APC), but > 50% inhibition was achieved with monoclonal antibody to lymphocyte function-associated antigen-3 (LFA-3). These results show that adult human cardiovascular endothelial cells are capable of causing allostimulation of resting CD4+ T cells, using a different second signal to classical APC. In view of these findings endothelial cells should be considered as APC following solid organ transplantation. PMID:8943718

  12. Proliferating Cell Nuclear Antigen (PCNA) Is Required for Cell Cycle-regulated Silent Chromatin on Replicated and Nonreplicated Genes*

    PubMed Central

    Miller, Andrew; Chen, Jiji; Takasuka, Taichi E.; Jacobi, Jennifer L.; Kaufman, Paul D.; Irudayaraj, Joseph M. K.; Kirchmaier, Ann L.

    2010-01-01

    In Saccharomyces cerevisiae, silent chromatin is formed at HMR upon the passage through S phase, yet neither the initiation of DNA replication at silencers nor the passage of a replication fork through HMR is required for silencing. Paradoxically, mutations in the DNA replication processivity factor, POL30, disrupt silencing despite this lack of requirement for DNA replication in the establishment of silencing. We tested whether pol30 mutants could establish silencing at either replicated or non-replicated HMR loci during S phase and found that pol30 mutants were defective in establishing silencing at HMR regardless of its replication status. Although previous studies tie the silencing defect of pol30 mutants to the chromatin assembly factors Asf1p and CAF-1, we found pol30 mutants did not exhibit a gross defect in packaging HMR into chromatin. Rather, the pol30 mutants exhibited defects in histone modifications linked to ASF1 and CAF-1-dependent pathways, including SAS-I- and Rtt109p-dependent acetylation events at H4-K16 and H3-K9 (plus H3-K56; Miller, A., Yang, B., Foster, T., and Kirchmaier, A. L. (2008) Genetics 179, 793–809). Additional experiments using FLIM-FRET revealed that Pol30p interacted with SAS-I and Rtt109p in the nuclei of living cells. However, these interactions were disrupted in pol30 mutants with defects linked to ASF1- and CAF-1-dependent pathways. Together, these results imply that Pol30p affects epigenetic processes by influencing the composition of chromosomal histone modifications. PMID:20813847

  13. Human NK cell development requires CD56-mediated motility and formation of the developmental synapse

    PubMed Central

    Mace, Emily M.; Gunesch, Justin T.; Dixon, Amera; Orange, Jordan S.

    2016-01-01

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34+ precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells, which we term the developmental synapse. Finally, we identify a role for CD56 in developmental synapse structure, NK cell motility and NK cell development. Thus, we define the developmental synapse leading to human NK cell functional maturation. PMID:27435370

  14. Human NK cell development requires CD56-mediated motility and formation of the developmental synapse.

    PubMed

    Mace, Emily M; Gunesch, Justin T; Dixon, Amera; Orange, Jordan S

    2016-01-01

    While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34(+) precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells, which we term the developmental synapse. Finally, we identify a role for CD56 in developmental synapse structure, NK cell motility and NK cell development. Thus, we define the developmental synapse leading to human NK cell functional maturation. PMID:27435370

  15. NK Cell-Specific Gata3 Ablation Identifies the Maturation Program Required for Bone Marrow Exit and Control of Proliferation.

    PubMed

    Ali, Alaa Kassim; Oh, Jun Seok; Vivier, Eric; Busslinger, Meinrad; Lee, Seung-Hwan

    2016-02-15

    NK cells are innate lymphocytes capable of eliciting an innate immune response to pathogens. NK cells develop and become mature in the bone marrow (BM) before they migrate out to peripheral organs. Although the developmental program leading to mature NK cells has been studied in the context of several transcription factors, the stage-specific role of GATA3 in NK cell development has been incompletely understood. Using NKp46-Cre-Gata3(fl/fl) mice in which Gata3 deficiency was induced as early as the immature stage of NK cell differentiation, we demonstrated that GATA3 is required for the NK cell maturation beyond the CD27 single-positive stage and is indispensable for the maintenance of liver-resident NK cells. The frequencies of NK cells from NKp46-Cre-Gata3(fl/fl) mice were found higher in the BM but lower in peripheral organs compared with control littermates, indicating that GATA3 controls the maturation program required for BM egress. Despite the defect in maturation, upon murine CMV infection, NK cells from NKp46-Cre-Gata3(fl/fl) mice expanded vigorously, achieving NK cell frequencies surpassing those in controls and therefore provided comparable protection. The heightened proliferation of NK cells from NKp46-Cre-Gata3(fl/fl) mice was cell intrinsic and associated with enhanced upregulation of CD25 expression. Taken together, our results demonstrate that GATA3 is a critical regulator for NK cell terminal maturation and egress out of the BM and that immature NK cells present in the periphery of NKp46-Cre-Gata3(fl/fl) mice can rapidly expand and provide a reservoir of NK cells capable of mounting an efficient cytotoxic response upon virus infection. PMID:26773150

  16. The requirement for DNAM-1, NKG2D, and NKp46 in the natural killer cell-mediated killing of myeloma cells.

    PubMed

    El-Sherbiny, Yasser M; Meade, Josephine L; Holmes, Tim D; McGonagle, Dennis; Mackie, Sarah L; Morgan, Ann W; Cook, Gordon; Feyler, Sylvia; Richards, Stephen J; Davies, Faith E; Morgan, Gareth J; Cook, Graham P

    2007-09-15

    Recent evidence suggests a role for natural killer (NK) cells in the control of multiple myeloma. We show that expression of the NK cell receptor DNAM-1 (CD226) is reduced on CD56(dim) NK cells from myeloma patients with active disease compared with patients in remission and healthy controls. This suggested that this receptor might play a role in NK-myeloma interactions. The DNAM-1 ligands Nectin-2 (CD112) and the poliovirus receptor (PVR; CD155) were expressed by most patient myeloma samples analyzed. NK killing of patient-derived myelomas expressing PVR and/or Nectin-2 was DNAM-1 dependent, revealing a functional role for DNAM-1 in myeloma cell killing. In myeloma cell lines, cell surface expression of PVR was associated with low levels of NKG2D ligands, whereas cells expressing high levels of NKG2D ligands did not express PVR protein or mRNA. Furthermore, NK cell-mediated killing of myeloma cell lines was dependent on either DNAM-1 or NKG2D but not both molecules. In contrast, the natural cytotoxicity receptor NKp46 was required for the killing of all myeloma cell lines analyzed. Thus, DNAM-1 is important in the NK cell-mediated killing of myeloma cells expressing the cognate ligands. The importance of NKp46, NKG2D, and DNAM-1 in myeloma killing mirrors the differential expression of NK cell ligands by myeloma cells, reflecting immune selection during myeloma disease progression. PMID:17875681

  17. Natural Killer Cell-Mediated Control of Infections Requires Production of Interleukin 15 by Type I IFN-Triggered Dendritic Cells

    PubMed Central

    Lucas, Mathias; Schachterle, William; Oberle, Karin; Aichele, Peter; Diefenbach, Andreas

    2007-01-01

    Summary Following infections, natural killer (NK) cells acquire effector functions presumably by interacting with accessory cells. The cellular and molecular signals required for NK cell priming in vivo remain poorly defined. Using a mouse model for the inducible ablation of dendritic cells (DC), we show that the in vivo priming of NK cell responses to viral and bacterial pathogens depended on the presence of CD11chigh DC. Following peripheral TLR stimulation, priming of NK cells required their recruitment to local lymph nodes and interaction with DC leading to the emergence of effector NK cells in the periphery. NK cell priming depended on the recognition of type I IFN signals by DC and the subsequent production and trans-presentation of IL-15 by DC to resting NK cells. CD11chigh DC-derived IL-15 was necessary and sufficient for the priming of NK cells. Our data define a unique in vivo role of DC for the priming of NK cells, revealing a striking and previously unappreciated homology to T lymphocytes of the adaptive immune system. PMID:17398124

  18. Membrane association of the CD3ε signaling domain is required for optimal T cell development and function1

    PubMed Central

    Bettini, Matthew L.; Guy, Clifford; Dash, Pradyot; Vignali, Kate M.; Hamm, David E.; Dobbins, Jessica; Gagnon, Etienne; Thomas, Paul G.; Wucherpfennig, Kai W.; Vignali, Dario A.A.

    2014-01-01

    The T cell receptor (TCR):CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). Here we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4−CD8− double negative-3 (DN3) to DN4 thymocyte transition, due to enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function. PMID:24899501

  19. Continuous inhibitory signaling by both SHP-1 and SHIP-1 pathways is required to maintain unresponsiveness of anergic B cells.

    PubMed

    Getahun, Andrew; Beavers, Nicole A; Larson, Sandy R; Shlomchik, Mark J; Cambier, John C

    2016-05-01

    Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. This unresponsiveness is rapidly reversible, requiring continuous BCR interaction with self-antigen and resultant regulatory signaling for its maintenance. Using adoptive transfer of anergic B cells with subsequent acute induction of gene deletion or expression, we demonstrate that the continuous activities of independent inhibitory signaling pathways involving the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 are required to maintain anergy. Acute breach of anergy by compromise of either of these pathways leads to rapid cell activation, proliferation, and generation of short-lived plasma cells that reside in extrafollicular foci. Results are consistent with predicted/observed reduction in the Lyn-SHIP-1-PTEN-SHP-1 axis function in B cells from systemic lupus erythematosus patients. PMID:27114609

  20. CTLA-4 expression on antigen-specific cells but not IL-10 secretion is required for oral tolerance.

    PubMed

    Fowler, Sanna; Powrie, Fiona

    2002-10-01

    CD4(+) T cells play a vital role in mediating the tolerance induced at mucosal sites following exposure to non-pathogenic stimuli, and further understanding of the precise mechanisms by which these cells prevent aberrant responses is required. We have developed a model using transfer of DO11.10 TCR-transgenic bone marrow into irradiated recipients in which it has been possible to track antigen-specific CD4(+) cells in mesenteric lymph nodes (mLN), Peyer's patches (PP) and lamina propria following primary exposure to antigen. Using this model we have demonstrated initial activation in all three gut-associated lymphoid tissue compartments characterized by increases in the frequency of transgenic cells expressing CD69 and CD25. These cells subsequently enter a state of hyporesponsiveness both locally in the mLN and PP and in the periphery following feeding and challenge. Investigating the role of CTLA-4 either using anti-CTLA-4 mAb or by generating chimeras using DO11.10xCTLA-4(-/-) mice as donors we have clearly shown that antigen-specific cells require the expression of this regulatory molecule for oral tolerance. In contrast, oral tolerance was intact in chimeras generated using DO11.10xIL-10(-/-) cells, indicating that secretion of this cytokine by antigen-specific cells is not required. PMID:12355454

  1. B cells are required for lupus nephritis in the polygenic, Fas-intact MRL model of systemic autoimmunity.

    PubMed

    Chan, O T; Madaio, M P; Shlomchik, M J

    1999-10-01

    B cells are required for both the expression of lupus nephritis and spontaneous T cell activation/memory cell accumulation in MRL-Faslpr mice (MRL/lpr). Autoimmunity in the MRL/lpr strain is the result of Fas-deficiency and multiple background genes; however, the precise roles of background genes vs Fas-deficiency have not been fully defined. Fas-deficiency (i.e., the lpr defect) is required in B cells for optimal autoantibody expression, raising the possibility that the central role for B cells in MRL/lpr mice may not extend to MRL/+ mice and, thus, to lupus models that do not depend on Fas-deficiency ("polygenic lupus"). To address this issue, B cell-deficient, Fas-intact MRL/+ mice (JHd-MRL/) were created; and disease was evaluated in aged animals (>9 mo). The JHd-MRL/+ animals did not develop nephritis or vasculitis at a time when the B cell-intact littermates had severe disease. In addition, while activated/memory CD4+ and CD8+ T cells accumulated in B cell-intact mice, such accumulation was substantially inhibited in the absence of B cells. This effect appeared to be restricted to the MRL strain because it was not seen in B cell-deficient BALB/c mice (JHd-BALB) of similar ages. The results indicate that B cells are essential in promoting systemic autoimmunity in a Fas-independent model. Therefore, B cells have an important role in pathogenesis, generalizable to lupus models that depend on multiple genes even when Fas expression is intact. The results provide further rationale for B cell suppression as therapy for systemic lupus erythematosus. PMID:10490951

  2. MicroRNA-181a/b-1 Is Not Required for Innate γδ NKT Effector Cell Development.

    PubMed

    Sandrock, Inga; Ziętara, Natalia; Łyszkiewicz, Marcin; Oberdörfer, Linda; Witzlau, Katrin; Krueger, Andreas; Prinz, Immo

    2015-01-01

    Thymic development of αβ T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1-/-mice, which show impaired agonistic T cell selection of invariant αβ NKT cells, were crossed to Tcrd-H2BeGFP reporter mice to monitor selection, intra-thymic expansion and differentiation of γδ T cells. We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αβ NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes. PMID:26673421

  3. MicroRNA-181a/b-1 Is Not Required for Innate γδ NKT Effector Cell Development

    PubMed Central

    Sandrock, Inga; Ziętara, Natalia; Łyszkiewicz, Marcin; Oberdörfer, Linda; Witzlau, Katrin; Krueger, Andreas; Prinz, Immo

    2015-01-01

    Thymic development of αβ T lymphocytes into invariant natural killer (NK) T cells depends on their selection via agonistic lipid antigen presented by CD1d. If successful, newly selected NKT cells gain effector functions already in the thymus. Some γδ T cell subsets also acquire effector functions in the thymus. However, it is not clear whether agonistic TCR stimulation is involved in thymic γδ T cell selection and development. Here we combine two genetic models to address this question. MiR-181a/b-1–/–mice, which show impaired agonistic T cell selection of invariant αβ NKT cells, were crossed to Tcrd-H2BeGFP reporter mice to monitor selection, intra-thymic expansion and differentiation of γδ T cells. We found that miR-181a/b-1-deficiency had no effect on numbers of thymic γδ T cell or on their differentiation towards an IL-17- or IFN-γ-producing effector phenotype. Also, the composition of peripheral lymph node γδ T cells was not affected by miR-181a/b-1-deficiency. Dendritic epidermal γδ T cells were normally present in knock-out animals. However, we observed elevated frequencies and numbers of γδ NKT cells in the liver, possibly because γδ NKT cells can expand and replace missing αβ NKT cells in peripheral niches. In summary, we investigated the role of miR-181a/b-1 for selection, intrathymic development and homeostasis of γδ T cells. We conclude that miR-181a/b-1-dependent modulation of T cell selection is not critically required for innate development of γδ NKT cells or of any other γδ T cell subtypes. PMID:26673421

  4. A virtual lymph node model to dissect the requirements for T-cell activation by synapses and kinapses.

    PubMed

    Moreau, Hélène D; Bogle, Gib; Bousso, Philippe

    2016-08-01

    The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1-2 days. We propose that the stability of T-cell-DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines. PMID:27089942

  5. Proper activation of MafA is required for optimal differentiation and maturation of pancreatic β-cells.

    PubMed

    El Khattabi, Ilham; Sharma, Arun

    2015-12-01

    A key therapeutic approach for the treatment of Type 1 diabetes (T1D) is transplantation of functional islet β-cells. Despite recent advances in generating stem cell-derived glucose-responsive insulin(+) cells, their further maturation to fully functional adult β-cells still remains a daunting task. Conquering this hurdle will require a better understanding of the mechanisms driving maturation of embryonic insulin(+) cells into adult β-cells, and the implementation of that knowledge to improve current differentiation protocols. Here, we will review our current understanding of β-cell maturation, and discuss the contribution of key β-cell transcription factor MafA, to this process. The fundamental importance of MafA in regulating adult β-cell maturation and function indicates that enhancing MafA expression may improve the generation of definitive β-cells for transplantation. Additionally, we suggest that the temporal control of MafA induction at a specific stage of β-cell differentiation will be the next critical challenge for achieving optimum maturation of β-cells. PMID:26696512

  6. Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration

    PubMed Central

    Obholzer, Nikolaus D.; Sun, Shan; Li, Wenyan; Petrillo, Marco; Dai, Pu; Zhou, Yi; Cotanche, Douglas A.; Megason, Sean G.; Li, Huawei; Chen, Zheng-Yi

    2016-01-01

    Unlike mammals, the non-mammalian vertebrate inner ear can regenerate the sensory cells, hair cells, either spontaneously or through induction after hair cell loss, leading to hearing recovery. The mechanisms underlying the regeneration are poorly understood. By microarray analysis on a chick model, we show that chick hair cell regeneration involves the activation of proliferation genes and downregulation of differentiation genes. Both MYC and FGF are activated in chick hair cell regeneration. Using a zebrafish lateral line neuromast hair cell regeneration model, we show that the specific inhibition of Myc or Fgf suppresses hair cell regeneration, demonstrating that both pathways are essential to the process. Rapid upregulation of Myc and delayed Fgf activation during regeneration suggest a role of Myc in proliferation and Fgf in differentiation. The dorsal-ventral pattern of fgfr1a in the neuromasts overlaps with the distribution of hair cell precursors. By laser ablation, we show that the fgfr1a-positive supporting cells are likely the hair cell precursors that directly give rise to new hair cells; whereas the anterior-posterior fgfr1a-negative supporting cells have heightened proliferation capacity, likely to serve as more primitive progenitor cells to replenish lost precursors after hair cell loss. Thus fgfr1a is likely to mark compartmentalized supporting cell subtypes with different capacities in renewal proliferation and hair cell regeneration. Manipulation of c-MYC and FGF pathways could be explored for mammalian hair cell regeneration. PMID:27351484

  7. Skin sensitization induced Langerhans' cell mobilization: variable requirements for tumour necrosis factor-α.

    PubMed

    Eaton, Laura H; Roberts, Ruth A; Kimber, Ian; Dearman, Rebecca J; Metryka, Aleksandra

    2015-01-01

    Upon antigen/allergen recognition, epidermal Langerhans' cells (LC) are mobilized and migrate to the local lymph node where they play a major role in initiating or regulating immune responses. It had been proposed that all chemical allergens induce LC migration via common cytokine signals delivered by TNF-α and IL-1β. Here the dependence of LC migration on TNF-α following treatment of mice with various chemical allergens has been investigated. It was found that under standard conditions the allergens oxazolone, paraphenylene diamine, and trimellitic anhydride, in addition to the skin irritant sodium lauryl sulfate, were unable to trigger LC mobilization in the absence of TNF-α signalling. In contrast, two members of the dinitrohalobenezene family (2,4-dinitrochlorobenzene [DNCB] and 2,4-dinitrofluorobenzene [DNFB]) promoted LC migration independently of TNF-R2 (the sole TNF-α receptor expressed by LC) and TNF-α although the presence of IL-1β was still required. However, increasing doses of oxazolone overcame the requirement of TNF-α for LC mobilization, whereas lower doses of DNCB were still able to induce LC migration in a TNF-α-independent manner. These novel findings demonstrate unexpected heterogeneity among chemical allergens and furthermore that LC can be induced to migrate from the epidermis via different mechanisms that are either dependent or independent of TNF-α. Although the exact mechanisms with regard to the signals that activate LC have yet to be elucidated, these differences may translate into functional speciation that will likely impact on the extent and quality of allergic sensitization. PMID:25039377

  8. Skin sensitization induced Langerhans’ cell mobilization: variable requirements for tumour necrosis factor-α

    PubMed Central

    Eaton, Laura H; Roberts, Ruth A; Kimber, Ian; Dearman, Rebecca J; Metryka, Aleksandra

    2015-01-01

    Upon antigen/allergen recognition, epidermal Langerhans’ cells (LC) are mobilized and migrate to the local lymph node where they play a major role in initiating or regulating immune responses. It had been proposed that all chemical allergens induce LC migration via common cytokine signals delivered by TNF-α and IL-1β. Here the dependence of LC migration on TNF-α following treatment of mice with various chemical allergens has been investigated. It was found that under standard conditions the allergens oxazolone, paraphenylene diamine, and trimellitic anhydride, in addition to the skin irritant sodium lauryl sulfate, were unable to trigger LC mobilization in the absence of TNF-α signalling. In contrast, two members of the dinitrohalobenezene family (2,4-dinitrochlorobenzene [DNCB] and 2,4-dinitrofluorobenzene [DNFB]) promoted LC migration independently of TNF-R2 (the sole TNF-α receptor expressed by LC) and TNF-α although the presence of IL-1β was still required. However, increasing doses of oxazolone overcame the requirement of TNF-α for LC mobilization, whereas lower doses of DNCB were still able to induce LC migration in a TNF-α-independent manner. These novel findings demonstrate unexpected heterogeneity among chemical allergens and furthermore that LC can be induced to migrate from the epidermis via different mechanisms that are either dependent or independent of TNF-α. Although the exact mechanisms with regard to the signals that activate LC have yet to be elucidated, these differences may translate into functional speciation that will likely impact on the extent and quality of allergic sensitization. PMID:25039377

  9. Functional ESCRT machinery is required for constitutive recycling of claudin-1 and maintenance of polarity in vertebrate epithelial cells.

    PubMed

    Dukes, Joseph D; Fish, Laura; Richardson, Judith D; Blaikley, Elizabeth; Burns, Samir; Caunt, Christopher J; Chalmers, Andrew D; Whitley, Paul

    2011-09-01

    Genetic screens in Drosophila have identified regulators of endocytic trafficking as neoplastic tumor suppressor genes. For example, Drosophila endosomal sorting complex required for transport (ESCRT) mutants lose epithelial polarity and show increased cell proliferation, suggesting that ESCRT proteins could function as tumor suppressors. In this study, we show for the for the first time to our knowledge that ESCRT proteins are required to maintain polarity in mammalian epithelial cells. Inhibition of ESCRT function caused the tight junction protein claudin-1 to accumulate in intracellular vesicles. In contrast E-cadherin and occludin localization was unaffected. We investigated the cause of this accumulation and show that claudin-1 is constitutively recycled in kidney, colon, and lung epithelial cells, identifying claudin-1 recycling as a newly described feature of diverse epithelial cell types. This recycling requires ESCRT function, explaining the accumulation of intracellular claudin-1 when ESCRT function is inhibited. We further demonstrate that small interfering RNA knockdown of the ESCRT protein Tsg101 causes epithelial monolayers to lose their polarized organization and interferes with the establishment of a normal epithelial permeability barrier. ESCRT knockdown also reduces the formation of correctly polarized three-dimensional cysts. Thus, in mammalian epithelial cells, ESCRT function is required for claudin-1 trafficking and for epithelial cell polarity, supporting the hypothesis that ESCRT proteins function as tumor suppressors. PMID:21757541

  10. CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection

    PubMed Central

    Ross, Ewan A; Coughlan, Ruth E; Flores-Langarica, Adriana; Bobat, Saeeda; Marshall, Jennifer L; Hussain, Khiyam; Charlesworth, James; Abhyankar, Nikita; Hitchcock, Jessica; Gil, Cristina; López-Macías, Constantino; Henderson, Ian R; Khan, Mahmood; Watson, Steve P; MacLennan, Ian C M; Buckley, Christopher D; Cunningham, Adam F

    2011-01-01

    Haematopoietic cells constitutively express CD31/PECAM1 a signalling, adhesion receptor associated with controlling responses to inflammatory stimuli. Although expressed on CD4+ T cells, its function on these cells is unclear. To address this we have used a model of systemic Salmonella infection that induces high levels of T cell activation and depends upon CD4+ T cells for resolution. Infection of CD31-deficient (CD31KO) mice demonstrates that these mice fail to control infection effectively. During infection, CD31KO mice have diminished numbers of total CD4+ T cells and IFN-γ-secreting Th1 cells. This is despite a higher proportion of CD31KO CD4+ T cells exhibiting an activated phenotype, and an undiminished capacity to prime normally and polarize to Th1. Reduced numbers of T cells reflected the increased propensity of naive and activated CD31KO T cells to undergo apoptosis after infection compared to wild-type (WT) T cells. Using adoptive transfer experiments we show that loss of CD31 on CD4+ T cells alone is sufficient to account for the defective CD31KO T cell accumulation. These data are consistent with CD31 helping to control T cell activation as in its absence T cells have a greater propensity to become activated, resulting in increased susceptibility to become apoptotic. The impact of CD31 loss on T cell homeostasis becomes most pronounced during severe, inflammatory and immunological stresses such as those caused by systemic Salmonella infection. This identifies a novel role for CD31 in regulating CD4 T cell homeostasis. PMID:21734076

  11. Tethering of ICAM on target cells is required for LFA-1-dependent NK cell adhesion and granule polarization

    PubMed Central

    Gross, Catharina C.; Brzostowski, Joseph A.; Liu, Dongfang; Long, Eric O.

    2013-01-01

    αLβ2 integrin (LFA-1) has an important role in the formation of T cell and NK cell cytotoxic immunological synapses and in target cell killing. Binding of LFA-1 to ICAM on target cells promotes not only adhesion, but also polarization of cytolytic granules in NK cells. Here we tested whether LFA-1-dependent NK cell responses are regulated by the distribution and mobility of ICAM at the surface of target cells. We show that depolymerization of F-actin in NK-sensitive target cells abrogated LFA-1-dependent conjugate formation and granule polarization in primary NK cells. Degranulation, which is not controlled by LFA-1, was not impaired. Fluorescence recovery after photobleaching experiments and particle tracking by total internal reflection fluorescence microscopy revealed that ICAM-1 and ICAM-2 were distributed in largely immobile clusters. ICAM clusters were maintained and became highly mobile after actin depolymerization. Moreover, reducing ICAM-2 mobility on an NK-resistant target cell through expression of ezrin, an adapter molecule that tethers proteins to the actin cytoskeleton, enhanced LFA-1-dependent adhesion and granule polarization. Finally, while NK cells kept moving over freely diffusible ICAM-1 on a lipid bilayer, they bound and spread over solid-phase ICAM-1. We conclude that tethering, rather than clustering of ICAM promotes proper signaling by LFA-1 in NK cells. Our findings suggest that the lateral diffusion of integrin ligands on cells may be an important determinant of susceptibility to lysis by cytotoxic lymphocytes. PMID:20675589

  12. TRAF3 is required for T cell-mediated immunity and T cell receptor/CD28 signaling1

    PubMed Central

    Xie, Ping; Kraus, Zachary J.; Stunz, Laura L.; Liu, Yan; Bishop, Gail A.

    2011-01-01

    We recently reported that TRAF3, a ubiquitously expressed adaptor protein, promotes mature B cell apoptosis. However, the specific function of TRAF3 in T cells has remained unclear. Here we report the generation and characterization of T cell-specific TRAF3−/− mice, in which the TRAF3 gene was deleted from thymocytes and T cells. Ablation of TRAF3 in the T cell-lineage did not affect the numbers or proportions of CD4+,CD8+ or double positive or negative thymocytes, or CD4 or CD8 T cell populations in secondary lymphoid organs except that the T cell specific TRAF3−/− mice had a two-fold increase in FoxP3+ T cells.. In striking contrast to mice lacking TRAF3 in B cells, the T cell TRAF3 deficient mice exhibited defective IgG1 responses to a T dependent antigen, and impaired T cell-mediated immunity to infection with Listeria monocytogenes. Surprisingly, we found that TRAF3 was recruited to the TCR/CD28 signaling complex upon co-stimulation, and that TCR/CD28-mediated proximal and distal signaling events were compromised by TRAF3 deficiency. These findings provide new insights into the roles played by TRAF3 in T cell activation and T cell-mediated immunity. PMID:21084666

  13. Properties and requirements for production of a macrophage product which suppresses steroid production by adrenocortical cells.

    PubMed Central

    Mathison, J C; La Forest, A C; Ulevitch, R J

    1984-01-01

    Lipopolysaccharide-treated murine peritoneal exudate macrophages (PEM) release a factor or factors into the supernatant that suppress adrenocorticotropic hormone-induced steroidogenesis in explanted rabbit adrenocortical cells (J. C. Mathison et al., J. Immunol. 130:2757-2762, 1983). To determine the requirements for suppression, PEM supernatants (30 microliters) were added to explanted rabbit adrenocortical cells in a final volume of 120 microliters with 10 mU of adrenocorticotropic hormone per ml, and after 18 h at 37 degrees C, steroid concentrations were measured by a fluorometric assay. Supernatant from proteose peptone-elicited C3HeB/FeJ PEM (5 X 10(6) PEM per 3.5-cm well, 10 micrograms of Salmonella minnesota Re595 LPS per ml, 18 h) suppressed steroid production ca. 50%, and kinetic studies demonstrated that the appearance of suppressive activity in the supernatant was gradual over 4 to 18 h. Release of suppressive activity was not associated with decreased viability of the PEM (assessed by fluorescein diacetate staining and measurement of lactic dehydrogenase in the supernatant). Suppression was not observed when the PEM supernatant was diluted 10-fold before addition to the adrenocortical cells, whereas supernatant concentrated 20-fold (prepared with a 10,000-molecular-weight-cutoff filter) produced 75 to 80% suppression. The suppressive activity was stable at pH 4, pH 11, or 70 degrees C for 30 min but was inactivated at 100 degrees C (10 min). Suppressive activity was also induced in C3HeB/FeJ PEM by O111:B4 lipopolysaccharide or heat-killed Listeria monocytogenes. In contrast, PEM from C3H/HeJ mice did not produce detectable suppressive activity in response to Re595 lipopolysaccharide or heat-killed L. monocytogenes. Thus, these results provide additional support for the inducible, selective release of a macrophage product that could affect the host response to lipopolysaccharide by regulation of the adrenocortical response to adrenocorticotropic

  14. An ARID Domain-Containing Protein within Nuclear Bodies Is Required for Sperm Cell Formation in Arabidopsis thaliana

    PubMed Central

    Zheng, Binglian; He, Hui; Zheng, Yanhua; Wu, Wenye; McCormick, Sheila

    2014-01-01

    In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, plays a key role in sperm cell formation by activating expression of several germline genes. But how DUO1 itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, that is specifically required for sperm cell formation in Arabidopsis. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of DUO1 activation. An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1. In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the arid1 mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on DUO1 through association with histone acetylation. PMID:25057814

  15. T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.

    PubMed

    Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

    2016-09-12

    Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies. PMID:27622331

  16. CD8+ T Cell Migration to the Skin Requires CD4+ Help in a Murine Model of Contact Hypersensitivity

    PubMed Central

    Fyhrquist, Nanna; Wolff, Henrik; Lauerma, Antti; Alenius, Harri

    2012-01-01

    The relative roles of CD4+ and CD8+ T cells in contact hypersensitivity responses have not been fully solved, and remain an important question. Using an adoptive transfer model, we investigated the role of the respective T cell subset. Magnetic bead separated CD4+ and CD8+ T cells from oxazolone sensitized C57BL/6 mice were transferred into RAG−/− mice, followed by hapten challenge and analysis of inflammatory parameters at 24 hours post exposure. The CD4+ T cell recipient mice developed partial contact hypersensitivity responses to oxazolone. CD8+ T cells caused significant amplification of the response in recipients of both CD4+ and CD8+ T cells including ear swelling, type 1 inflammatory mediators, and cell killing. Unexpectedly, CD8+ T cells were not sufficient to mediate contact hypersensitivity, although abundantly present in the lymph nodes in the CD8+ T cell reconstituted mice. There were no signs of inflammation at the site of hapten exposure, indicating impaired recruitment of CD8+ T cells in the absence of CD4+ T cells. These data show that CD4+ T cells mediate contact hypersensitivity to oxazolone, but CD8+ T cells contribute with the most potent effector mechanisms. Moreover, our results suggest that CD4+ T cell function is required for the mobilization of CD8+ effector T cells to the site of hapten exposure. The results shed new light on the relative importance of CD4+ and CD8+ T cells during the effector phase of contact hypersensitivity. PMID:22916101

  17. Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion.

    PubMed

    Nithipatikom, Kasem; Isbell, Marilyn A; Lindholm, Paul F; Kajdacsy-Balla, Andre; Kaul, Sushma; Campell, William B

    2002-01-01

    The PC-3 Low Invasive cells and the PC-3 High Invasive cells were used to investigate the correlation of the COX-2 expression and its arachidonic acid metabolites, prostaglandins, with their invasiveness through Matrigel using a Boyden chamber assay. The COX-2 expression in PC-3 High Invasive cells was approximately 3-fold higher than in PC-3 Low Invasive cells while the COX-1 expression was similar in both cell sublines. When incubated with arachidonic acid, PGE2 was the major prostaglandin produced by these cells. PC-3 High Invasive cells produced PGE2 approximately 2.5-fold higher than PC-3 Low Invasive cells. PGD2 was the second most abundant prostaglandin produced by these cells. Both indomethacin (a nonspecific COX inhibitor) and NS-398 (a specific COX-2 inhibitor) inhibited the production of prostaglandins and the cell invasion. PGE2 alone did not induce the cell invasion of PC-3 Low Invasive cells. However, PGE2 reversed the inhibition of cell invasion by NS-398 and enhanced the cell invasion of the PC-3 High Invasive cells. In contrast, PGD2 slightly inhibited the cell invasion. These results suggest that in the PC-3 Low Invasive cells, COX-2-derived PGE2 may not be sufficient to induce cell invasion while in the PC-3 High Invasive cells, PGE2 may be sufficient to act as an enhancer for the cell invasion. Further, PGD2 may represent a weak inhibitor and counteracts the effect of PGE2 in the cell invasion. PMID:12498388

  18. The SHIP2 interactor Myo1c is required for cell migration in 1321 N1 glioblastoma cells.

    PubMed

    Edimo, William's Elong; Ramos, Ana Raquel; Ghosh, Somadri; Vanderwinden, Jean-Marie; Erneux, Christophe

    2016-08-01

    The phosphoinositide 5-phosphatases consist of several enzymes that have been shown to modulate cell migration and invasion. SHIP2, one family member, is known to interact with growth factor receptors and cytoskeletal proteins. In a human model of glioblastoma 1321 N1 cells, we recently identified Myo1c as a new interactor of SHIP2. This was shown in a complex of proteins also containing filamin A. We show here that SHIP2 localization at lamellipodia and ruffles is impaired in Myo1c depleted cells. In the absence of Myo1c, N1 cells tend to associate to form clusters. Cell migration is very much reduced in Myo1c depleted cells, concomitantly with a decrease in FAK Tyr397 phosphorylation, focal adhesion length and PI(4,5)P2 immunostaining. In N1 cells, Myo1c is thus important for lamellipodia formation to assemble a protein complex containing SHIP2 to facilitate cell migration. PMID:27246739

  19. Virus uncoating is required for apoptosis induction in cultured mammalian cells infected with African horse sickness virus.

    PubMed

    Vermaak, Elaine; Theron, Jacques

    2015-07-01

    Infection of cultured mammalian cells with African horse sickness virus (AHSV) is known to induce cell death. To date, the trigger(s) of this response, the apoptotic pathways activated during AHSV infection and the functional consequences of apoptosis on the virus replication cycle have yet to be characterized. This study demonstrated that extracellular treatment of BHK-21 cells with both of the AHSV4 outer capsid proteins, VP2 and VP5, was sufficient to trigger apoptosis. Whether steps in AHSV4 replication subsequent to viral attachment were required for AHSV4-induced apoptosis was also investigated. Apoptosis was induced in BHK-21 cells infected with UV-inactivated AHSV4 and in ribavirin-treated cells infected with AHSV4. However, both AHSV4- and VP2/VP5-stimulated apoptotic responses were inhibited in the presence of the endosomal acidification inhibitors ammonium chloride and chloroquine. These results indicated that uncoating of AHSV4 virions, but not viral transcription or subsequent steps in viral replication, was required for AHSV4 to induce apoptosis in BHK-21 cells. Furthermore, this study showed that both the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways were induced following AHSV4 infection. The inhibition of caspase activity in AHSV4-infected cells did not diminish AHSV4 replication, but reduced the release and dissemination of progeny viral particles. Taken together, the data indicated that uncoating of AHSV virions was required for apoptosis induction, and that apoptosis enhanced virus spread and release. PMID:25783475

  20. All Three Rows of Outer Hair Cells Are Required for Cochlear Amplification.

    PubMed

    Murakoshi, Michio; Suzuki, Sho; Wada, Hiroshi

    2015-01-01

    In the mammalian auditory system, the three rows of outer hair cells (OHCs) located in the cochlea are thought to increase the displacement amplitude of the organ of Corti. This cochlear amplification is thought to contribute to the high sensitivity, wide dynamic range, and sharp frequency selectivity of the hearing system. Recent studies have shown that traumatic stimuli, such as noise exposure and ototoxic acid, cause functional loss of OHCs in one, two, or all three rows. However, the degree of decrease in cochlear amplification caused by such functional losses remains unclear. In the present study, a finite element model of a cross section of the gerbil cochlea was constructed. Then, to determine effects of the functional losses of OHCs on the cochlear amplification, changes in the displacement amplitude of the basilar membrane (BM) due to the functional losses of OHCs were calculated. Results showed that the displacement amplitude of the BM decreases significantly when a single row of OHCs lost its function, suggesting that all three rows of OHCs are required for cochlear amplification. PMID:26295049

  1. Group 3 innate lymphoid cells continuously require the transcription factor GATA3 after commitment

    PubMed Central

    Zhong, Chao; Cui, Kairong; Wilhelm, Christoph; Hu, Gangqing; Mao, Kairui; Belkaid, Yasmine; Zhao, Keji; Zhu, Jinfang

    2015-01-01

    The transcription factor GATA3 is indispensable for the development of all interleukin-7 receptor α (IL-7Rα)-expressing innate lymphoid cells (ILCs). However, the functional role of low GATA3 expression in committed ILC3s has not been identified. We report that GATA3 regulates homeostasis of ILC3s by controlling IL-7Rα expression. In addition, GATA3 is critical for the development of the NKp46+ ILC3 subset by regulating the balance between the transcription factors T-bet and RORγt. Alhough GATA3 positively regulates NKp46+ ILC3 subset-specific genes, it negatively regulates CCR6+ ILC3 subset lymphoid tissue inducer (LTi)-specific genes in NKp46+ ILC3s. Furthermore, GATA3 is required for IL-22 production in both LTi and NKp46+ ILC3s. Thus, despite its low expression, GATA3 is critical for the homeostasis, development and function of ILC3 subsets. PMID:26595886

  2. Nup50 is required for cell differentiation and exhibits transcription-dependent dynamics.

    PubMed

    Buchwalter, Abigail L; Liang, Yun; Hetzer, Martin W

    2014-08-15

    The nuclear pore complex (NPC) plays a critical role in gene expression by mediating import of transcription regulators into the nucleus and export of RNA transcripts to the cytoplasm. Emerging evidence suggests that in addition to mediating transport, a subset of nucleoporins (Nups) engage in transcriptional activation and elongation at genomic loci that are not associated with NPCs. The underlying mechanism and regulation of Nup mobility on and off nuclear pores remain unclear. Here we show that Nup50 is a mobile Nup with a pronounced presence both at the NPC and in the nucleoplasm that can move between these different localizations. Strikingly, the dynamic behavior of Nup50 in both locations is dependent on active transcription by RNA polymerase II and requires the N-terminal half of the protein, which contains importin α- and Nup153-binding domains. However, Nup50 dynamics are independent of importin α, Nup153, and Nup98, even though the latter two proteins also exhibit transcription-dependent mobility. Of interest, depletion of Nup50 from C2C12 myoblasts does not affect cell proliferation but inhibits differentiation into myotubes. Taken together, our results suggest a transport-independent role for Nup50 in chromatin biology that occurs away from the NPC. PMID:24943837

  3. Sorbent Material Property Requirements for On-Board Hydrogen Storage for Automotive Fuel Cell Systems.

    SciTech Connect

    Ahluwalia, R. K.; Peng, J-K; Hua, T. Q.

    2015-05-25

    Material properties required for on-board hydrogen storage in cryogenic sorbents for use with automotive polymer electrolyte membrane (PEM) fuel cell systems are discussed. Models are formulated for physical, thermodynamic and transport properties, and for the dynamics of H-2 refueling and discharge from a sorbent bed. A conceptual storage configuration with in-bed heat exchanger tubes, a Type-3 containment vessel, vacuum insulation and requisite balance-of-plant components is developed to determine the peak excess sorption capacity and differential enthalpy of adsorption for 5.5 wt% system gravimetric capacity and 55% well-to-tank (WTT) efficiency. The analysis also determines the bulk density to which the material must be compacted for the storage system to reach 40 g.L-1 volumetric capacity. Thermal transport properties and heat transfer enhancement methods are analyzed to estimate the material thermal conductivity needed to achieve 1.5 kg.min(-1) H-2 refueling rate. Operating temperatures and pressures are determined for 55% WTT efficiency and 95% usable H-2. Needs for further improvements in material properties are analyzed that would allow reduction of storage pressure to 50 bar from 100 bar, elevation of storage temperature to 175-200 K from 150 K, and increase of WTT efficiency to 57.5% or higher.

  4. Cellular Defense and Sensory Cell Survival Require Distinct Functions of ebi in Drosophila

    PubMed Central

    Lim, Young-Mi; Yagi, Yoshimasa; Tsuda, Leo

    2015-01-01

    The innate immune response and stress-induced apoptosis are well-established signaling pathways related to cellular defense. NF-κB and AP-1 are redox-sensitive transcription factors that play important roles in those pathways. Here we show that Ebi, a Drosophila homolog of the mammalian co-repressor molecule transducin β-like 1 (TBL1), variously regulates the expression of specific genes that are targets of redox-sensitive transcription factors. In response to different stimuli, Ebi activated gene expression to support the acute immune response in fat bodies, whereas Ebi repressed genes that are involved in apoptosis in photoreceptor cells. Thus, Ebi seems to act as a regulatory switch for genes that are activated or repressed in response to different external stimuli. Our results offer clear in vivo evidence that the Ebi-containing co-repressor complex acts in a distinct manner to regulate transcription that is required for modulating the output of various processes during Drosophila development. PMID:26524764

  5. WASp-dependent actin cytoskeleton stability at the dendritic cell immunological synapse is required for extensive, functional T cell contacts.

    PubMed

    Malinova, Dessislava; Fritzsche, Marco; Nowosad, Carla R; Armer, Hannah; Munro, Peter M G; Blundell, Michael P; Charras, Guillaume; Tolar, Pavel; Bouma, Gerben; Thrasher, Adrian J

    2016-05-01

    The immunological synapse is a highly structured and molecularly dynamic interface between communicating immune cells. Although the immunological synapse promotes T cell activation by dendritic cells, the specific organization of the immunological synapse on the dendritic cell side in response to T cell engagement is largely unknown. In this study, confocal and electron microscopy techniques were used to investigate the role of dendritic cell actin regulation in immunological synapse formation, stabilization, and function. In the dendritic cell-restricted absence of the Wiskott-Aldrich syndrome protein, an important regulator of the actin cytoskeleton in hematopoietic cells, the immunological synapse contact with T cells occupied a significantly reduced surface area. At a molecular level, the actin network localized to the immunological synapse exhibited reduced stability, in particular, of the actin-related protein-2/3-dependent, short-filament network. This was associated with decreased polarization of dendritic cell-associated ICAM-1 and MHC class II, which was partially dependent on Wiskott-Aldrich syndrome protein phosphorylation. With the use of supported planar lipid bilayers incorporating anti-ICAM-1 and anti-MHC class II antibodies, the dendritic cell actin cytoskeleton organized into recognizable synaptic structures but interestingly, formed Wiskott-Aldrich syndrome protein-dependent podosomes within this area. These findings demonstrate that intrinsic dendritic cell cytoskeletal remodeling is a key regulatory component of normal immunological synapse formation, likely through consolidation of adhesive interaction and modulation of immunological synapse stability. PMID:26590149

  6. A virtual lymph node model to dissect the requirements for T-cell activation by synapses and kinapses

    PubMed Central

    Moreau, Hélène D; Bogle, Gib; Bousso, Philippe

    2016-01-01

    The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1–2 days. We propose that the stability of T-cell–DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines. PMID:27089942

  7. Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine.

    PubMed

    Cetinbas, Naniye Mallı; Sudderth, Jessica; Harris, Robert C; Cebeci, Aysun; Negri, Gian L; Yılmaz, Ömer H; DeBerardinis, Ralph J; Sorensen, Poul H

    2016-01-01

    Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by (13)C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis. PMID:27605385

  8. Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine

    PubMed Central

    Cetinbas, Naniye Mallı; Sudderth, Jessica; Harris, Robert C.; Cebeci, Aysun; Negri, Gian L.; Yılmaz, Ömer H.; DeBerardinis, Ralph J.; Sorensen, Poul H.

    2016-01-01

    Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis. PMID:27605385

  9. Requirement of CD30 expression on CD4 T cells in the pathogenesis of experimental autoimmune encephalomyelitis.

    PubMed

    Shinoda, Koji; Sun, Xun; Oyamada, Akiko; Yamada, Hisakata; Kira, Jun-Ichi; Yoshikai, Yasunobu

    2016-02-15

    CD30, a member of the tumor necrosis factor receptor superfamily, is expressed preferentially by effector or memory helper T cells. Here we show that experimental autoimmune encephalomyelitis (EAE) is ameliorated with markedly reduced induction of antigen-specific Th1 and Th17 cells in CD30 knockout mice. Passive EAE indicated that CD30 on non-hematopoietic parenchymal cell is not required and mixed bone marrow chimera experiments revealed that CD30 signaling on CD4 T cells amplified the development of antigen-specific and encephalitogenic CD4 T cells. Thus, CD30 expression on CD4 T cells is critically involved in the pathogenesis of central nervous system autoimmunity. PMID:26857493

  10. The hsp70 inhibitor VER155008 induces paraptosis requiring de novo protein synthesis in anaplastic thyroid carcinoma cells.

    PubMed

    Kim, Si Hyoung; Kang, Jun Goo; Kim, Chul Sik; Ihm, Sung-Hee; Choi, Moon Gi; Yoo, Hyung Joon; Lee, Seong Jin

    2014-11-01

    In this study, we evaluated the effect of the hsp70 inhibitor VER155008 on survival of anaplastic thyroid carcinoma (ATC) cells. In ATC cells, VER155008 increased the percentages of dead cells and vacuolated cells. VER155008 did not lead to the cleavage of caspase-3 protein regardless of pretreatment with z-VAD-fmk. VER155008 increased LC3-II protein levels but the protein levels were not changed by autophagy inhibitors. VER155008 caused the dilatation of endoplasmic reticulum (ER), and the increased mRNA levels of Bip and CHOP, suggesting paraptosis. VER155008-induced paraptosis was attenuated by pretreatment with cycloheximide. In conclusion, VER155008 induces paraptosis characterized by cytoplasmic vacuolation, independence of caspase, dilatation of ER and induction of ER stress markers in ATC cells. Moreover, VER155008-induced paraptosis requires de novo protein synthesis in ATC cells. PMID:25450359

  11. Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance

    PubMed Central

    Mizutani, Tatsuaki; Neugebauer, Nina; Putz, Eva M.; Moritz, Nadine; Simma, Olivia; Zebedin-Brandl, Eva; Gotthardt, Dagmar; Warsch, Wolfgang; Eckelhart, Eva; Kantner, Hans-Peter; Kalinke, Ulrich; Lienenklaus, Stefan; Weiss, Siegfried; Strobl, Birgit; Müller, Mathias; Sexl, Veronika; Stoiber, Dagmar

    2012-01-01

    Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNβ-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1f/f CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1-/- NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1f/f animals), maturation was not altered. However, NK cells derived from Ifnar1f/f Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1f/f Ncr1-iCre compared with Ifnar1f/f control mice. Moreover, the ability of Ifnar1f/f Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance. PMID:23170251

  12. Invariant NKT Cell Development Requires a Full Complement of Functional CD3 ζ Immunoreceptor Tyrosine-Based Activation Motifs

    PubMed Central

    Becker, Amy M.; Blevins, Jon S.; Tomson, Farol L.; Eitson, Jennifer L.; Medeiros, Jennifer J.; Yarovinsky, Felix; Norgard, Michael V.; van Oers, Nicolai S. C.

    2010-01-01

    Invariant NKT (iNKT) cells regulate early immune responses to infections, in part because of their rapid release of IFN-γ and IL-4. iNKT cells are proposed to reduce the severity of Lyme disease following Borrelia burgdorferi infection. Unlike conventional T cells, iNKT cells express an invariant αβ TCR that recognizes lipids bound to the MHC class I-like molecule, CD1d. Furthermore, these cells are positively selected following TCR interactions with glycolipid/CD1d complexes expressed on CD4+CD8+ thymocytes. Whereas conventional T cell development can proceed with as few as 4/10 CD3 immunoreceptor tyrosine-based activation motifs (ITAMs), little is known about the ITAM requirements for iNKT cell selection and expansion. We analyzed iNKT cell development in CD3 ζ transgenic lines with various tyrosine-to-phenylalanine substitutions (YF) that eliminated the functions of the first (YF1,2), third (YF5,6), or all three (YF1–6) CD3 ζ ITAMs. iNKT cell numbers were significantly reduced in the thymus, spleen, and liver of all YF mice compared with wild type mice. The reduced numbers of iNKT cells resulted from significant reductions in the expression of the early growth response 2 and promyelocytic leukemia zinc finger transcription factors. In the mice with few to no iNKT cells, there was no difference in the severity of Lyme arthritis compared with wild type controls, following infections with the spirochete B. burgdorferi. These findings indicate that a full complement of functional CD3 ζ ITAMs is required for effective iNKT cell development. The Journal of Immunology, 2010, 184: 6822–6832. PMID:20483726

  13. Timing of Tissue-specific Cell Division Requires a Differential Onset of Zygotic Transcription during Metazoan Embryogenesis.

    PubMed

    Wong, Ming-Kin; Guan, Daogang; Ng, Kaoru Hon Chun; Ho, Vincy Wing Sze; An, Xiaomeng; Li, Runsheng; Ren, Xiaoliang; Zhao, Zhongying

    2016-06-10

    Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development. PMID:27056332

  14. Human mast cell degranulation and preformed TNF secretion require mitochondrial translocation to exocytosis sites: Relevance to atopic dermatitis

    PubMed Central

    Zhang, Bodi; Alysandratos, Konstantinos-Dionysios; Angelidou, Asimenia; Asadi, Shahrzad; Sismanopoulos, Nikolaos; Delivanis, Danae-Anastasia; Weng, Zuyi; Miniati, Alexandra; Vasiadi, Magdalini; Katsarou-Katsari, Alexandra; Miao, Benchun; Leeman, Susan E.; Kalogeromitros, Dimitrios; Theoharides, Theoharis C.

    2012-01-01

    Background Mast cells derive from hematopoietic cell precursors and participate in tissue allergic, immune, and inflammatory processes. They secrete many mediators, including preformed TNF, in response to allergic, neuropeptide, and environmental triggers. However, regulation of mast cell degranulation is not well understood. Objective We investigated the role of mitochondrial dynamics in degranulation of human cultured mast cells. Methods Human umbilical cord blood–derived mast cells (hCBMCs) and Laboratory of Allergic Diseases 2 (LAD2) mast cells were examined by confocal and differential interference contrast microscopy during activation by IgE/antigen and substance P (SP). Mast cells in control and atopic dermatitis (AD) skin were evaluated by transmission electron microscopy. LAD2 cells were pretreated with mitochondrial division inhibitor, a dynamin-related protein 1 (Drp1) inhibitor, and small interfering RNA for Drp1, which is necessary for mitochondrial fission and translocation. Calcineurin and Drp1 gene expression was analyzed in stimulated LAD2 cells and AD skin biopsies. Results Stimulation of hCBMCs with IgE/antigen or LAD2 cells with SP leads to rapid (30 minutes) secretion of preformed TNF. Degranulation is accompanied by mitochondrial translocation from a perinuclear location to exocytosis sites. Extracellular calcium depletion prevents these effects, indicating calcium requirement. The calcium-dependent calcineurin and Drp1 are activated 30 minutes after SP stimulation. Reduction of Drp1 activity by mitochondrial division inhibitor and decrease of Drp1 expression using small interfering RNA inhibit mitochondrial translocation, degranulation, and TNF secretion. Mitochondrial translocation is also evident by transmission electron microscopy in skin mast cells from AD biopsies, in which gene expression of calcineurin, Drp1, and SP is higher than in normal skin. Conclusion Human mast cell degranulation requires mitochondrial dynamics, also

  15. A Septin Requirement Differentiates Autonomous- and Contact-Facilitated T Cell Proliferation

    PubMed Central

    Mujal, Adriana M.; Gilden, Julia K.; Gérard, Audrey; Kinoshita, Makoto; Krummel, Matthew F.

    2015-01-01

    T cell proliferation is initiated by T cell antigen receptor (TCR) triggering and/or by soluble growth factors. In characterizing T cells lacking the septin cytoskeleton, we found that successful cell division has discrete septin-dependent and -independent pathways. Septin-deficient T cells failed cytokinesis when prompted by pharmacological activation or cytokines. In contrast, cell division was independent of septins when cell-cell contacts, such as those from antigen-presenting cells, provided a niche. This septin-independent pathway was mediated by phosphatidylinositol-3-OH kinase activation through a combination of integrins and co-stimulatory signals. We could differentiate cytokine- versus antigen-driven expansion in vivo and thus demonstrate that targeting septins has strong potential to moderate detrimental bystander or homeostatic cytokine-driven proliferation without influencing expansion driven by conventional antigen-presentation. PMID:26692174

  16. Regeneration of Sensory Hair Cells Requires Localized Interactions between the Notch and Wnt Pathways.

    PubMed

    Romero-Carvajal, Andrés; Navajas Acedo, Joaquín; Jiang, Linjia; Kozlovskaja-Gumbrienė, Agnė; Alexander, Richard; Li, Hua; Piotrowski, Tatjana

    2015-08-10

    In vertebrates, mechano-electrical transduction of sound is accomplished by sensory hair cells. Whereas mammalian hair cells are not replaced when lost, in fish they constantly renew and regenerate after injury. In vivo tracking and cell fate analyses of all dividing cells during lateral line hair cell regeneration revealed that support and hair cell progenitors localize to distinct tissue compartments. Importantly, we find that the balance between self-renewal and differentiation in these compartments is controlled by spatially restricted Notch signaling and its inhibition of Wnt-induced proliferation. The ability to simultaneously study and manipulate individual cell behaviors and multiple pathways in vivo transforms the lateral line into a powerful paradigm to mechanistically dissect sensory organ regeneration. The striking similarities to other vertebrate stem cell compartments uniquely place zebrafish to help elucidate why mammals possess such low capacity to regenerate hair cells. PMID:26190147

  17. KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata

    PubMed Central

    Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

    2016-01-01

    The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

  18. KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata.

    PubMed

    Tanaka, Yutaka; Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

    2016-01-01

    The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

  19. Intact vinculin protein is required for control of cell shape, cell mechanics, and rac-dependent lamellipodia formation

    NASA Technical Reports Server (NTRS)

    Goldmann, Wolfgang H.; Ingber, Donald E.

    2002-01-01

    Studies were carried out using vinculin-deficient F9 embryonic carcinoma (gamma229) cells to analyze the relationship between structure and function within the focal adhesion protein vinculin, in the context of control of cell shape, cell mechanics, and movement. Atomic force microscopy studies revealed that transfection of the head (aa 1-821) or tail (aa 811-1066) domain of vinculin, alone or together, was unable to fully reverse the decrease in cell stiffness, spreading, and lamellipodia formation caused by vinculin deficiency. In contrast, replacement with intact vinculin completely restored normal cell mechanics and spreading regardless of whether its tyrosine phosphorylation site was deleted. Constitutively active rac also only induced extension of lamellipodia when microinjected into cells that expressed intact vinculin protein. These data indicate that vinculin's ability to physically couple integrins to the cytoskeleton, to mechanically stabilize cell shape, and to support rac-dependent lamellipodia formation all appear to depend on its intact three-dimensional structure.

  20. B-lymphocyte responses to trinitrophenyl-conjugated Ficoll: requirement for T lymphocytes and Ia-bearing adherent cells.

    PubMed Central

    Letvin, N L; Benacerraf, B; Germain, R N

    1981-01-01

    These studies were done to characterize the cellular requirements for B-lymphocyte responses to the haptenated polysaccharide trinitrophenyl-conjugated Ficoll. By using an in vitro microculture system, it was demonstrated that hapten-specific anti-trinitrophenyl-conjugated Ficoll plaque-forming cell responses by B lymphocytes require Ia-bearing adherent accessory cells and Thy 1+ Lyt 1+2- nylon wool-nonadherent (T) lymphocytes. Such T cells could be primed in vivo with nonderivatized Ficoll to show carrier-specific helper cell function for derivatized Ficoll responses in vitro. The implications of these findings for our understanding of b-lymphocyte triggering by so-called T-independent antigens are discussed. PMID:6975477

  1. Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation

    PubMed Central

    Solaimani Kartalaei, Parham; Yamada-Inagawa, Tomoko; Vink, Chris S.; de Pater, Emma; van der Linden, Reinier; Marks-Bluth, Jonathon; van der Sloot, Anthon; van den Hout, Mirjam; Yokomizo, Tomomasa; van Schaick-Solernó, M. Lucila; Delwel, Ruud; Pimanda, John E.; van IJcken, Wilfred F.J.

    2015-01-01

    Hematopoietic stem cells (HSCs) are generated via a natural transdifferentiation process known as endothelial to hematopoietic cell transition (EHT). Because of small numbers of embryonal arterial cells undergoing EHT and the paucity of markers to enrich for hemogenic endothelial cells (ECs [HECs]), the genetic program driving HSC emergence is largely unknown. Here, we use a highly sensitive RNAseq method to examine the whole transcriptome of small numbers of enriched aortic HSCs, HECs, and ECs. Gpr56, a G-coupled protein receptor, is one of the most highly up-regulated of the 530 differentially expressed genes. Also, highly up-regulated are hematopoietic transcription factors, including the “heptad” complex of factors. We show that Gpr56 (mouse and human) is a target of the heptad complex and is required for hematopoietic cluster formation during EHT. Our results identify the processes and regulators involved in EHT and reveal the surprising requirement for Gpr56 in generating the first HSCs. PMID:25547674

  2. Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Jorgensen, Niklas Rye; Teilmann, Stefan Cuoni; Henriksen, Zanne; Civitelli, Roberto; Sorensen, Ole Helmer; Steinberg, Thomas H.

    2003-01-01

    The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in "fast" ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in "slow" ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

  3. Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

    PubMed

    Sasseville, Maxime; Ritter, Lesley J; Nguyen, Thao M; Liu, Fang; Mottershead, David G; Russell, Darryl L; Gilchrist, Robert B

    2010-09-15

    Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation. PMID:20736313

  4. Connexin43 Is Required for the Maintenance of Multipotency in Skin-Derived Stem Cells

    PubMed Central

    Dyce, Paul W.; Li, Dan; Barr, Kevin J.

    2014-01-01

    Expression of the gap junction protein, connexin43 (Cx43), begins early during embryogenesis and is maintained in many different cell types. Several stem cell populations have been shown to express Cx43 and to form functional gap junctions. While it is clear that Cx43 is critical to the function of many organs, whether the same is true for stem cells has not been clearly demonstrated. Recently, stem cells isolated from newborn mouse skin were shown to form oocyte-like cells (OLCs) in vitro, hence the present study focussed on the role Cx43 plays in the proliferation and differentiation of these cells. The stem cells express Cx43 and those from knockout mice (Cx43 KO) exhibited significantly reduced cell–cell coupling. Loss of Cx43 reduced the rate of cellular migration [Cx43 KO, 1.57±0.65 radial cell units (RCU); wildtype (WT), 5.57±0.37 RCU] but increased the proliferation rate of the stem cells (Cx43 KO, 29.40%±2.02%; WT, 12.76%±1.50%). The expression of the pluripotency markers OCT4 and Nanog were found to be reduced in the Cx43 KO population, suggesting an inhibition of differentiation potential. To test the differentiation ability, the stem cells were induced to form neuronal cell types in vitro. While both the WT and KO cells were able to form GFAP-positive astrocytic cells, only WT stem cells were able to form βIII tubulin-positive neurons. Similarly, the ability of the stem cells to form OLCs was ablated by the loss of Cx43. These data reveal a role for Cx43 in maintaining multipotency within the skin-derived stem cell population. PMID:24694074

  5. Downregulation of FOXP1 is required during germinal center B-cell function

    PubMed Central

    Sagardoy, Ainara; Martinez-Ferrandis, Jose I.; Roa, Sergio; Bunting, Karen L.; Aznar, María Angela; Elemento, Olivier; Shaknovich, Rita; Fontán, Lorena; Fresquet, Vicente; Perez-Roger, Ignacio; Robles, Eloy F.; De Smedt, Linde; Sagaert, Xavier

    2013-01-01

    B-cell maturation and germinal center (GC) formation are dependent on the interplay between BCL6 and other transcriptional regulators. FOXP1 is a transcription factor that regulates early B-cell development, but whether it plays a role in mature B cells is unknown. Analysis of human tonsillar B-cell subpopulations revealed that FOXP1 shows the opposite expression pattern to BCL6, suggesting that FOXP1 regulates the transition from resting follicular B cell to activated GC B cell. Chromatin immunoprecipitation-on-chip and gene expression assays on B cells indicated that FOXP1 acts as a transcriptional activator and repressor of genes involved in the GC reaction, half of which are also BCL6 targets. To study FOXP1 function in vivo, we developed transgenic mice expressing human FOXP1 in lymphoid cells. These mice exhibited irregular formation of splenic GCs, showing a modest increase in naïve and marginal-zone B cells and a significant decrease in GC B cells. Furthermore, aberrant expression of FOXP1 impaired transcription of noncoding γ1 germline transcripts and inhibited efficient class switching to the immunoglobulin G1 isotype. These studies show that FOXP1 is physiologically downregulated in GC B cells and that aberrant expression of FOXP1 impairs mechanisms triggered by B-cell activation, potentially contributing to B-cell lymphomagenesis. PMID:23580662

  6. Porcine Induced Pluripotent Stem Cells Require LIF and Maintain Their Developmental Potential in Early Stage of Embryos

    PubMed Central

    Cheng, De; Guo, Yanjie; Li, Zhenzhen; Liu, Yajun; Gao, Xing; Gao, Yi; Cheng, Xiang; Hu, Junhe; Wang, Huayan

    2012-01-01

    Porcine induced pluripotent stem (piPS) cell lines have been generated recently by using a cocktail of defined transcription factors, however, the features of authentic piPS cells have not been agreed upon and most of published iPS clones did not meet the stringent requirements of pluripotency. Here, we report the generation of piPS cells from fibroblasts using retrovirus carrying four mouse transcription factors (mOct4, mSox2, mKlf4 and mc-Myc, 4F). Multiple LIF-dependent piPS cell lines were generated and these cells showed the morphology similar to mouse embryonic stem cells and other pluripotent stem cells. In addition to the routine characterization, piPS cells were injected into porcine pre-compacted embryos to generate chimera embryos and nuclear transfer (NT) embryos. The results showed that piPS cells retain the ability to integrate into inner and outer layers of the blastocysts, and support the NT embryos development to blastocysts. The generations of chimera embryos and NT embryos derived from piPS clones are a practical means to determine the quality of iPS cells ex vivo. PMID:23251622

  7. Na,K-ATPase β-Subunit Is Required for Epithelial Polarization, Suppression of Invasion, and Cell Motility

    PubMed Central

    Rajasekaran, Sigrid A.; Palmer, Lawrence G.; Quan, Karina; Harper, Jeffrey F.; Ball, William J.; Bander, Neil H.; Soler, Alejandro Peralta; Rajasekaran, Ayyappan K.

    2001-01-01

    The cell adhesion molecule E-cadherin has been implicated in maintaining the polarized phenotype of epithelial cells and suppression of invasiveness and motility of carcinoma cells. Na,K-ATPase, consisting of an α- and β-subunit, maintains the sodium gradient across the plasma membrane. A functional relationship between E-cadherin and Na,K-ATPase has not previously been described. We present evidence that the Na,K-ATPase plays a crucial role in E-cadherin–mediated development of epithelial polarity, and suppression of invasiveness and motility of carcinoma cells. Moloney sarcoma virus-transformed Madin-Darby canine kidney cells (MSV-MDCK) have highly reduced levels of E-cadherin and β1-subunit of Na,K-ATPase. Forced expression of E-cadherin in MSV-MDCK cells did not reestablish epithelial polarity or inhibit the invasiveness and motility of these cells. In contrast, expression of E-cadherin and Na,K-ATPase β1-subunit induced epithelial polarization, including the formation of tight junctions and desmosomes, abolished invasiveness, and reduced cell motility in MSV-MDCK cells. Our results suggest that E-cadherin–mediated cell-cell adhesion requires the Na,K-ATPase β-subunit's function to induce epithelial polarization and suppress invasiveness and motility of carcinoma cells. Involvement of the β1-subunit of Na,K-ATPase in the polarized phenotype of epithelial cells reveals a novel link between the structural organization and vectorial ion transport function of epithelial cells. PMID:11179415

  8. The Mi-2-like Smed-CHD4 gene is required for stem cell differentiation in the planarian Schmidtea mediterranea.

    PubMed

    Scimone, M Lucila; Meisel, Joshua; Reddien, Peter W

    2010-04-01

    Freshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of the action of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. We identified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, as required for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference (RNAi), neoblast numbers were initially normal, despite an inability of the animals to regenerate. However, the proliferative response of neoblasts to amputation or growth stimulation in Smed-CHD4(RNAi) animals was diminished. Smed-CHD4(RNAi) animals displayed a dramatic reduction in the numbers of certain neoblast progeny cells. Smed-CHD4 was required for the formation of these neoblast progeny cells. Together, these results indicate that Smed-CHD4 is required for neoblasts to produce progeny cells committed to differentiation in order to control tissue turnover and regeneration and suggest a crucial role for CHD4 proteins in stem cell differentiation. PMID:20223763

  9. The Mi-2-like Smed-CHD4 gene is required for stem cell differentiation in the planarian Schmidtea mediterranea

    PubMed Central

    Scimone, M. Lucila; Meisel, Joshua; Reddien, Peter W.

    2010-01-01

    Freshwater planarians are able to regenerate any missing part of their body and have extensive tissue turnover because of the action of dividing cells called neoblasts. Neoblasts provide an excellent system for in vivo study of adult stem cell biology. We identified the Smed-CHD4 gene, which is predicted to encode a chromatin-remodeling protein similar to CHD4/Mi-2 proteins, as required for planarian regeneration and tissue homeostasis. Following inhibition of Smed-CHD4 with RNA interference (RNAi), neoblast numbers were initially normal, despite an inability of the animals to regenerate. However, the proliferative response of neoblasts to amputation or growth stimulation in Smed-CHD4(RNAi) animals was diminished. Smed-CHD4(RNAi) animals displayed a dramatic reduction in the numbers of certain neoblast progeny cells. Smed-CHD4 was required for the formation of these neoblast progeny cells. Together, these results indicate that Smed-CHD4 is required for neoblasts to produce progeny cells committed to differentiation in order to control tissue turnover and regeneration and suggest a crucial role for CHD4 proteins in stem cell differentiation. PMID:20223763

  10. Murine complement receptor 1 is required for germinal center B cell maintenance but not initiation

    PubMed Central

    Donius, Luke R.; Weis, Janis J.; Weis, John H.

    2014-01-01

    Germinal centers are the anatomic sites for the generation of high affinity immunoglobulin expressing plasma cells and memory B cells. The germinal center B cells that are precursors of these cells circulate between the light zone B cell population that interact with antigen laden follicular dendritic cells (FDC) and the proliferative dark zone B cell population. Antigen retention by follicular dendritic cells is dependent on Fc receptors and complement receptors, and complement receptor 1 (Cr1) is the predominant complement receptor expressed by FDC. The newly created Cr1KO mouse was used to test the effect of Cr1-deficiency on the kinetics of the germinal center reaction and the generation of IgM and switched memory B cell formation. Immunization of Cr1KO mice with a T cell-dependent antigen resulted in the normal initial expansion of B cells with a germinal center phenotype however these cells were preferentially lost in the Cr1KO animal over time (days). Bone marrow chimera animals documented the surprising finding that the loss of germinal center B cell maintenance was linked to the expression of Cr1 on B cells, not the FDC. Cr1-deficiency further resulted in antigen-specific IgM titer and IgM memory B cell reductions, but not antigen-specific IgG after 35-37 days. Investigations of nitrophenyl (NP)-specific IgG demonstrated that Cr1 is not necessary for affinity maturation during the response to particulate antigen. These data, along with those generated in our initial description of the Cr1KO animal describe unique functions of Cr1 on the surface of both B cells and FDC. PMID:24636730

  11. Nitric oxide production is not required for dihydrosphingosine-induced cell death in tobacco BY-2 cells

    PubMed Central

    Da Silva, Daniel; Lachaud, Christophe; Cotelle, Valérie; Brière, Christian; Grat, Sabine; Mazars, Christian

    2011-01-01

    Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium-dependent programmed cell death (PCD) in tobacco BY-2 cells. We have recently shown that DHS triggers a production of H2O2, via the activation of NADPH oxidase(s). However, this production of H2O2 is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H2O2, this NO production is not necessary for cell death induction. PMID:21455022

  12. BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties.

    PubMed

    Rendl, Michael; Polak, Lisa; Fuchs, Elaine

    2008-02-15

    Hair follicle (HF) formation is initiated when epithelial stem cells receive cues from specialized mesenchymal dermal papilla (DP) cells. In culture, DP cells lose their HF-inducing properties, but during hair growth in vivo, they reside within the HF bulb and instruct surrounding epithelial progenitors to orchestrate the complex hair differentiation program. To gain insights into the molecular program that maintains DP cell fate, we previously purified DP cells and four neighboring populations and defined their cell-type-specific molecular signatures. Here, we exploit this information to show that the bulb microenvironment is rich in bone morphogenetic proteins (BMPs) that act on DP cells to maintain key signature features in vitro and hair-inducing activity in vivo. By employing a novel in vitro/in vivo hybrid knockout assay, we ablate BMP receptor 1a in purified DP cells. When DPs cannot receive BMP signals, they lose signature characteristics in vitro and fail to generate HFs when engrafted with epithelial stem cells in vivo. These results reveal that BMP signaling, in addition to its key role in epithelial stem cell maintenance and progenitor cell differentiation, is essential for DP cell function, and suggest that it is a critical feature of the complex epithelial-mesenchymal cross-talk necessary to make hair. PMID:18281466

  13. Connexin43 (GJA1) is required in the population of dividing cells during fin regeneration

    PubMed Central

    Hoptak-Solga, Angela D.; Nielsen, Sarah; Jain, Isha; Thummel, Ryan; Hyde, David R.; Iovine, M. Kathryn

    2008-01-01

    In zebrafish, mutations in the gap junction gene connexin43 lead to short bony fin ray segments that give rise to the short fin phenotype. The sofb123 mutant exhibits fins that are half the length of wild-type fins and have reduced levels of cx43 mRNA. We find that sofb123 regenerating fins exhibit reduced levels of cell proliferation. Interestingly, the number of dividing cells per unit length of fin growth is similar between wild-type and mutant fins, suggesting that the number of cells that enter the cell cycle is specifically affected in sofb123. Expression of cx43 is identified in mitotic cells, which further suggests that Cx43 may contribute to establishing or maintaining the population of dividing cells. Indeed, missense alleles exhibiting high or low levels of gap junctional communication reveal a correlation between defects in direct cell-cell communication, cell proliferation, and segment length. Finally, targeted gene knockdown of cx43 in adult regenerating fins recapitulates the sofb123phenotype, revealing that the loss of Cx43 is sufficient to reduce both cell proliferation and segment length. We hypothesize that the level of gap junctional intercellular communication among dividing cells regulates the level of cell proliferation and ultimately regulates bone growth. PMID:18406403

  14. Vinculin is required for cell polarization, migration, and extracellular matrix remodeling in 3D collagen.

    PubMed

    Thievessen, Ingo; Fakhri, Nikta; Steinwachs, Julian; Kraus, Viola; McIsaac, R Scott; Gao, Liang; Chen, Bi-Chang; Baird, Michelle A; Davidson, Michael W; Betzig, Eric; Oldenbourg, Rudolf; Waterman, Clare M; Fabry, Ben

    2015-11-01

    Vinculin is filamentous (F)-actin-binding protein enriched in integrin-based adhesions to the extracellular matrix (ECM). Whereas studies in 2-dimensional (2D) tissue culture models have suggested that vinculin negatively regulates cell migration by promoting cytoskeleton-ECM coupling to strengthen and stabilize adhesions, its role in regulating cell migration in more physiologic, 3-dimensional (3D) environments is unclear. To address the role of vinculin in 3D cell migration, we analyzed the morphodynamics, migration, and ECM remodeling of primary murine embryonic fibroblasts (MEFs) with cre/loxP-mediated vinculin gene disruption in 3D collagen I cultures. We found that vinculin promoted 3D cell migration by increasing directional persistence. Vinculin was necessary for persistent cell protrusion, cell elongation, and stable cell orientation in 3D collagen, but was dispensable for lamellipodia formation, suggesting that vinculin-mediated cell adhesion to the ECM is needed to convert actin-based cell protrusion into persistent cell shape change and migration. Consistent with this finding, vinculin was necessary for efficient traction force generation in 3D collagen without affecting myosin II activity and promoted 3D collagen fiber alignment and macroscopical gel contraction. Our results suggest that vinculin promotes directionally persistent cell migration and tension-dependent ECM remodeling in complex 3D environments by increasing cell-ECM adhesion and traction force generation. PMID:26195589

  15. A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division

    PubMed Central

    Li, Yubing; Liu, Dianyi; López-Paz, Cristina; Olson, Bradley JSC; Umen, James G

    2016-01-01

    Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a ‘counting’ mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control. DOI: http://dx.doi.org/10.7554/eLife.10767.001 PMID:27015111

  16. A new class of cyclin dependent kinase in Chlamydomonas is required for coupling cell size to cell division.

    PubMed

    Li, Yubing; Liu, Dianyi; López-Paz, Cristina; Olson, Bradley Jsc; Umen, James G

    2016-01-01

    Proliferating cells actively control their size by mechanisms that are poorly understood. The unicellular green alga Chlamydomonas reinhardtii divides by multiple fission, wherein a 'counting' mechanism couples mother cell-size to cell division number allowing production of uniform-sized daughters. We identified a sizer protein, CDKG1, that acts through the retinoblastoma (RB) tumor suppressor pathway as a D-cyclin-dependent RB kinase to regulate mitotic counting. Loss of CDKG1 leads to fewer mitotic divisions and large daughters, while mis-expression of CDKG1 causes supernumerous mitotic divisions and small daughters. The concentration of nuclear-localized CDKG1 in pre-mitotic cells is set by mother cell size, and its progressive dilution and degradation with each round of cell division may provide a link between mother cell-size and mitotic division number. Cell-size-dependent accumulation of limiting cell cycle regulators such as CDKG1 is a potentially general mechanism for size control. PMID:27015111

  17. Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

    PubMed

    Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

    2014-07-01

    The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. PMID:24906928

  18. Preoperative platelet transfusions and perioperative red blood cell requirements in patients with thrombocytopenia undergoing noncardiac surgery

    PubMed Central

    Warner, Matthew A.; Jia, Qing; Clifford, Leanne; Wilson, Gregory; Brown, Michael J.; Hanson, Andrew C.; Schroeder, Darrell R.; Kor, Daryl J.

    2016-01-01

    BACKGROUND Perioperative hemorrhage impacts patient outcomes and health care resource utilization, yet the risks of transfusion therapies are significant. In patients with preoperative thrombocytopenia, the effects of prophylactic preoperative platelet (PLT) transfusion on perioperative bleeding complications remain uncertain. STUDY DESIGN AND METHODS This is a retrospective cohort study of noncardiac surgical patients between January 1, 2008, and December 31, 2011. Propensity-adjusted analyses were used to evaluate associations between preoperative thrombocytopenia, preoperative PLT transfusion, and the outcomes of interest, with a primary outcome of perioperative red blood cell (RBC) transfusion. RESULTS A total of 13,978 study participants were included; 860 (6.2%) had a PLT count of not more than 100 × 109/L with 71 (8.3%) receiving PLTs preoperatively. Administration of PLTs was associated with higher rates of perioperative RBC transfusion (66.2% vs. 49.1%, p 0.0065); however, in propensity-adjusted analysis there was no significant difference between groups (odds ratio [OR] [95% confidence interval {95% CI}], 1.68 [0.95–2.99]; p =0.0764]. Patients receiving PLTs had higher rates of intensive care unit (ICU) admission (OR [95% CI], 1.95 [1.10–3.46]; p =0.0224) and longer hospital lengths of stay (estimate [95% bootstrap CI], 7.2 [0.8–13.9] days; p =0.0006) in propensity-adjusted analyses. CONCLUSION Preoperative PLT transfusion did not attenuate RBC requirements in patients with thrombocytopenia undergoing noncardiac surgery. Moreover, preoperative PLT transfusion was associated with increased ICU admission rates and hospital duration. These findings suggest that more conservative management of preoperative thrombocytopenia may be warranted. PMID:26559936

  19. ABCB5 is a limbal stem cell gene required for corneal development and repair

    PubMed Central

    Ksander, Bruce R.; Kolovou, Paraskevi E.; Wilson, Brian J.; Saab, Karim R.; Guo, Qin; Ma, Jie; McGuire, Sean P.; Gregory, Meredith S.; Vincent, William J. B.; Perez, Victor L.; Cruz-Guilloty, Fernando; Kao, Winston W. Y.; Call, Mindy K.; Tucker, Budd A.; Zhan, Qian; Murphy, George F.; Lathrop, Kira L.; Alt, Clemens; Mortensen, Luke J.; Lin, Charles P.; Zieske, James D.; Frank, Markus H.; Frank, Natasha Y.

    2014-01-01

    Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency. PMID:25030174

  20. Cell proliferation, DNA repair, and p53 function are not required for programmed death of prostatic glandular cells induced by androgen ablation.

    PubMed Central

    Berges, R R; Furuya, Y; Remington, L; English, H F; Jacks, T; Isaacs, J T

    1993-01-01

    Androgen ablation induces programmed death of androgen-dependent prostatic glandular cells, resulting in fragmentation of their genomic DNA and the cells themselves into apoptotic bodies. Twenty percent of prostatic glandular cells undergo programmed death per day between day 2 and 5 after castration. During this same period, < 1% of prostatic glandular cells enter the S phase of the cell cycle, documenting that > 95% of these die in G0. During the programmed death of these G0 glandular cells, a futile DNA repair process is induced secondary to the DNA fragmentation. This futile DNA repair is not required, however, since inhibition of this process by > 90% with an appropriately timed hydroxy-urea dosing regimen had no effect upon the extent of the programmed death of these cells after castration. Likewise, p53 gene expression is not required since the same degree of cell death occurred in prostates and seminal vesicles after castration of wild-type and p53-deficient mice. PMID:8415631

  1. Phoenix Is Required for Mechanosensory Hair Cell Regeneration in the Zebrafish Lateral Line

    PubMed Central

    Behra, Martine; Bradsher, John; Sougrat, Rachid; Gallardo, Viviana; Allende, Miguel L.; Burgess, Shawn M.

    2009-01-01

    In humans, the absence or irreversible loss of hair cells, the sensory mechanoreceptors in the cochlea, accounts for a large majority of acquired and congenital hearing disorders. In the auditory and vestibular neuroepithelia of the inner ear, hair cells are accompanied by another cell type called supporting cells. This second cell population has been described as having stem cell-like properties, allowing efficient hair cell replacement during embryonic and larval/fetal development of all vertebrates. However, mammals lose their regenerative capacity in most inner ear neuroepithelia in postnatal life. Remarkably, reptiles, birds, amphibians, and fish are different in that they can regenerate hair cells throughout their lifespan. The lateral line in amphibians and in fish is an additional sensory organ, which is used to detect water movements and is comprised of neuroepithelial patches, called neuromasts. These are similar in ultra-structure to the inner ear's neuroepithelia and they share the expression of various molecular markers. We examined the regeneration process in hair cells of the lateral line of zebrafish larvae carrying a retroviral integration in a previously uncharacterized gene, phoenix (pho). Phoenix mutant larvae develop normally and display a morphologically intact lateral line. However, after ablation of hair cells with copper or neomycin, their regeneration in pho mutants is severely impaired. We show that proliferation in the supporting cells is strongly decreased after damage to hair cells and correlates with the reduction of newly formed hair cells in the regenerating phoenix mutant neuromasts. The retroviral integration linked to the phenotype is in a novel gene with no known homologs showing high expression in neuromast supporting cells. Whereas its role during early development of the lateral line remains to be addressed, in later larval stages phoenix defines a new class of proteins implicated in hair cell regeneration. PMID:19381250

  2. Wasp, the Drosophila Wiskott-Aldrich Syndrome Gene Homologue, Is Required for Cell Fate Decisions Mediated by Notch Signaling

    PubMed Central

    Ben-Yaacov, Sari; Le Borgne, Roland; Abramson, Irit; Schweisguth, Francois; Schejter, Eyal D.

    2001-01-01

    Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway. PMID:11149916

  3. Repression of the integrated papillomavirus E6/E7 promoter is required for growth suppression of cervical cancer cells.

    PubMed

    Francis, D A; Schmid, S I; Howley, P M

    2000-03-01

    The human papillomavirus (HPV) E2 protein is an important regulator of viral E6 and E7 gene expression. E2 can repress the viral promoter for E6 and E7 expression as well as block progression of the cell cycle in cancer cells harboring the DNA of "high-risk" HPV types. Although the phenomenon of E2-mediated growth arrest of HeLa cells and other HPV-positive cancer cells has been well documented, the specific mechanism by which E2 affects cellular proliferation has not yet been elucidated. Here, we show that bovine papillomavirus (BPV) E2-induced growth arrest of HeLa cells requires the repression of the E6 and E7 promoter. This repression is specific for E2TA and not E2TR, a BPV E2 variant that lacks the N-terminal transactivation domain. We demonstrate that expression of HPV16 E6 and E7 from a heterologous promoter that is not regulated by E2 rescues HeLa cells from E2-mediated growth arrest. Our data indicate that the pathway of E2-mediated growth arrest of HeLa cells requires repression of E6 and E7 expression through an activity specified by the transactivation domain of E2TA. PMID:10684283

  4. Endodermal cell–cell contact is required for the spatial control of Casparian band development in Arabidopsis thaliana

    PubMed Central

    Martinka, Michal; Dolan, Liam; Pernas, Monica; Abe, Jun; Lux, Alexander

    2012-01-01

    Background and Aims Apoplasmic barriers in plants fulfil important roles such as the control of apoplasmic movement of substances and the protection against invasion of pathogens. The aim of this study was to describe the development of apoplasmic barriers (Casparian bands and suberin lamellae) in endodermal cells of Arabidopsis thaliana primary root and during lateral root initiation. Methods Modifications of the endodermal cell walls in roots of wild-type Landsberg erecta (Ler) and mutants with defective endodermal development – scarecrow-3 (scr-3) and shortroot (shr) – of A. thaliana plants were characterized by light, fluorescent, confocal laser scanning, transmission and cryo-scanning electron microscopy. Key Results In wild-type plant roots Casparian bands initiate at approx. 1600 µm from the root cap junction and suberin lamellae first appear on the inner primary cell walls at approx. 7000–8000 µm from the root apex in the region of developing lateral root primordia. When a single cell replaces a pair of endodermal and cortical cells in the scr-3 mutant, Casparian band-like material is deposited ectopically at the junction between this ‘cortical’ cell and adjacent pericycle cells. Shr mutant roots with an undeveloped endodermis deposit Casparian band-like material in patches in the middle lamellae of cells of the vascular cylinder. Endodermal cells in the vicinity of developing lateral root primordia develop suberin lamellae earlier, and these are thicker, compared wih the neighbouring endodermal cells. Protruding primordia are protected by an endodermal pocket covered by suberin lamellae. Conclusions The data suggest that endodermal cell–cell contact is required for the spatial control of Casparian band development. Additionally, the endodermal cells form a collet (collar) of short cells covered by a thick suberin layer at the base of lateral root, which may serve as a barrier constituting a ‘safety zone’ protecting the vascular cylinder

  5. T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells.

    PubMed

    Arbore, Giuseppina; West, Erin E; Spolski, Rosanne; Robertson, Avril A B; Klos, Andreas; Rheinheimer, Claudia; Dutow, Pavel; Woodruff, Trent M; Yu, Zu Xi; O'Neill, Luke A; Coll, Rebecca C; Sher, Alan; Leonard, Warren J; Köhl, Jörg; Monk, Pete; Cooper, Matthew A; Arno, Matthew; Afzali, Behdad; Lachmann, Helen J; Cope, Andrew P; Mayer-Barber, Katrin D; Kemper, Claudia

    2016-06-17

    The NLRP3 inflammasome controls interleukin-1β maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described. We found that the NLRP3 inflammasome assembles in human CD4(+) T cells and initiates caspase-1-dependent interleukin-1β secretion, thereby promoting interferon-γ production and T helper 1 (T(H)1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to "innate immune cells" but is an integral component of normal adaptive T(H)1 responses. PMID:27313051

  6. Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing.

    PubMed

    Peramo, Antonio; Feinberg, Stephen E; Marcelo, Cynthia L

    2013-09-01

    Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they are produced in large numbers, they are highly suitable for use in epithelial cell banking. PMID:23149549

  7. Hhex is Required at Multiple Stages of Adult Hematopoietic Stem and Progenitor Cell Differentiation

    PubMed Central

    Goodings, Charnise; Smith, Elizabeth; Mathias, Elizabeth; Elliott, Natalina; Cleveland, Susan M.; Tripathi, Rati M.; Layer, Justin H.; Chen, Xi; Guo, Yan; Shyr, Yu; Hamid, Rizwan; Du, Yang; Davé, Utpal P.

    2015-01-01

    Hhex encodes a homeodomain transcription factor that is widely expressed in hematopoietic stem and progenitor cell populations. Its enforced expression induces T-cell leukemia and we have implicated it as an important oncogene in early T-cell precursor leukemias where it is immediately downstream of an LMO2-associated protein complex. Conventional Hhex knockouts cause embryonic lethality precluding analysis of adult hematopoiesis. Thus, we induced highly efficient conditional knockout (cKO) using vav-Cre transgenic mice. Hhex cKO mice were viable and born at normal litter sizes. At steady state, we observed a defect in B-cell development that we localized to the earliest B-cell precursor, the pro-B-cell stage. Most remarkably, bone marrow transplantation using Hhex cKO donor cells revealed a more profound defect in all hematopoietic lineages. In contrast, sublethal irradiation resulted in normal myeloid cell repopulation of the bone marrow but markedly impaired repopulation of T- and B-cell compartments. We noted that Hhex cKO stem and progenitor cell populations were skewed in their distribution and showed enhanced proliferation compared to WT cells. Our results implicate Hhex in the maintenance of LT-HSCs and in lineage allocation from multipotent progenitors especially in stress hematopoiesis. PMID:25968920

  8. Insulin-secreting β cells require a post-genomic concept

    PubMed Central

    Jiang, Fang-Xu; Morahan, Grant

    2016-01-01

    Pancreatic insulin-secreting β cells are essential in maintaining normal glucose homeostasis accomplished by highly specialized transcription of insulin gene, of which occupies up to 40% their transcriptome. Deficiency of these cells causes diabetes mellitus, a global public health problem. Although tremendous endeavors have been made to generate insulin-secreting cells from human pluripotent stem cells (i.e., primitive cells capable of giving rise to all cell types in the body), a regenerative therapy to diabetes has not yet been established. Furthermore, the nomenclature of β cells has become inconsistent, confusing and controversial due to the lack of standardized positive controls of developmental stage-matched in vivo cells. In order to minimize this negative impact and facilitate critical research in this field, a post-genomic concept of pancreatic β cells might be helpful. In this review article, we will briefly describe how β cells were discovered and islet lineage is developed that may help understand the cause of nomenclatural controversy, suggest a post-genomic definition and finally provide a conclusive remark on future research of this pivotal cell. PMID:27226815

  9. Prmt5 is required for germ cell survival during spermatogenesis in mice

    PubMed Central

    Wang, Yanbo; Zhu, Tianxiang; Li, Qiuling; Liu, Chunyi; Han, Feng; Chen, Min; Zhang, Lianjun; Cui, Xiuhong; Qin, Yan; Bao, Shilai; Gao, Fei

    2015-01-01

    During germ cell development, epigenetic modifications undergo extensive remodeling. Abnormal epigenetic modifications usually result in germ cell loss and reproductive defect. Prmt5 (Protein arginine methyltransferase 5) encodes a protein arginine methyltransferase which has been demonstrated to play important roles in germ cell development during embryonic stages. In the present study, we found that Prmt5 was also abundantly expressed in male germ cells after birth. Inactivation of this gene by crossing with Stra8-Cre transgenic mice resulted in germ cell loss during spermatogenesis. Further study revealed that the germ cell development was grossly normal before P10. However, most of the germ cells in Prmt5Δ/f; Stra8-Cre mice were blocked at meiotic stage. The expression of meiosis associated genes was reduced in Prmt5Δ/f; Stra8-Cre testes compared to control testes at P10. γH2AX was detected in sex body of control germ cells at P12, whereas multiple foci were observed in Prmt5-deficient germ cells. Further study revealed that H4R3me2s was virtually absent in germ cells after Prmt5 inactivation. The results of this study indicate that Prmt5 also plays important roles in germ cell development during spermatogenesis. PMID:26072710

  10. Insulin-secreting β cells require a post-genomic concept.

    PubMed

    Jiang, Fang-Xu; Morahan, Grant

    2016-05-25

    Pancreatic insulin-secreting β cells are essential in maintaining normal glucose homeostasis accomplished by highly specialized transcription of insulin gene, of which occupies up to 40% their transcriptome. Deficiency of these cells causes diabetes mellitus, a global public health problem. Although tremendous endeavors have been made to generate insulin-secreting cells from human pluripotent stem cells (i.e., primitive cells capable of giving rise to all cell types in the body), a regenerative therapy to diabetes has not yet been established. Furthermore, the nomenclature of β cells has become inconsistent, confusing and controversial due to the lack of standardized positive controls of developmental stage-matched in vivo cells. In order to minimize this negative impact and facilitate critical research in this field, a post-genomic concept of pancreatic β cells might be helpful. In this review article, we will briefly describe how β cells were discovered and islet lineage is developed that may help understand the cause of nomenclatural controversy, suggest a post-genomic definition and finally provide a conclusive remark on future research of this pivotal cell. PMID:27226815

  11. Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

    PubMed

    Thomas, Andrew J; Hailey, Dale W; Stawicki, Tamara M; Wu, Patricia; Coffin, Allison B; Rubel, Edwin W; Raible, David W; Simon, Julian A; Ou, Henry C

    2013-03-01

    Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line. PMID:23467357

  12. Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line

    PubMed Central

    Thomas, Andrew J.; Hailey, Dale W.; Stawicki, Tamara M.; Wu, Patricia; Coffin, Allison B.; Rubel, Edwin W.; Raible, David W.; Simon, Julian A.; Ou, Henry C.

    2013-01-01

    Cisplatin, one of the most commonly used anti-cancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analogue of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line. PMID:23467357

  13. MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic, but Not Syngeneic Recipient Mice

    PubMed Central

    Hirata, Yuichi; Li, Hao-Wei; Takahashi, Kazuko; Ishii, Hiroshi; Sykes, Megan; Fujisaki, Joji

    2015-01-01

    NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs. PMID:26544200

  14. T cells expressing the V beta 1 T-cell receptor are required for IgA production in the chicken.

    PubMed Central

    Cihak, J; Hoffmann-Fezer, G; Ziegler-Heibrock, H W; Stein, H; Kaspers, B; Chen, C H; Cooper, M D; Lösch, U

    1991-01-01

    While alpha beta T cells in mammals may express one of many variable (V) gene families in the beta locus, chickens have only two V beta gene families. The avian V beta 2+ T cells are recognized by the T-cell receptor 3 (TCR3) monoclonal antibody and V beta 1+ T cells are recognized by the TCR2 antibody, which we used to selectively suppress development of V beta 1+ T cells in order to examine their functional role. Suppression was accomplished by multiple injections of anti-TCR2 antibodies beginning in embryonic life and perpetuated by thymectomy 8 days after hatching. Young birds thus depleted of V beta 1+ T cells had greater than normal numbers of V beta 2+ T cells and appeared as healthy as thymectomized and untreated controls. While production of IgM and IgG antibodies was unimpaired, IgA antibody production was severely compromised in the V beta 1-depleted birds. The levels of secretory IgA in bile and lung lavage fluid were reduced 1000- to 10,000-fold and secretory IgA antibodies were not produced in response to mucosal immunization. B-cell production of IgA antibodies thus appears to require T cells expressing the V beta 1 genes, whereas T cells that express the V beta 2 genes lack this capacity. Images PMID:1835793