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Sample records for juxtaglomerular cells requires

  1. Nonfunctioning Juxtaglomerular Cell Tumor

    PubMed Central

    Sakata, Ryoko; Shimoyamada, Hiroaki; Yanagisawa, Masahiro; Murakami, Takayuki; Makiyama, Kazuhide; Nakaigawa, Noboru; Inayama, Yoshiaki; Ohashi, Kenichi; Nagashima, Yoji; Yao, Masahiro; Kubota, Yoshinobu

    2013-01-01

    The juxtaglomerular cell tumor (JGCT) is a rare renal tumor characterized by excessive renin secretion causing intractable hypertension and hypokalemia. However, asymptomatic nonfunctioning JGCT is extremely rare. Here, we report a case of nonfunctioning JGCT in a 31-year-old woman. The patient presented with a left renal tumor without hypertension or hypokalemia. Under a clinical diagnosis of renal cell carcinoma, radical nephrectomy was performed. The tumor was located in the middle portion adjacent to the renal pelvis, measuring 2 cm in size. Pathologically, the tumor was composed of cuboidal cells forming a solid arrangement, immunohistochemically positive for renin. Based on these findings, the tumor was diagnosed as JGCT. In cases with hyperreninism, preoperative diagnosis of JGCT is straightforward but difficult in nonfunctioning case. Generally, JGCT presents a benign biological behavior. Therefore, we should take nonfunctioning JGCT into the differential diagnoses for renal tumors, especially in younger patients to avoid excessive surgery. PMID:23607027

  2. Juxtaglomerular cell tumor: MR findings.

    PubMed

    Agrawal, R; Jafri, S Z; Gibson, D P; Bis, K G; Ali-Reza

    1995-01-01

    Juxtaglomerular (JG) cell tumor is a rare benign neoplasm of the kidney that typically presents with hypertension, secondary hyperaldosteronism, hypocalcemia, and hyperreninism. We describe a case of JG cell tumor diagnosed with MRI.

  3. Hypotonicity-Induced Renin Exocytosis from Juxtaglomerular Cells Requires Aquaporin-1 and Cyclooxygenase-2

    PubMed Central

    Madsen, Kirsten; Svenningsen, Per; Hansen, Pernille B.L.; Gulaveerasingam, Ambika; Jørgensen, Finn; Aalkjær, Christian; Skøtt, Ole; Jensen, Boye L.

    2009-01-01

    The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (Cm) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased Cm significantly and enhanced outward current. Indomethacin, PLA2 inhibition, and an antagonist of prostaglandin transport impaired the Cm and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2−/ − and AQP1−/ − mice, hypotonicity increased neither Cm nor outward current, but 0.1-μM PGE2 increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced Cm and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA2/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin. PMID:19628672

  4. Dendritic Arborization Patterns of Small Juxtaglomerular Cell Subtypes within the Rodent Olfactory Bulb

    PubMed Central

    Bywalez, Wolfgang G.; Ona-Jodar, Tiffany; Lukas, Michael; Ninkovic, Jovica; Egger, Veronica

    2017-01-01

    Within the glomerular layer of the rodent olfactory bulb, numerous subtypes of local interneurons contribute to early processing of incoming sensory information. Here we have investigated dopaminergic and other small local juxtaglomerular cells in rats and mice and characterized their dendritic arborization pattern with respect to individual glomeruli by fluorescent labeling via patching and reconstruction of dendrites and glomerular contours from two-photon imaging data. Dopaminergic neurons were identified in a transgenic mouse line where the expression of dopamine transporter (DAT) was labeled with GFP. Among the DAT+ cells we found a small short-axon cell (SAC) subtype featuring hitherto undescribed dendritic specializations. These densely ramifying structures clasped mostly around somata of other juxtaglomerular neurons, which were also small, non-dopaminergic and to a large extent non-GABAergic. Clasping SACs were observed also in wild-type mice and juvenile rats. In DAT+ SAC dendrites, single backpropagating action potentials evoked robust calcium entry throughout both clasping and non-clasping compartments. Besides clasping SACs, most other small neurons either corresponded to the classical periglomerular cell type (PGCs), which was never DAT+, or were undersized cells with a small dendritic tree and low excitability. Aside from the presence of clasps in SAC dendrites, many descriptors of dendritic morphology such as the number of dendrites and the extent of branching were not significantly different between clasping SACs and PGCs. However, a detailed morphometric analysis in relation to glomerular contours revealed that the dendrites of clasping SACs arborized mostly in the juxtaglomerular space and never entered more than one glomerulus (if at all), whereas most PGC dendrites were restricted to their parent glomerulus, similar to the apical tufts of mitral cells. These complementary arborization patterns might underlie a highly complementary functional

  5. Juxtaglomerular cell tumour as a curable cause of hypertension: case presentation.

    PubMed

    Sierra, Jeremías T; Rigo, Diego; Arancibia, Agustín; Mukdsi, Jorge; Nicolai, Silvia; Ortiz, M Elvira

    2015-01-01

    Arterial hypertension is a highly prevalent disease and its secondary causes must always be kept in mind because the treatment and prognosis differ between these and essential hypertension. Here we present the first reported case in Argentina of a 21-year-old patient with arterial hypertension and hypokalaemia due to a renin-secreting juxtaglomerular cell tumour, which was diagnosed after seven years of development.

  6. Spatial distribution of synapses on tyrosine hydroxylase-expressing juxtaglomerular cells in the mouse olfactory glomerulus.

    PubMed

    Kiyokage, Emi; Kobayashi, Kazuto; Toida, Kazunori

    2017-04-01

    Olfactory sensory axons converge in specific glomeruli where they form excitatory synapses onto dendrites of mitral/tufted (M/T) and juxtaglomerular (JG) cells, including periglomerular (PG), external tufted (ET), and superficial-short axon cells. JG cells consist of heterogeneous subpopulations with different neurochemical, physiological, and morphological properties. Among JG cells, previous electron microscopic (EM) studies have shown that the majority of synaptic inputs to tyrosine hydroxylase (TH)-immunoreactive neurons were asymmetrical synapses from olfactory nerve (ON) terminals. However, recent physiological results revealed that 70% of dopaminergic/γ-aminobutyric acid (GABA)ergic neurons received polysynaptic inputs via ET cells, whereas the remaining 30% received monosynaptic ON inputs. To understand the discrepancies between EM and physiological data, we used serial EM analysis combined with confocal laser scanning microscope images to examine the spatial distribution of synapses on dendrites using mice expressing enhanced green fluorescent protein under the control of the TH promoter. The majority of synaptic inputs to TH-expressing JG cells were from ON terminals, and they preferentially targeted distal dendrites from the soma. On the other hand, the numbers of non-ON inputs were fewer and targeted proximal dendrites. Furthermore, individual TH-expressing JG cells formed serial synapses, such as M/T→TH→another presumed M/T or ON→TH→presumed M/T, but not reciprocal synapses. Serotonergic fibers also associated with somatic regions of TH neurons, displaying non-ON profiles. Thus, fewer proximal non-ON synapses provide more effective inputs than large numbers of distal ON synapses and may occur on the physiologically characterized population of dopaminergic-GABAergic neurons (70%) that receive their most effective inputs indirectly via an ON→ET→TH circuit. J. Comp. Neurol. 525:1059-1074, 2017. © 2017 Wiley Periodicals, Inc.

  7. Radiology of juxtaglomerular tumors

    SciTech Connect

    Dunnick, N.R.; Hartman, D.S.; Ford, K.K.; Davis, C.J. Jr.; Amis, E.S. Jr.

    1983-05-01

    Nine cases of proven juxtaglomerular tumor of the kidney are reviewed. Each patient presented with hypertension; elevated peripheral renin levels were found in four patients. As in past studies, this tumor occurred more frequently in women (7/9 cases). Although the patients tended to be younger (mean age, 31 years) than those with essential hypertension, all but two patients were more than 20 years of age. In all cases, the tumor was solitary, well-defined, and curable by surgery. The tumor was identified by excretory urography in 5/8 patients who underwent this procedure. A solid renal mass was detected in each of the seven patients examined by ultrasound. Since the tumor tends to be isodense with normal renal parenchyma, it is sometimes not seen on computed tomography without intravenouse contrast material. Arteriography revealed a hypovascular mass in each of the nine patients. The combination of a hypovascular solid renal mass in a patient with elevated renin but no renal artery lesions should suggest the diagnosis of a juxtaglomerular cell tumor.

  8. Potassium Currents of Olfactory Bulb Juxtaglomerular Cells: Characterization, Simulation, and Implications for Plateau Potential Firing

    PubMed Central

    Masurkar, Arjun V.; Chen, Wei R.

    2011-01-01

    Odor identity is encoded by the activity of olfactory bulb glomeruli, which receive primary sensory input and transfer it to projection neurons. Juxtaglomerular cells (JGCs) may influence glomerular processing via firing of long lasting plateau potentials. Though inward currents have been investigated, little is known regarding potassium current contribution to JGC plateau potentials. We pursued study of these currents, with the overarching goal of creating components for a computational model of JGC plateau potential firing. In conditions minimizing calcium-activated potassium current (IK(Ca)), we used whole cell voltage clamp and in vitro slice preparations to characterize three potassium currents in rat JGCs. The prominent component Ikt1 displayed rapid kinetics (τ10%−90% rise 0.6–2ms, τinactivation 5–10ms) and was blocked by high concentration 4-AP (5mM) and TEA (40mM). It had half maximal activation at −10mV (V½max) and little inactivation at rest. Ikt2, with slower kinetics (τ10%−90% rise 11–15ms, τinactivation 100–300ms), was blocked by low concentration 4-AP (0.5mM) and TEA (5mM). The V½max was 0mV and inactivation was also minimal at rest. Sustained current Ikt3 showed sensitivity to low concentration 4-AP and TEA, and had V½max of +10mV. Further experiments, in conditions of physiologic calcium buffering, suggested that IK(Ca) contributed to Ikt3 with minimal effect on plateau potential evolution. We transformed these characterizations into Hodgkin-Huxley models that robustly mimicked experimental data. Further simulation demonstrated that Ikt1 would be most efficiently activated by plateau potential waveforms, predicting a critical role in shaping JGC firing. These studies demonstrated that JGCs possess a unique potassium current profile, with delayed rectifier (Ikt3), atypical A-current (Ikt1), and D-current (Ikt2) in accordance with known expression patterns in OB glomeruli. Our simulations also provide an initial framework for

  9. Juxtaglomerular cell CaSR stimulation decreases renin release via activation of the PLC/IP(3) pathway and the ryanodine receptor.

    PubMed

    Ortiz-Capisano, M Cecilia; Reddy, Mahendranath; Mendez, Mariela; Garvin, Jeffrey L; Beierwaltes, William H

    2013-02-01

    The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through G(q) and/or G(i). Activation of G(q) stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP(3)), releasing calcium from intracellular stores. G(i) stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP(3) and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n = 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 ± 7% of control (P < 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 ± 11% of control). The IP(3) inhibitor 2-APB also reversed inhibition of renin from 56 ± 6 to 104 ± 11% of control (P < 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 ± 8 to 118 ± 22% of control (P < 0.01). Combining inhibition of IP(3) and RyR was not additive. G(i) inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 ± 12 to 41 ± 8% of control, P < 0.001). We conclude stimulating JG cell CaSR activates G(q), initiating the PLC/IP(3) pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition.

  10. Fluid flow in the juxtaglomerular interstitium visualized in vivo.

    PubMed

    Rosivall, László; Mirzahosseini, Shahrokh; Toma, Ildikó; Sipos, Arnold; Peti-Peterdi, János

    2006-12-01

    Earlier electron microscopy studies demonstrated morphological signs of fluid flow in the juxtaglomerular apparatus (JGA), including fenestrations of the afferent arteriole (AA) endothelium facing renin granular cells. We aimed to directly visualize fluid flow in the JGA, the putative function of the fenestrated endothelium, using intravital multiphoton microscopy of Munich-Wistar rats and C57BL6 mice. Renin content of the AA correlated strongly with the length of the fenestrated, filtering AA segment. Fluorescence of the extracellular fluid marker lucifer yellow (LY) injected into the cannulated femoral vein in bolus was followed in the renal cortex by real-time imaging. LY was detected in the interstitium around the JG AA before the plasma LY filtered into Bowman's capsule and early proximal tubule. The fluorescence intensity of LY in the JGA interstitium was 17.9 +/- 3.5% of that in the AA plasma (n = 6). The JGA fluid flow was oscillatory, consisting of two components: a fast (one every 5-10 s) and a slow (one every 45-50 s) oscillation, most likely due to the rapid transmission of both the myogenic and tubuloglomerular feedback (TGF)-mediated hemodynamic changes. LY was also detected in the distal tubular lumen about 2-5 s later than in the AA, indicating the flow of JGA interstitial fluid through the macula densa. In the isolated microperfused JGA, blocking the early proximal tubule with a micropipette caused significant increases in MD cell volume by 62 +/- 4% (n = 4) and induced dilation of the intercellular lateral spaces. In summary, significant and dynamic fluid flow exists in the JGA which may help filter the released renin into the renal interstitium (endocrine function). It may also modulate TGF and renin signals in the JGA (hemodynamic function).

  11. Two-photon excitation fluorescence imaging of the living juxtaglomerular apparatus.

    PubMed

    Peti-Peterdi, János; Morishima, Shigeru; Bell, P Darwin; Okada, Yasunobu

    2002-07-01

    Recently, multiphoton excitation fluorescence microscopy has been developed that offers important advantages over confocal imaging, particularly for in vivo visualization of thick tissue samples. We used this state-of-the-art technique to capture high-quality images and study the function of otherwise inaccessible cell types and complex cell structures of the juxtaglomerular apparatus (JGA) in living preparations of the kidney. This structure has multiple cell types that exhibit a complex array of functions, which regulate the process of filtrate formation and renal hemodynamics. We report, for the first time, on high-resolution three-dimensional morphology and Z-sectioning through isolated, perfused kidney glomeruli, tubules, and JGA. Time-series images show how alterations in tubular fluid composition cause striking changes in single-cell volume of the unique macula densa tubular epithelium in situ and how they also affect glomerular filtration through alterations in associated structures within the JGA. In addition, calcium imaging of the glomerulus and JGA demonstrates the utility of this system in capturing the complexity of events and effects that are exerted by the specific hypertensive autacoid angiotensin II. This imaging approach to the study of isolated, perfused live tissue with multiphoton microscopy may be applied to other biological systems in which multiple cell types form a functionally integrated syncytium.

  12. Connexin45 is expressed in the juxtaglomerular apparatus and is involved in the regulation of renin secretion and blood pressure.

    PubMed

    Hanner, Fiona; von Maltzahn, Julia; Maxeiner, Stephan; Toma, Ildiko; Sipos, Arnold; Krüger, Olaf; Willecke, Klaus; Peti-Peterdi, János

    2008-08-01

    Connexin (Cx) proteins are known to play a role in cell-to-cell communication via intercellular gap junction channels or transiently open hemichannels. Previous studies have identified several connexin isoforms in the juxtaglomerular apparatus (JGA), but the vascular connexin isoform Cx45 has not yet been studied in this region. The present work aimed to identify in detail the localization of Cx45 in the JGA and to suggest a functional role for Cx45 in the kidney using conditions where Cx45 expression or function was altered. Using mice that express lacZ coding DNA under the control of the Cx45 promoter, we observed beta-galactosidase staining in cortical vasculature and glomeruli, with specific localization to the JGA region. Renal vascular localization of Cx45 was further confirmed with the use of conditional Cx45-deficient (Cx45fl/fl:Nestin-Cre) mice, which express enhanced green fluorescence protein (EGFP) instead of Cx45 only in cells that, during development, expressed the intermediate filament nestin. EGFP fluorescence was found in the afferent and efferent arteriole smooth muscle cells, in the renin-producing juxtaglomerular cells, and in the extra- and intraglomerular mesangium. Cx45fl/fl:Nestin-Cre mice exhibited increased renin expression and activity, as well as higher systemic blood pressure. The propagation of mechanically induced calcium waves was slower in cultured vascular smooth muscle cells (VSMCs) from Cx45fl/fl:Nestin-Cre mice and in control VSMC treated with a Cx45 gap mimetic peptide that inhibits Cx45 gap junctional communication. VSMCs allowed the cell-to-cell passage of the gap junction permeable dye Lucifer yellow, and calcium wave propagation was not altered by addition of the ATP receptor blocker suramin, suggesting that Cx45 regulates calcium wave propagation via direct gap junction coupling. In conclusion, the localization of Cx45 to the JGA and functional data from Cx45fl/fl:Nestin-Cre mice suggest that Cx45 is involved in the

  13. Disruption of Npr1 gene differentially regulates the juxtaglomerular and distal tubular renin levels in null mutant mice

    PubMed Central

    Prieto, Minolfa C; Das, Subhankar; Somanna, Naveen K; Harrison-Bernard, Lisa M; Navar, L Gabriel; Pandey, Kailash N

    2012-01-01

    Atrial natriuretic peptide (ANP) exerts an inhibitory effect on juxtaglomerular (JG) renin synthesis and release by activating guanylyl cyclase/ natriuretic peptide receptor-A (GC-A/NPRA). Renin has also been localized in connecting tubule cells; however, the effect of ANP/NPRA signaling on tubular renin has not been determined. In the present study, we determined the role of NPRA in regulating both JG and tubular renin using Npr1 (coding for NPRA) gene-disrupted mice, which exhibit a hypertensive phenotype. Renin-positive immunoreactivity in Npr1-/- homozygous null mutant mice was significantly reduced compared with Npr1+/+ wild-type mice (23% vs 69% renin-positive glomeruli). However, after chronic diuretic treatment, Npr1-/- mice showed an increment of JG renin immunoreactivity compared with Npr1+/+ mice (70% vs 81% renin-positive glomeruli). There were no significant differences in the distal tubule renin between Npr1+/+ and Npr1-/- mice. However, after diuretic treatment, Npr1-/- mice showed a significant decrease in renin immunoreactivity in principal cells of cortical collecting ducts (p<0.05). The increased JG renin immunoreactivity after reduction in blood pressure in diuretic-treated Npr1-/- mice, demonstrates an inhibitory action of ANP/NPRA system on JG renin; however, a decreased expression of distal tubular renin suggests a differential effect of ANP/NPRA signaling on JG and distal tubular renin. PMID:23071870

  14. Lipofuscin-like granules of the juxtaglomerular apparatus of the kidney. The diagnostic significance of a quasi-normal subcellular structure incidentally encountered in the course of routine ultrastructural evaluation of renal biopsies.

    PubMed

    Bisceglia, Michele; Carosi, Illuminato; D'Errico, Michele; Di Giorgio, Giuseppe; Castagnoli, Anna; Pasquinelli, Gianandrea

    2011-02-15

    Lipofuscin-like granules, first described by Biava and West in 1965, are a subcellular, quasi-physiologic finding mainly seen in the smooth muscle cells of renal arterioles, but also in juxtaglomerular cells and the lacis cells of human kidneys. They increase in number in subjects affected by arterial hypertension and diabetes. They do not correlate with a specific primary renal disease. Lipofuscin-like granules are not related to renin granules. The world literature on this subject is almost non-existent, and the awareness of this finding or its clinical significance among either pathologists or nephrologists is very poor. We incidentally observed these lipofuscin-like granules in 8 cases during the routine electron microscope examination of 440 renal biopsies, and report herein on their ultrastructural features. Six of these 8 patients were affected by arterial hypertension, one of whom was also concomitantly affected by diabetes mellitus. These lipofuscin-like granules appear as dense bodies with a lipid component, a coarsely granular matrix, and a crystalloid component which may appear in a band or dot pattern, according to the plane of sectioning. The pathologist has to be aware of these lipofuscin-like granules in order not to confuse them with the semicircularly organized (fingerprint) linear immune deposits associated with some specific glomerulopathies.

  15. 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.

  16. 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.

  17. Requirements for high-efficiency solar cells

    NASA Technical Reports Server (NTRS)

    Sah, C. T.

    1986-01-01

    Minimum recombination and low injection level are essential for high efficiency. Twenty percent AM1 efficiency requires a dark recombination current density of 2 x 10 to the minus 13th power A/sq cm and a recombination center density of less than 10 to the 10th power /cu cm. Recombination mechanisms at thirteen locations in a conventional single crystalline silicon cell design are reviewed. Three additional recombination locations are described at grain boundaries in polycrystalline cells. Material perfection and fabrication process optimization requirements for high efficiency are outlined. Innovative device designs to reduce recombination in the bulk and interfaces of single crystalline cells and in the grain boundary of polycrystalline cells are reviewed.

  18. Reovirus Cell Entry Requires Functional Microtubules

    PubMed Central

    Mainou, Bernardo A.; Zamora, Paula F.; Ashbrook, Alison W.; Dorset, Daniel C.; Kim, Kwang S.; Dermody, Terence S.

    2013-01-01

    ABSTRACT Mammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells. PMID:23820395

  19. Laminin is required for Schwann cell morphogenesis.

    PubMed

    Yu, Wei-Ming; Chen, Zu-Lin; North, Alison J; Strickland, Sidney

    2009-04-01

    Development of the peripheral nervous system requires radial axonal sorting by Schwann cells (SCs). To accomplish sorting, SCs must both proliferate and undergo morphogenetic changes such as process extension. Signaling studies reveal pathways that control either proliferation or morphogenesis, and laminin is essential for SC proliferation. However, it is not clear whether laminin is also required for SC morphogenesis. By using a novel time-lapse live-cell-imaging technique, we demonstrated that laminins are required for SCs to form a bipolar shape as well as for process extension. These morphological deficits are accompanied by alterations in signaling pathways. Phosphorylation of Schwannomin at serine 518 and activation of Rho GTPase Cdc42 and Rac1 were all significantly decreased in SCs lacking laminins. Inhibiting Rac1 and/or Cdc42 activities in cultured SCs attenuated laminin-induced myelination, whereas forced activation of Rac1 and/or Cdc42 in vivo improved sorting and hypomyelinating phenotypes in SCs lacking laminins. These findings indicate that laminins play a pivotal role in regulating SC cytoskeletal signaling. Coupled with previous results demonstrating that laminin is critical for SC proliferation, this work identifies laminin signaling as a central regulator coordinating the processes of proliferation and morphogenesis in radial axonal sorting.

  20. Growth requirements of human mammary epithelial cells in culture.

    PubMed

    Taylor-Papadimitriou, J; Shearer, M; Stoker, M G

    1977-12-15

    Colony-forming epithelial cells can be separated from the non-dividing "foam cells" in human milk by differential adhesion to glass and freezing. The growth of such partially purified mammary epithelial cells is stimulated by co-culture with non-dividing feeder cells. Foam cells, mitomycin-treated mouse fibroblast lines and human mammary fibroblasts and calf lens epithelial cells are all effective in promoting mammary epithelial cell growth. Contact between epithelial cells and feeders is not required for the growth-promoting effect. The mitogenic effect of epidermal growth factor on mammary epithelial cells also requires feeder cell activity.

  1. T cell activation requires force generation

    PubMed Central

    Hu, Kenneth H.

    2016-01-01

    Triggering of the T cell receptor (TCR) integrates both binding kinetics and mechanical forces. To understand the contribution of the T cell cytoskeleton to these forces, we triggered T cells using a novel application of atomic force microscopy (AFM). We presented antigenic stimulation using the AFM cantilever while simultaneously imaging with optical microscopy and measuring forces on the cantilever. T cells respond forcefully to antigen after calcium flux. All forces and calcium responses were abrogated upon treatment with an F-actin inhibitor. When we emulated the forces of the T cell using the AFM cantilever, even these actin-inhibited T cells became activated. Purely mechanical stimulation was not sufficient; the exogenous forces had to couple through the TCR. These studies suggest a mechanical–chemical feedback loop in which TCR-triggered T cells generate forceful contacts with antigen-presenting cells to improve access to antigen. PMID:27241914

  2. Glial chain migration requires pioneer cells.

    PubMed

    Aigouy, Benoît; Lepelletier, Léa; Giangrande, Angela

    2008-11-05

    The migration of glial chains along the nerve entails directional and coordinated movement. Despite its importance in the formation of the nervous system, this process remains poorly understood, because of the difficulty of manipulating identified cells. Using confocal time-lapse and cell ablation in the whole animal, we provide direct evidence for a discrete number of Drosophila peripheral glial cells acting as pioneers and guiding the rest of the migratory chain. These cells are in direct contact with several follower cells through a very long and stable cytoplasmic extension. The presence of pioneer cells and homotypic interactions at the tip of the chain allows coordinated movement and the formation of a continuous sheath around the nerve. These in vivo data open novel perspectives for understanding the cellular bases of vertebrate glial migration in physiological and pathological conditions.

  3. 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.

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

    PubMed Central

    Hordichok, Andrew J.; Steyger, Peter S.

    2007-01-01

    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 (6 hours), and allowed to recover (18 hours). 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

  5. Differential requirements for survivin in hematopoietic cell development.

    PubMed

    Gurbuxani, Sandeep; Xu, Yanfei; Keerthivasan, Ganesan; Wickrema, Amittha; Crispino, John D

    2005-08-09

    Although erythroid cells and megakaryocytes arise from a common progenitor, their terminal maturation follows very different paths; erythroid cells undergo cell-cycle exit and enucleation, whereas megakaryocytes continue to progress through the cell cycle but skip late stages of mitosis to become polyploid cells. In our efforts to identify genes that participate in this process, we discovered that survivin, a member of the inhibitor of apoptosis family that also has an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express survivin throughout their maturation, whereas megakaryocytes express approximately 4-fold lower levels of survivin mRNA and no detectable protein. To investigate the role of survivin in these lineages, we overexpressed or knocked down survivin from mouse bone marrow cells and then examined erythroid and megakaryocyte development. These studies revealed that overexpression of survivin antagonized megakaryocyte growth, maturation, and polyploidization but had no effect on erythroid development. This block in polyploidization was accompanied by increased expression of p21 and decreased expression of megakaryocyte genes such as von Willebrand factor and beta(1)-tubulin. In contrast, a reduction in survivin expression interfered with the formation of erythroid cells but not megakaryocytes. Last, consistent with the requirement for survivin in the survival of proliferating cells, survivin-deficient hematopoietic progenitors failed to give rise to either erythroid or megakaryocytic colonies. Together, these studies show that whereas survivin expression is essential for megakaryocyte and erythroid progenitors, its down-regulation is required for terminal differentiation of megakaryocytes.

  6. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI.

    PubMed

    FREIFELDER, D; MAALOE, O

    1964-10-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987-990. 1964.-Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process.

  7. ENERGY REQUIREMENT FOR THYMINELESS DEATH IN CELLS OF ESCHERICHIA COLI

    PubMed Central

    Freifelder, David; Maaløe, Ole

    1964-01-01

    Freifelder, David (University of California, Berkeley), and Ole Maaløe. Energy requirement for thymineless death in cells of Escherichia coli. J. Bacteriol. 88:987–990. 1964.—Thymineless death in thymine-requiring Escherichia coli is arrested immediately and reversibly by nitrogenation if the bacterial population is growing in a medium containing a carbon source that can only be metabolized aerobically. The mechanism of death, therefore, involves a metabolic process. PMID:14219063

  8. Requirements for invasion of epithelial cells by Actinobacillus actinomycetemcomitans.

    PubMed Central

    Sreenivasan, P K; Meyer, D H; Fives-Taylor, P M

    1993-01-01

    Actinobacillus actinomycetemcomitans, an oral bacterium implicated in human periodontal disease, was recently demonstrated to invade cultured epithelial cells (D. H. Meyer, P. K. Sreenivasan, and P. M. Fives-Taylor, Infect. Immun. 59:2719-2726, 1991). This report characterizes the requirements for invasion of KB cells by A. actinomycetemcomitans. The roles of bacterial and host factors were investigated by using selective agents that influence specific bacterial or host cell functions. Inhibition of bacterial protein synthesis decreased invasion, suggesting the absence of a preformed pool of proteins involved in A. actinomycetemcomitans invasion. Inhibition of bacterial and eukaryotic energy synthesis also decreased invasion, confirming that A. actinomycetemcomitans invasion is an active process. Bacterial adherence to KB cells was indicated by scanning electron microscopy of infected KB cells. Further, the addition of A. actinomycetemcomitans-specific serum to the bacterial inoculum reduced invasion substantially, suggesting a role for bacterial attachment in invasion. Many of the adherent bacteria invaded the epithelial cells under optimal conditions. Inhibitors of receptor-mediated endocytosis inhibited invasion by A. actinomycetemcomitans. Like that of many facultatively intracellular bacteria, A. actinomycetemcomitans invasion was not affected by eukaryotic endosomal acidification. These are the first published observations describing the requirements for epithelial cell invasion by a periodontopathogen. They demonstrate that A. actinomycetemcomitans utilizes a mechanism similar to those used by many but not all invasive bacteria to gain entry into eukaryotic cells. Images PMID:8454326

  9. Identifying Francisella tularensis genes required for growth in host cells.

    PubMed

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

    2015-08-01

    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.

  10. 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

  11. ERBB3 is required for metastasis formation of melanoma cells

    PubMed Central

    Tiwary, S; Preziosi, M; Rothberg, P G; Zeitouni, N; Corson, N; Xu, L

    2014-01-01

    Melanoma is curable when it is at an early phase but is lethal once it becomes metastatic. The recent development of BRAFV600E inhibitors (BIs) showed great promise in treating metastatic melanoma, but resistance developed quickly in the treated patients, and these inhibitors are not effective on melanomas that express wild-type BRAF. Alternative therapeutic strategies for metastatic melanoma are urgently needed. Here we report that ERBB3, a member of the epidermal growth factor receptor family, is required for the formation of lung metastasis from both the BI-sensitive melanoma cell line, MA-2, and the BI-resistant melanoma cell line, 451Lu-R. Further analyses revealed that ERBB3 does not affect the initial seeding of melanoma cells in lung but is required for their further development into overt metastases, indicating that ERBB3 might be essential for the survival of melanoma cells after they reach the lung. Consistent with this, the ERBB3 ligand, NRG1, is highly expressed in mouse lungs and induces ERBB3-depdnent phosphorylation of AKT in both MA-2 and 451Lu-R cells in vitro. These findings suggest that ERBB3 may serve as a target for treating metastatic melanomas that are resistant to BIs. In support of this, administration of the pan-ERBB inhibitor, canertinib, significantly suppresses the metastasis formation of BI-resistant melanoma cell lines. PMID:25000258

  12. 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

  13. 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

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

    PubMed Central

    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

    2016-01-01

    ABSTRACT 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

  15. Establishment of Human Papillomavirus Infection Requires Cell Cycle Progression

    PubMed Central

    Pyeon, Dohun; Pearce, Shane M.; Lank, Simon M.; Ahlquist, Paul; Lambert, Paul F.

    2009-01-01

    Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have

  16. Antigen persistence is required for dendritic cell licensing and CD8+ T cell cross-priming.

    PubMed

    Jusforgues-Saklani, Hélène; Uhl, Martin; Blachère, Nathalie; Lemaître, Fabrice; Lantz, Olivier; Bousso, Philippe; Braun, Deborah; Moon, James J; Albert, Matthew L

    2008-09-01

    It has been demonstrated that CD4(+) T cells require Ag persistence to achieve effective priming, whereas CD8(+) T cells are on "autopilot" after only a brief exposure. This finding presents a disturbing conundrum as it does not account for situations in which CD8(+) T cells require CD4(+) T cell help. We used a physiologic in vivo model to study the requirement of Ag persistence for the cross-priming of minor histocompatibility Ag-specific CD8(+) T cells. We report inefficient cross-priming in situations in which male cells are rapidly cleared. Strikingly, the failure to achieve robust CD8(+) T cell activation is not due to a problem with cross-presentation. In fact, by providing "extra help" in the form of dendritic cells (DCs) loaded with MHC class II peptide, it was possible to achieve robust activation of CD8(+) T cells. Our data suggest that the "licensing" of cross-presenting DCs does not occur during their initial encounter with CD4(+) T cells, thus accounting for the requirement for Ag persistence and suggesting that DCs make multiple interactions with CD8(+) T cells during the priming phase. These findings imply that long-lived Ag is critical for efficient vaccination protocols in which the CD8(+) T cell response is helper-dependent.

  17. Epidermal Growth Factor Receptor Cell Survival Signaling Requires Phosphatidylcholine Biosynthesis

    PubMed Central

    Crook, Matt; Upadhyay, Awani; Ido, Liyana J.; Hanna-Rose, Wendy

    2016-01-01

    Identification of pro-cell survival signaling pathways has implications for cancer, cardiovascular, and neurodegenerative disease. We show that the Caenorhabditis elegans epidermal growth factor receptor LET-23 (LET-23 EGFR) has a prosurvival function in counteracting excitotoxicity, and we identify novel molecular players required for this prosurvival signaling. uv1 sensory cells in the C. elegans uterus undergo excitotoxic death in response to activation of the OSM-9/OCR-4 TRPV channel by the endogenous agonist nicotinamide. Activation of LET-23 EGFR can effectively prevent this excitotoxic death. We investigate the roles of signaling pathways known to act downstream of LET-23 EGFR in C. elegans and find that the LET-60 Ras/MAPK pathway, but not the IP3 receptor pathway, is required for efficient LET-23 EGFR activity in its prosurvival function. However, activation of LET-60 Ras/MAPK pathway does not appear to be sufficient to fully mimic LET-23 EGFR activity. We screen for genes that are required for EGFR prosurvival function and uncover a role for phosphatidylcholine biosynthetic enzymes in EGFR prosurvival function. Finally, we show that exogenous application of phosphatidylcholine is sufficient to prevent some deaths in this excitotoxicity model. Our work implicates regulation of lipid synthesis downstream of EGFR in cell survival and death decisions. PMID:27605519

  18. Prenylation is required for polar cell elongation, cell adhesion, and differentiation in Physcomitrella patens.

    PubMed

    Thole, Julie M; Perroud, Pierre-Francois; Quatrano, Ralph S; Running, Mark P

    2014-05-01

    Protein prenylation is required for a variety of growth and developmental processes in flowering plants. Here we report the consequences of loss of function of all known prenylation subunits in the moss Physcomitrella patens. As in Arabidopsis, protein farnesyltransferase and protein geranylgeranyltransferase type I are not required for viability. However, protein geranylgeranyltransferase type I activity is required for cell adhesion, polar cell elongation, and cell differentiation. Loss of protein geranylgeranyltransferase activity results in colonies of round, single-celled organisms that resemble unicellular algae. The loss of protein farnesylation is not as severe but also results in polar cell elongation and differentiation defects. The complete loss of Rab geranylgeranyltransferase activity appears to be lethal in P. patens. Labeling with antibodies to cell wall components support the lack of polarity establishment and the undifferentiated state of geranylgeranyltransferase type I mutant plants. Our results show that prenylated proteins play key roles in P. patens development and differentiation processes.

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

    PubMed

    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

    2015-05-01

    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 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 Src homology 2 domain containing leukocyte protein of 76 kDa (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 phospholipase C γ (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. Because 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 adhesion and degranulation promoting adapter protein or CT10 regulator of kinase/CT10 regulator of kinase-like, which are required for TCR-mediated integrin activation, inhibited Tconv proliferation to a similar extent as wild-type Tregs. Together, these data suggest that TCR-mediated PLCγ activation, but not integrin activation, is required for Tregs to inhibit Tconv proliferation.

  20. Alloantigen presentation by B cells: analysis of the requirement for B-cell activation.

    PubMed Central

    Wilson, J L; Cunningham, A C; Kirby, J A

    1995-01-01

    This paper describes a model for investigation of the functional implications of B-cell activation for antigen presentation. Mixed lymphocyte cultures were used to assess the ability of freshly isolated B cells, mitogen-activated B cells and Epstein-Barr virus (EBV)-transformed B-cell lines to stimulate the activation and proliferation of allogeneic T cells under a variety of experimental conditions. It was found that resting B cells presented antigen poorly, while activated cells were highly immunogenic. Paraformaldehyde fixation completely eliminated antigen presentation by resting B cells, despite constitutive expression of class II MHC antigens. However, fixation had little effect on antigen presentation by activated B cells that expressed B7-1 and B7-2 in addition to class II major histocompatibility complex (MHC) molecules. Arrest of B-cell activation by serial fixation after treatment with F(ab')2 fragments of goat anti-human IgM produced cells with variable antigen-presenting capacity. Optimal antigen presentation was observed for cells fixed 72 hr after the initiation of B-cell activation. Although both B7-1 and B7-2 antigen expression increased after B-cell activation, it was found that the rate of T-cell proliferation correlated most closely with B7-2 expression. Stimulation of T cells by fixed activated B lymphocytes could be blocked by antibodies directed at class II MHC molecules, indicating involvement of the T-cell antigen receptor. In addition, T-cell proliferation was inhibited by antibodies specific for B7-1 and B7-2 and by the fusion protein CTLA4-Ig, demonstrating a requirement for CD28 signal transduction. The sole requirement of B7 family expression for antigen presentation by B lymphocytes was shown by demonstration of T-cell stimulation by fixed resting B cells in the presence of CD28 antibody as a source of artificial costimulation. PMID:8550066

  1. Pancreatic β cell identity requires continual repression of non–β cell programs

    PubMed Central

    Gutiérrez, Giselle Domínguez; Bender, Aaron S.; Cirulli, Vincenzo; Mastracci, Teresa L.; Kelly, Stephen M.; Tsirigos, Aristotelis; Kaestner, Klaus H.

    2016-01-01

    Loss of β cell identity, the presence of polyhormonal cells, and reprogramming are emerging as important features of β cell dysfunction in patients with type 1 and type 2 diabetes. In this study, we have demonstrated that the transcription factor NKX2.2 is essential for the active maintenance of adult β cell identity as well as function. Deletion of Nkx2.2 in β cells caused rapid onset of a diabetic phenotype in mice that was attributed to loss of insulin and downregulation of many β cell functional genes. Concomitantly, NKX2.2-deficient murine β cells acquired non–β cell endocrine features, resulting in populations of completely reprogrammed cells and bihormonal cells that displayed hybrid endocrine cell morphological characteristics. Molecular analysis in mouse and human islets revealed that NKX2.2 is a conserved master regulatory protein that controls the acquisition and maintenance of a functional, monohormonal β cell identity by directly activating critical β cell genes and actively repressing genes that specify the alternative islet endocrine cell lineages. This study demonstrates the highly volatile nature of the β cell, indicating that acquiring and sustaining β cell identity and function requires not only active maintaining of the expression of genes involved in β cell function, but also continual repression of closely related endocrine gene programs. PMID:27941248

  2. Single cell wound generates electric current circuit and cell membrane potential variations that requires calcium influx.

    PubMed

    Luxardi, Guillaume; Reid, Brian; Maillard, Pauline; Zhao, Min

    2014-07-24

    Breaching of the cell membrane is one of the earliest and most common causes of cell injury, tissue damage, and disease. If the compromise in cell membrane is not repaired quickly, irreversible cell damage, cell death and defective organ functions will result. It is therefore fundamentally important to efficiently repair damage to the cell membrane. While the molecular aspects of single cell wound healing are starting to be deciphered, its bio-physical counterpart has been poorly investigated. Using Xenopus laevis oocytes as a model for single cell wound healing, we describe the temporal and spatial dynamics of the wound electric current circuitry and the temporal dynamics of cell membrane potential variation. In addition, we show the role of calcium influx in controlling electric current circuitry and cell membrane potential variations. (i) Upon wounding a single cell: an inward electric current appears at the wound center while an outward electric current is observed at its sides, illustrating the wound electric current circuitry; the cell membrane is depolarized; calcium flows into the cell. (ii) During cell membrane re-sealing: the wound center current density is maintained for a few minutes before decreasing; the cell membrane gradually re-polarizes; calcium flow into the cell drops. (iii) In conclusion, calcium influx is required for the formation and maintenance of the wound electric current circuitry, for cell membrane re-polarization and for wound healing.

  3. 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.

  4. Mast cells are required for phototolerance induction and scratching abatement.

    PubMed

    Schweintzger, Nina A; Bambach, Isabella; Reginato, Eleonora; Mayer, Gerlinde; Limón-Flores, Alberto Y; Ullrich, Stephen E; Byrne, Scott N; Wolf, Peter

    2015-07-01

    Dermal mast cells protect the skin from inflammatory effects of ultraviolet (UV) radiation and are required for UV-induced immune suppression. We sought to determine a potential mechanistic role of mast cells in reducing the sensitivity to UV radiation (i.e. phototolerance induction) through photohardening. We administered single UV exposures as well as a chronic UV irradiation regime to mast cell-deficient Kit(W-Sh/W-Sh) mice and their controls. The chronic irradiation protocol was similar to that given for prophylaxis in certain photodermatoses in humans. Compared to controls, UV-exposed Kit(W-Sh/W-Sh) mice were more susceptible to epidermal hyperplasia and dermal oedema which was linked to blood vessel dilation. Unexpectedly, Kit(W-Sh/W-Sh) mice exhibited an excessive scratching behaviour following broadband UVB plus UVA or solar simulated UV irradiation at doses far below their minimal skin-swelling dose. Protection from this UV-induced scratching phenotype was dependent on mast cells, as engraftment of bone marrow-derived cultured mast cells abated it entirely. Kit(W-Sh/W-Sh) mice were entirely resistant to phototolerance induction by photohardening treatment. Compared to controls, these mice also showed reduced numbers of regulatory T cells and neutrophils in the skin 24 h after UV irradiation. While it is well known that mast cell-deficient mice are resistant to UV-induced immune suppression, we have discovered that they are prone to develop photo-itch and are more susceptible to UV-induced epidermal hyperplasia and skin oedema.

  5. TORC1 is required to balance cell proliferation and cell death in planarians.

    PubMed

    Tu, Kimberly C; Pearson, Bret J; Sánchez Alvarado, Alejandro

    2012-05-15

    Multicellular organisms are equipped with cellular mechanisms that enable them to replace differentiated cells lost to normal physiological turnover, injury, and for some such as planarians, even amputation. This process of tissue homeostasis is generally mediated by adult stem cells (ASCs), tissue-specific stem cells responsible for maintaining anatomical form and function. To do so, ASCs must modulate the balance between cell proliferation, i.e. in response to nutrients, and that of cell death, i.e. in response to starvation or injury. But how these two antagonistic processes are coordinated remains unclear. Here, we explore the role of the core components of the TOR pathway during planarian tissue homeostasis and regeneration and identified an essential function for TORC1 in these two processes. RNAi-mediated silencing of TOR in intact animals resulted in a significant increase in cell death, whereas stem cell proliferation and stem cell maintenance were unaffected. Amputated animals failed to increase stem cell proliferation after wounding and displayed defects in tissue remodeling. Together, our findings suggest two distinct roles for TORC1 in planarians. TORC1 is required to modulate the balance between cell proliferation and cell death during normal cell turnover and in response to nutrients. In addition, it is required to initiate appropriate stem cell proliferation during regeneration and for proper tissue remodeling to occur to maintain scale and proportion.

  6. BMP2 rescues deficient cell migration in Tgfbr3(-/-) epicardial cells and requires Src kinase.

    PubMed

    Allison, Patrick; Espiritu, Daniella; Camenisch, Todd D

    2016-05-03

    During embryogenesis, the epicardium undergoes proliferation, migration, and differentiation into several cardiac cell types which contribute to the coronary vessels. The type III transforming growth factor-β receptor (TGFβR3) is required for epicardial cell invasion and development of coronary vasculature in vivo. Bone Morphogenic Protein-2 (BMP2) is a driver of epicardial cell migration. Utilizing a primary epicardial cell line derived from Tgfbr3(+/+) and Tgfbr3(-/-) mouse embryos, we show that Tgfbr3(-/-) epicardial cells are deficient in BMP2 mRNA expression. Tgfbr3(-/-) epicardial cells are deficient in 2-dimensional migration relative to Tgfbr3(+/+) cells; BMP2 induces cellular migration to Tgfbr3(+/+) levels without affecting proliferation. We further demonstrate that Src kinase activity is required for BMP2 driven Tgfbr3(-/-) migration. BMP2 also requires Src for filamentous actin polymerization in Tgfbr3(-/-) epicardial cells. Taken together, our data identifies a novel pathway in epicardial cell migration required for development of the coronary vessels.

  7. Pronephric Tubulogenesis Requires Daam1-Mediated Planar Cell Polarity Signaling

    PubMed Central

    Gomez de la Torre Canny, Sol; Jang, Chuan-Wei; Cho, Kyucheol; Ji, Hong; Wagner, Daniel S.; Jones, Elizabeth A.; Habas, Raymond

    2011-01-01

    Canonical β-catenin-mediated Wnt signaling is essential for the induction of nephron development. Noncanonical Wnt/planar cell polarity (PCP) pathways contribute to processes such as cell polarization and cytoskeletal modulation in several tissues. Although PCP components likely establish the plane of polarization in kidney tubulogenesis, whether PCP effectors directly modulate the actin cytoskeleton in tubulogenesis is unknown. Here, we investigated the roles of Wnt PCP components in cytoskeletal assembly during kidney tubule morphogenesis in Xenopus laevis and zebrafish. We found that during tubulogenesis, the developing pronephric anlagen expresses Daam1 and its interacting Rho-GEF (WGEF), which compose one PCP/noncanonical Wnt pathway branch. Knockdown of Daam1 resulted in reduced expression of late pronephric epithelial markers with no apparent effect upon early markers of patterning and determination. Inhibiting various points in the Daam1 signaling pathway significantly reduced pronephric tubulogenesis. These data indicate that pronephric tubulogenesis requires the Daam1/WGEF/Rho PCP pathway. PMID:21804089

  8. 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).

  9. Critical Requirement of GABPα for Normal T Cell Development*

    PubMed Central

    Yu, Shuyang; Zhao, Dong-Mei; Jothi, Raja; Xue, Hai-Hui

    2010-01-01

    GA binding protein (GABP) consists of GABPα and GABPβ subunits. GABPα is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPβ does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor α chain (IL-7Rα) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPα conditional knock-out mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPα with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative 3 (DN3) stage, and resulted in reduced expression of T cell receptor (TCR) β chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPα is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPα expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of prearranged TCR but not IL-7Rα can alleviate the DN3 block in GABPα-targeted mice. Our observations collectively indicate that in addition to regulating IL-7Rα expression, GABP is critically required for TCR rearrangements and hence normal T cell development. PMID:20139079

  10. Mitochondrial metabolism in hematopoietic stem cells requires functional FOXO3

    PubMed Central

    Rimmelé, Pauline; Liang, Raymond; Bigarella, Carolina L; Kocabas, Fatih; Xie, Jingjing; Serasinghe, Madhavika N; Chipuk, Jerry; Sadek, Hesham; Zhang, Cheng Cheng; Ghaffari, Saghi

    2015-01-01

    Hematopoietic stem cells (HSC) are primarily dormant but have the potential to become highly active on demand to reconstitute blood. This requires a swift metabolic switch from glycolysis to mitochondrial oxidative phosphorylation. Maintenance of low levels of reactive oxygen species (ROS), a by-product of mitochondrial metabolism, is also necessary for sustaining HSC dormancy. Little is known about mechanisms that integrate energy metabolism with hematopoietic stem cell homeostasis. Here, we identify the transcription factor FOXO3 as a new regulator of metabolic adaptation of HSC. ROS are elevated in Foxo3−/− HSC that are defective in their activity. We show that Foxo3−/− HSC are impaired in mitochondrial metabolism independent of ROS levels. These defects are associated with altered expression of mitochondrial/metabolic genes in Foxo3−/− hematopoietic stem and progenitor cells (HSPC). We further show that defects of Foxo3−/− HSC long-term repopulation activity are independent of ROS or mTOR signaling. Our results point to FOXO3 as a potential node that couples mitochondrial metabolism with HSC homeostasis. These findings have critical implications for mechanisms that promote malignant transformation and aging of blood stem and progenitor cells. PMID:26209246

  11. Cell cycle progression dictates the requirement for BCL2 in natural killer cell survival.

    PubMed

    Viant, Charlotte; Guia, Sophie; Hennessy, Robert J; Rautela, Jai; Pham, Kim; Bernat, Claire; Goh, Wilford; Jiao, Yuhao; Delconte, Rebecca; Roger, Michael; Simon, Vanina; Souza-Fonseca-Guimaraes, Fernando; Grabow, Stephanie; Belz, Gabrielle T; Kile, Benjamin T; Strasser, Andreas; Gray, Daniel; Hodgkin, Phillip D; Beutler, Bruce; Vivier, Eric; Ugolini, Sophie; Huntington, Nicholas D

    2017-02-01

    Natural killer (NK) cells are innate lymphoid cells with antitumor functions. Using an N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a strain with an NK cell deficiency caused by a hypomorphic mutation in the Bcl2 (B cell lymphoma 2) gene. Analysis of these mice and the conditional deletion of Bcl2 in NK cells revealed a nonredundant intrinsic requirement for BCL2 in NK cell survival. In these mice, NK cells in cycle were protected against apoptosis, and NK cell counts were restored in inflammatory conditions, suggesting a redundant role for BCL2 in proliferating NK cells. Consistent with this, cycling NK cells expressed higher MCL1 (myeloid cell leukemia 1) levels in both control and BCL2-null mice. Finally, we showed that deletion of BIM restored survival in BCL2-deficient but not MCL1-deficient NK cells. Overall, these data demonstrate an essential role for the binding of BCL2 to BIM in the survival of noncycling NK cells. They also favor a model in which MCL1 is the dominant survival protein in proliferating NK cells.

  12. 9 CFR 113.52 - Requirements for cell lines 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 cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  13. 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...

  14. 9 CFR 113.52 - Requirements for cell lines 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 cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  15. 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...

  16. 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...

  17. 9 CFR 113.52 - Requirements for cell lines 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 cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  18. 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...

  19. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 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. 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... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  2. What makes cells move: requirements and obstacles for spontaneous cell motility.

    PubMed

    Binamé, Fabien; Pawlak, Geraldine; Roux, Pierre; Hibner, Urszula

    2010-04-01

    Movement of individual cells and of cellular cohorts, chains or sheets requires physical forces that are established through interactions of cells with their environment. In vivo, migration occurs extensively during embryonic development and in adults during wound healing and tumorigenesis. In order to identify the molecular events involved in cell movement, in vitro systems have been developed. These have contributed to the definition of a number of molecular pathways put into play in the course of migratory behaviours, such as mesenchymal and amoeboid movement. More recently, our knowledge of migratory modes has been enriched by analyses of cells exploring and moving through three-dimensional (3D) matrices. While the cells' morphologies differ in 2D and 3D environments, the basic mechanisms that put a cellular body into motion are remarkably similar. Thus, in both 2D and 3D, the polarity of the migrating cell is initially defined by a specific subcellular localization of signalling molecules and components of molecular machines required for motion. While the polarization can be initiated either in response to extracellular signalling or be a chance occurrence, it is reinforced and sustained by positive feedback loops of signalling molecules. Second, adhesion to a substratum is necessary to generate forces that will propel the cell engaged in either mesenchymal or ameboid migration. For collective cell movement, intercellular coordination constitutes an additional requirement: a cell cohort remains stationary if individual cells pull in opposite directions. Finally, the availability of space to move into is a general requirement to set cells into motion. Lack of free space is probably the main obstacle for migration of most healthy cells in an adult multicellular organism. Thus, the requirements for cell movement are both intrinsic to the cell, involving coordinated signalling and interactions with molecular machines, and extrinsic, imposed by the physicochemical

  3. Fuel processing requirements and techniques for fuel cell propulsion power

    SciTech Connect

    Kumar, R.; Ahmed, S.; Yu, M.

    1993-08-01

    Fuels for fuel cells in transportation systems are likely to be methanol, natural gas, hydrogen, propane, or ethanol. Fuels other than hydrogen wig need to be reformed to hydrogen on-board the vehicle. The fuel reformer must meet stringent requirements for weight and volume, product quality, and transient operation. It must be compact and lightweight, must produce low levels of CO and other byproducts, and must have rapid start-up and good dynamic response. Catalytic steam reforming, catalytic or noncatalytic partial oxidation reforming, or some combination of these processes may be used. This paper discusses salient features of the different kinds of reformers and describes the catalysts and processes being examined for the oxidation reforming of methanol and the steam reforming of ethanol. Effective catalysts and reaction conditions for the former have been identified; promising catalysts and reaction conditions for the latter are being investigated.

  4. An unconventional myosin required for cell polarization and chemotaxis.

    PubMed

    Breshears, Laura M; Wessels, Deborah; Soll, David R; Titus, Margaret A

    2010-04-13

    MyTH/FERM (myosin tail homology 4/band 4.1, ezrin, radixin, and moesin) myosins have roles in cellular adhesion, extension of actin-filled projections such as filopodia and stereocilia, and directional migration. The amoeba Dictyostelium discoideum expresses a simple complement of MyTH/FERM myosins, a class VII (M7) myosin required for cell-substrate adhesion and a unique myosin named MyoG. Mutants lacking MyoG exhibit a wide range of normal actin-based behaviors, including chemotaxis to folic acid, but have a striking defect in polarization and chemotaxis to cAMP. Although the myoG mutants respond to cAMP stimulation by increasing persistence and weakly increasing levels of cortical F-actin, they do not polarize; instead, they maintain a round shape and move slowly and randomly when exposed to a chemotactic gradient. The mutants also fail to activate and localize PI3K to the membrane closest to the source of chemoattractant. These data reveal a role for a MyTH/FERM myosin in mediating early chemotactic signaling and suggest that MyTH/FERM proteins have conserved roles in signaling and the generation of cell polarity.

  5. 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.

  6. Bacterial Cell Enlargement Requires Control of Cell Wall Stiffness Mediated by Peptidoglycan Hydrolases

    PubMed Central

    Wheeler, Richard; Turner, Robert D.; Bailey, Richard G.; Salamaga, Bartłomiej; Mesnage, Stéphane; Mohamad, Sharifah A. S.; Hayhurst, Emma J.; Horsburgh, Malcolm; Hobbs, Jamie K.

    2015-01-01

    ABSTRACT Most bacterial cells are enclosed in a single macromolecule of the cell wall polymer, peptidoglycan, which is required for shape determination and maintenance of viability, while peptidoglycan biosynthesis is an important antibiotic target. It is hypothesized that cellular enlargement requires regional expansion of the cell wall through coordinated insertion and hydrolysis of peptidoglycan. Here, a group of (apparent glucosaminidase) peptidoglycan hydrolases are identified that are together required for cell enlargement and correct cellular morphology of Staphylococcus aureus, demonstrating the overall importance of this enzyme activity. These are Atl, SagA, ScaH, and SagB. The major advance here is the explanation of the observed morphological defects in terms of the mechanical and biochemical properties of peptidoglycan. It was shown that cells lacking groups of these hydrolases have increased surface stiffness and, in the absence of SagB, substantially increased glycan chain length. This indicates that, beyond their established roles (for example in cell separation), some hydrolases enable cellular enlargement by making peptidoglycan easier to stretch, providing the first direct evidence demonstrating that cellular enlargement occurs via modulation of the mechanical properties of peptidoglycan. PMID:26220963

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

    NASA Astrophysics Data System (ADS)

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

    2013-06-01

    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.

  8. Simvastatin requires activation in accessory cells to modulate T-cell responses in asthma and COPD.

    PubMed

    Knobloch, Jürgen; Yakin, Yakup; Körber, Sandra; Grensemann, Barbara; Bendella, Zeynep; Boyaci, Niyazi; Gallert, Willem-Jakob; Yanik, Sarah Derya; Jungck, David; Koch, Andrea

    2016-10-05

    T-cell-dependent airway and systemic inflammation triggers the progression of chronic obstructive pulmonary disease (COPD) and asthma. Retrospective studies suggest that simvastatin has anti-inflammatory effects in both diseases but it is unclear, which cell types are targeted. We hypothesized that simvastatin modulates T-cell activity. Circulating CD4+ and CD8+ T-cells, either pure, co-cultured with monocytes or alveolar macrophages (AM) or in peripheral blood mononuclear cells (PBMCs), were ex vivo activated towards Th1/Tc1 or Th2/Tc2 and incubated with simvastatin. Markers for Th1/Tc1 (IFNγ) and Th2/Tc2 (IL-5, IL-13) were measured by ELISA; with PBMCs this was done comparative between 11 healthy never-smokers, 11 current smokers without airflow limitation, 14 smokers with COPD and 11 never-smokers with atopic asthma. T-cell activation induced IFNγ, IL-5 and IL-13 in the presence and absence of accessory cells. Simvastatin did not modulate cytokine expression in pure T-cell fractions. β-hydroxy-simvastatin acid (activated simvastatin) suppressed IL-5 and IL-13 in pure Th2- and Tc2-cells. Simvastatin suppressed IL-5 and IL-13 in Th2-cells co-cultivated with monocytes or AM, which was partially reversed by the carboxylesterase inhibitor benzil. Simvastatin suppressed IL-5 production of Th2/Tc2-cells in PBMCs without differences between cohorts and IL-13 stronger in never-smokers and asthma compared to COPD. Simvastatin induced IFNγ in Th1/Tc1-cells in PBMCs of all cohorts except asthmatics. Simvastatin requires activation in accessory cells likely by carboxylesterase to suppress IL-5 and IL-13 in Th2/Tc2-cells. The effects on Il-13 are partially reduced in COPD. Asthma pathogenesis prevents simvastatin-induced IFNγ up-regulation. Simvastatin has anti-inflammatory effects that could be of interest for asthma therapy.

  9. Distinct Requirements for HIV-Cell Fusion and HIV-mediated Cell-Cell Fusion*

    PubMed Central

    Kondo, Naoyuki; Marin, Mariana; Kim, Jeong Hwa; Desai, Tanay M.; Melikyan, Gregory B.

    2015-01-01

    Whether HIV-1 enters cells by fusing with the plasma membrane or with endosomes is a subject of active debate. The ability of HIV-1 to mediate fusion between adjacent cells, a process referred to as “fusion-from-without” (FFWO), shows that this virus can fuse with the plasma membrane. To compare FFWO occurring at the cell surface with HIV-cell fusion through a conventional entry route, we designed an experimental approach that enabled the measurements of both processes in the same sample. The following key differences were observed. First, a very small fraction of viruses fusing with target cells participated in FFWO. Second, whereas HIV-1 fusion with adherent cells was insensitive to actin inhibitors, post-CD4/coreceptor binding steps during FFWO were abrogated. A partial dependence of HIV-cell fusion on actin remodeling was observed in CD4+ T cells, but this effect appeared to be due to the actin dependence of virus uptake. Third, deletion of the cytoplasmic tail of HIV-1 gp41 dramatically enhanced the ability of the virus to promote FFWO, while having a modest effect on virus-cell fusion. Distinct efficiencies and actin dependences of FFWO versus HIV-cell fusion are consistent with the notion that, except for a minor fraction of particles that mediate fusion between the plasma membranes of adjacent cells, HIV-1 enters through an endocytic pathway. We surmise, however, that cell-cell contacts enabling HIV-1 fusion with the plasma membrane could be favored at the sites of high density of target cells, such as lymph nodes. PMID:25589785

  10. Genetic Requirements for the Transformation of Human Cells

    DTIC Science & Technology

    2004-07-01

    apoptosis indicate that EIAACR2 can be functionally complemented cyclin E expression (A. Samuelson and S. Lowe, personal communication.) We therefore 4 SEGER...oncogenes such as c-myc or adenovirus El A sensi- moting factors, are not restricted by cell-cell contact, and are tizes primary cells to apoptosis (Debbas...which many cells undergo apoptosis or senes- cence. Cells that emerged from this crisis event IP-actin become telomerase-positive (ERM P.C.). The in

  11. Cell-autonomous requirements for Dlg-1 for lens epithelial cell structure and fiber cell morphogenesis.

    PubMed

    Rivera, Charlene; Yamben, Idella F; Shatadal, Shalini; Waldof, Malinda; Robinson, Michael L; Griep, Anne E

    2009-09-01

    Cell polarity and adhesion are thought to be key determinants in organismal development. In Drosophila, discs large (dlg) has emerged as an important regulator of epithelial cell proliferation, adhesion, and polarity. Herein, we investigated the role of the mouse homolog of dlg (Dlg-1) in the development of the mouse ocular lens. Tissue-specific ablation of Dlg-1 throughout the lens early in lens development led to an expansion and disorganization of the epithelium that correlated with changes in the distribution of adhesion and polarity factors. In the fiber cells, differentiation defects were observed. These included alterations in cell structure and the disposition of cell adhesion/cytoskeletal factors, delay in denucleation, and reduced levels of alpha-catenin, pERK1/2, and MIP26. These fiber cell defects were recapitulated when Dlg-1 was disrupted only in fiber cells. These results suggest that Dlg-1 acts in a cell autonomous manner to regulate epithelial cell structure and fiber cell differentiation.

  12. Isolation of Chinese hamster ovary cell mutants requiring the continuous presence of taxol for cell division

    PubMed Central

    1983-01-01

    Chinese hamster ovary (CHO) cell mutants resistant to the cytotoxic effects of taxol and requiring the drug for normal growth were isolated in a single step. One of these mutant cell lines, Tax-18, fails to divide in the absence of taxol; instead, the cells become larger, rounder, flatter, and multinucleated. Analysis by flow cytometry indicates that during taxol deprivation there is an accumulation of cells in G2 + M phase but that the cells are able to leak through the block in the absence of cell division and further increase their DNA content beyond the tetraploid amount. This interpretation is confirmed by karyotype analysis and by time-lapse studies that show cells rounded for mitosis two to five times longer than in wild-type cultures or in Tax-18 cultures grown in taxol. The cells finally attempt to undergo cytokinesis, fail, and spread out again, but as larger cells than before. Tax-18 has a normal growth rate and morphology when grown in taxol even at concentrations three to five times below the selecting concentration of the drug. The cells, however, have increased sensitivity to microtubule-disrupting drugs such as colcemid, griseofulvin, and D2O. The mutation for taxol auxotrophy behaves recessively in somatic cell hybridization experiments, and the phenotypic reversion rate is approximately 10(-5) in a nonmutagenized population. Both alpha- and beta-tubulin are present in apparently normal amounts and with normal electrophoretic mobilities on two- dimensional gels. The results suggest that Tax-18 lacks a factor necessary for mitosis and that taxol may be able to substitute for this factor. PMID:6134736

  13. Six3 is required for ependymal cell maturation.

    PubMed

    Lavado, Alfonso; Oliver, Guillermo

    2011-12-01

    Ependymal cells are part of the neurogenic niche in the adult subventricular zone of the lateral ventricles, where they regulate neurogenesis and neuroblast migration. Ependymal cells are generated from radial glia cells during embryonic brain development and acquire their final characteristics postnatally. The homeobox gene Six3 is expressed in ependymal cells during the formation of the lateral wall of the lateral ventricles in the brain. Here, we show that Six3 is necessary for ependymal cell maturation during postnatal stages of brain development. In its absence, ependymal cells fail to suppress radial glia characteristics, resulting in a defective lateral wall, abnormal neuroblast migration and differentiation, and hydrocephaly.

  14. Natural killer cells require monocytic Gr-1(+)/CD11b(+) myeloid cells to eradicate orthotopically engrafted glioma cells.

    PubMed

    Baker, Gregory J; Chockley, Peter; Zamler, Daniel; Castro, Maria G; Lowenstein, Pedro R

    2016-06-01

    Malignant gliomas are resistant to natural killer (NK) cell immune surveillance. However, the mechanisms used by these cancers to suppress antitumor NK cell activity remain poorly understood. We have recently reported on a novel mechanism of innate immune evasion characterized by the overexpression of the carbohydrate-binding protein galectin-1 by both mouse and rat malignant glioma. Here, we investigate the cytokine profile of galectin-1-deficient GL26 cells and describe the process by which these tumors are targeted by the early innate immune system in RAG1(-/-) and C57BL/6J mice. Our data reveal that galectin-1 knockdown in GL26 cells heightens their inflammatory status leading to the rapid recruitment of Gr-1(+)/CD11b(+) myeloid cells and NK1.1(+) NK cells into the brain tumor microenvironment, culminating in tumor clearance. We show that immunodepletion of Gr-1(+) myeloid cells in RAG1(-/-) mice permits the growth of galectin-1-deficient glioma despite the presence of NK cells, thus demonstrating an essential role for myeloid cells in the clearance of galectin-1-deficient glioma. Further characterization of tumor-infiltrating Gr-1(+)/CD11b(+) cells reveals that these cells also express CCR2 and Ly-6C, markers consistent with inflammatory monocytes. Our results demonstrate that Gr-1(+)/CD11b(+) myeloid cells, often referred to as myeloid-derived suppressor cells (MDSCs), are required for antitumor NK cell activity against galectin-1-deficient GL26 glioma. We conclude that glioma-derived galectin-1 represents an important factor in dictating the phenotypic behavior of monocytic Gr-1(+)/CD11b(+) myeloid cells. Galectin-1 suppression may be a valuable treatment approach for clinical glioma by promoting their innate immune-mediated recognition and clearance through the concerted effort of innate myeloid and lymphoid cell lineages.

  15. Requirements for effective antitumor responses of TCR transduced T cells.

    PubMed

    de Witte, Moniek A; Jorritsma, Annelies; Kaiser, Andrew; van den Boom, Marly D; Dokter, Maarten; Bendle, Gavin M; Haanen, John B A G; Schumacher, Ton N M

    2008-10-01

    Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.

  16. 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...

  17. 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...

  18. 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...

  19. Haematopoietic stem cells require a highly regulated protein synthesis rate.

    PubMed

    Signer, Robert A J; Magee, Jeffrey A; Salic, Adrian; Morrison, Sean J

    2014-05-01

    Many aspects of cellular physiology remain unstudied in somatic stem cells, for example, there are almost no data on protein synthesis in any somatic stem cell. Here we set out to compare protein synthesis in haematopoietic stem cells (HSCs) and restricted haematopoietic progenitors. We found that the amount of protein synthesized per hour in HSCs in vivo was lower than in most other haematopoietic cells, even if we controlled for differences in cell cycle status or forced HSCs to undergo self-renewing divisions. Reduced ribosome function in Rpl24(Bst/+) mice further reduced protein synthesis in HSCs and impaired HSC function. Pten deletion increased protein synthesis in HSCs but also reduced HSC function. Rpl24(Bst/+) cell-autonomously rescued the effects of Pten deletion in HSCs; blocking the increase in protein synthesis, restoring HSC function, and delaying leukaemogenesis. Pten deficiency thus depletes HSCs and promotes leukaemia partly by increasing protein synthesis. Either increased or decreased protein synthesis impairs HSC function.

  20. Reprogramming Human Endothelial to Hematopoietic Cells Requires Vascular Induction

    PubMed Central

    Sandler, Vladislav M.; Lis, Raphael; Liu, Ying; Kedem, Alon; James, Daylon; Elemento, Olivier; Butler, Jason M.; Scandura, Joseph M.; Rafii, Shahin

    2014-01-01

    Summary Generating engraftable human hematopoietic cells from autologous tissues promises new therapies for blood diseases. Directed differentiation of pluripotent stem cells yields hematopoietic cells that poorly engraft. Here, we devised a method to phenocopy the vascular-niche microenvironment of hemogenic cells, thereby enabling reprogramming of human endothelial cells (ECs) into engraftable hematopoietic cells without transition through a pluripotent intermediate. Highly purified non-hemogenic human umbilical vein-ECs (HUVECs) or adult dermal microvascular ECs (hDMECs) were transduced with transcription factors (TFs), FOSB, GFI1, RUNX1, and SPI1 (FGRS), and then propagated on serum-free instructive vascular niche monolayers to induce outgrowth of hematopoietic colonies containing cells with functional and immunophenotypic features of multipotent progenitor cells (MPP). These reprogrammed ECs- into human-MPPs (rEC-hMPPs) acquire colony-forming cell (CFC) potential and durably engraft in immune-deficient mice after primary and secondary transplantation, producing long-term rEC-hMPP-derived myeloid (granulocytic/monocytic, erythroid, megakaryocytic) and lymphoid (NK, B) progeny. Conditional expression of FGRS transgenes, combined with vascular-induction, activates endogenous FGRS genes endowing rEC-hMPPs with a transcriptional and functional profile similar to self-renewing MPPs. Our approach underscores the role of inductive cues from vascular-niche in orchestrating and sustaining hematopoietic specification and may prove useful for engineering autologous hematopoietic grafts to treat inherited and acquired blood disorders. PMID:25030167

  1. Glutathione Peroxidase 4 Is Required for Maturation of Photoreceptor Cells*

    PubMed Central

    Ueta, Takashi; Inoue, Tatsuya; Furukawa, Takahisa; Tamaki, Yasuhiro; Nakagawa, Yasuhito; Imai, Hirotaka; Yanagi, Yasuo

    2012-01-01

    Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells. PMID:22207760

  2. 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

    2015-08-07

    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.

  3. 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

  4. YY1 Is Required for Germinal Center B Cell Development.

    PubMed

    Banerjee, Anupam; Sindhava, Vishal; 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.

  5. 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.

  6. 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

  7. Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium

    PubMed Central

    Bastide, Pauline; Darido, Charbel; Pannequin, Julie; Kist, Ralf; Robine, Sylvie; Marty-Double, Christiane; Bibeau, Frédéric; Scherer, Gerd; Joubert, Dominique; Hollande, Frédéric; Blache, Philippe; Jay, Philippe

    2007-01-01

    The HMG-box transcription factor Sox9 is expressed in the intestinal epithelium, specifically, in stem/progenitor cells and in Paneth cells. Sox9 expression requires an active β-catenin–Tcf complex, the transcriptional effector of the Wnt pathway. This pathway is critical for numerous aspects of the intestinal epithelium physiopathology, but processes that specify the cell response to such multipotential signals still remain to be identified. We inactivated the Sox9 gene in the intestinal epithelium to analyze its physiological function. Sox9 inactivation affected differentiation throughout the intestinal epithelium, with a disappearance of Paneth cells and a decrease of the goblet cell lineage. Additionally, the morphology of the colon epithelium was severely altered. We detected general hyperplasia and local crypt dysplasia in the intestine, and Wnt pathway target genes were up-regulated. These results highlight the central position of Sox9 as both a transcriptional target and a regulator of the Wnt pathway in the regulation of intestinal epithelium homeostasis. PMID:17698607

  8. Drosophila dyskerin is required for somatic stem cell homeostasis.

    PubMed

    Vicidomini, Rosario; Petrizzo, Arianna; di Giovanni, Annamaria; Cassese, Laura; Lombardi, Antonella Anna; Pragliola, Caterina; Furia, Maria

    2017-03-23

    Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, we used this organism to investigate the specific role played by dyskerin in somatic stem cell maintenance. To this aim, we focussed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, a few dispersed precursors found in the depleted midguts can maintain stem identity and the ability to divide asymmetrically, nor show cell-growth defects or undergo apoptosis. Instead, their loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction.

  9. 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.

  10. Piwi is required in multiple cell types to control germline stem cell lineage development in the Drosophila ovary.

    PubMed

    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.

  11. LagC is required for cell-cell interactions that are essential for cell-type differentiation in Dictyostelium.

    PubMed

    Dynes, J L; Clark, A M; Shaulsky, G; Kuspa, A; Loomis, W F; Firtel, R A

    1994-04-15

    Strain AK127 is a developmental mutant of Dictyostelium discoideum that was isolated by restriction enzyme-mediated integration (REMI). Mutant cells aggregate normally but are unable to proceed past the loose aggregate stage. The cloned gene, lagC (loose aggregate C), encodes a novel protein of 98 kD that contains an amino-terminal signal sequence and a putative carboxy-terminal transmembrane domain. The mutant strain AK127 shows no detectable lagC transcript upon Northern analysis, indicating that the observed phenotype is that of a null allele. Expression of the lagC cDNA in AK127 cells complements the arrest at the loose aggregate stage, indicating that the mutant phenotype results from disruption of the lagC gene. In wild-type cells, lagC mRNA is induced at the loose aggregate stage and is expressed through the remainder of development. lagC- null cells aggregate but then disaggregate and reaggregate to form small granular mounds. Mature spores are produced at an extremely low efficiency (< 0.1% of wild type), appearing only after approximately 72 hr, whereas wild-type strains produce mature spores by 26 hr. lagC- null cells accumulate reduced levels of transcripts for the prestalk-enriched genes rasD and CP2 and do not express the DIF-induced prestalk-specific gene ecmA or the cAMP-induced prespore-specific gene SP60 to significant levels. In chimeric organisms resulting from the coaggregation of lagC- null and wild-type cells, cell-type-specific gene expression is rescued in the lagC- null cells; however, lagC- prespore cells are localized to the posterior of the prespore region and do not form mature spores, suggesting that LagC protein has both no cell-autonomous and cell-autonomous functions. Overexpression of lagC from an actin promoter in both wild-type and lagC- cells causes a delay at the tight aggregate stage, the first stage requiring LagC activity. These results suggest that the LagC protein functions as a nondiffusible cell-cell signaling molecule

  12. FIP200 is required for the cell-autonomous maintenance of fetal hematopoietic stem cells.

    PubMed

    Liu, Fei; Lee, Jae Y; Wei, Huijun; Tanabe, Osamu; Engel, James D; Morrison, Sean J; Guan, Jun-Lin

    2010-12-02

    Little is known about whether autophagic mechanisms are active in hematopoietic stem cells (HSCs) or how they are regulated. FIP200 (200-kDa FAK-family interacting protein) plays important roles in mammalian autophagy and other cellular functions, but its role in hematopoietic cells has not been examined. Here we show that conditional deletion of FIP200 in hematopoietic cells leads to perinatal lethality and severe anemia. FIP200 was cell-autonomously required for the maintenance and function of fetal HSCs. FIP200-deficient HSC were unable to reconstitute lethally irradiated recipients. FIP200 ablation did not result in increased HSC apoptosis, but it did increase the rate of HSC proliferation. Consistent with an essential role for FIP200 in autophagy, FIP200-null fetal HSCs exhibited both increased mitochondrial mass and reactive oxygen species. These data identify FIP200 as a key intrinsic regulator of fetal HSCs and implicate a potential role for autophagy in the maintenance of fetal hematopoiesis and HSCs.

  13. Specific requirement for CD3ɛ in T cell development

    PubMed Central

    DeJarnette, Jan B.; Sommers, Connie L.; Huang, Kun; Woodside, Kenneth J.; Emmons, Rebecca; Katz, Kenneth; Shores, Elizabeth W.; Love, Paul E.

    1998-01-01

    T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3γ, -δ, -ɛ, and ζ). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3γ, -δ, or ζ results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3ɛ−/− mice, and thymocyte development is arrested at the early CD4−CD8− stage. Although these results suggest that CD3ɛ is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3γ and CD3δ genes also is reduced in CD3ɛ−/− mice. Thus, it is unclear whether the phenotype of CD3ɛ−/− mice reflects the collective effects of CD3γ, -δ, and -ɛ deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3ɛ gene via Cre/loxP-mediated recombination, we generated mice that lack CD3ɛ yet retain normal expression of the closely linked CD3γ and CD3δ genes. These (CD3ɛΔ/Δ) mice exhibited an early arrest in T cell development, similar to that of CD3ɛ−/− mice. Moreover, the developmental defect could be rescued by expression of a CD3ɛ transgene. These results identify an essential role for CD3ɛ in T cell development not shared by the CD3γ, CD3δ, or ζ-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity. PMID:9843989

  14. Transferrin is required for early T-cell differentiation.

    PubMed

    Macedo, M Fatima; de Sousa, Maria; Ned, Renee M; Mascarenhas, Claudia; Andrews, Nancy C; Correia-Neves, Margarida

    2004-08-01

    Transferrin, the major plasma iron carrier, mediates iron entry into cells through interaction with its receptor. Several in vitro studies have demonstrated that transferrin plays an essential role in lymphocyte division, a role attributed to its iron transport function. In the present study we used hypotransferrinaemic (Trf(hpx/hpx)) mice to investigate the possible involvement of transferrin in T lymphocyte differentiation in vivo. The absolute number of thymocytes was substantially reduced in Trf(hpx/hpx) mice, a result that could not be attributed to increased apoptosis. Moreover, the proportions of the four major thymic subpopulations were maintained and the percentage of dividing cells was not reduced. A leaky block in the differentiation of CD4(-) CD8(-) CD3(-) CD44(-) CD25(+) (TN3) into CD4(-) CD8(-) CD3(-) CD44(-) CD25(-) (TN4) cells was observed. In addition, a similar impairment of early thymocyte differentiation was observed in mice with reduced levels of transferrin receptor. The present study demonstrates, for the first time, that transferrin itself or a pathway triggered by the interaction of transferrin with its receptor is essential for normal early T-cell differentiation in vivo.

  15. Th17 cells are not required for maintenance of IL-17A producing γδ T cells in vivo

    PubMed Central

    Gupta, Pawan K.; Wagner, Sarah R.; Wu, Qiang; Shilling, Rebecca A.

    2016-01-01

    γδ T cells producing IL-17A (γδT17) are thought to develop spontaneously in the thymus and to be maintained in the periphery. Previous studies suggested a role for Th17 cells in the maintenance of γδT17 via the expression of TGFβ1. However, we have previously found that Th17 cells were not required for expansion of γδT17 cells after lung transplant in a mouse model. Using mice deficient in STAT3 in CD4+ T cells, which are unable to develop Th17 cells, we investigated the requirement for Th17 cells and TGFβ1 to maintain γδT17 cells in the lung and lymphoid tissues. At steady state, we found no defect in γδT17 cells in the thymus or periphery of these mice. Further, STAT3-deficient CD4+ T cells produced significantly higher levels of TGFβ1 than wild-type CD4+ T cells under Th17 differentiation conditions in vitro. To determine whether STAT3-deficient CD4+ T cells could expand γδT17 cells in vivo, we used TCRβ−/− mice, which are known to have a defect in γδT17 cells that can be rescued by Th17 cells. However, adoptive transfer of wild-type Th17 cells or bulk CD4+ T cells did not expand γδT17 cells in TCRβ−/− mice. In contrast, IFN-γ+ γδ T cells preferentially expanded, particularly in the lungs. Interestingly, we found in vivo and in vitro that TGFβ1 may negatively regulate the pool of γδT17 cells. Our data suggest that Th17 cells and TGFβ1 are not required for the maintenance of γδT17 cells. PMID:27649780

  16. A conformational switch controls cell wall-remodelling enzymes required for bacterial cell division.

    PubMed

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

    2012-08-01

    Remodelling of the peptidoglycan (PG) exoskeleton is intimately tied to the growth and division of bacteria. Enzymes that hydrolyse 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 orthologue 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 autoinhibition governed by conserved subcomplexes within the cytokinetic ring. Analogous conformational control mechanisms are likely to be part of a general strategy used to control PG hydrolases present within multienzyme PG-remodelling machines.

  17. Structural requirement(s) of N-phenylthioureas and benzaldehyde thiosemicarbazones as inhibitors of melanogenesis in melanoma B 16 cells.

    PubMed

    Thanigaimalai, P; Hoang, Tuan Anh Le; Lee, Ki-Cheul; Bang, Seong-Cheol; Sharma, Vinay K; Yun, Cheong-Yong; Roh, Eunmiri; Hwang, Bang-Yeon; Kim, Youngsoo; Jung, Sang-Hun

    2010-05-01

    In order to define the structural requirements of phenylthiourea (PTU), a series of thiourea and thiosemicarbazone analogs were prepared and evaluated as inhibitors of melanogenesis in melanoma B16 cells. The most potent analog was 2-(4-tert-butylbenzylidene)hydrazinecarbothioamide (1u) with an IC(50) value of 2.7 microM in inhibition of melanogenesis. The structure for potent inhibitory activity of these derivatives are required with the direct connection of pi-planar structure to thiourea without steric hinderance in PTU derivatives and the hydrophobic substituent at para position in case of semicarbazones.

  18. Design requirements for high-efficiency high concentration ratio space solar cells

    NASA Technical Reports Server (NTRS)

    Rauschenbach, H.; Patterson, R.

    1980-01-01

    A miniaturized Cassegrainian concentrator system concept was developed for low cost, multikilowatt space solar arrays. The system imposes some requirements on solar cells which are new and different from those imposed for conventional applications. The solar cells require a circular active area of approximately 4 mm in diameter. High reliability contacts are required on both front and back surfaces. The back area must be metallurgically bonded to a heat sink. The cell should be designed to achieve the highest practical efficiency at 100 AMO suns and at 80 C. The cell design must minimize losses due to nonuniform illumination intensity and nonnormal light incidence. The primary radiation concern is the omnidirectional proton environment.

  19. Tentative and transient natural killer cell polarization balances the requirements for discriminatory recognition and cytolytic efficacy.

    PubMed

    Sinai, Parisa; Roybal, Kole T; Wülfing, Christoph

    2010-11-01

    Natural killer (NK) cells are immune cells that lyse virally infected and tumor cells. Initially, their cytolytic capability is induced by cytokines. Subsequently, in their decision whether to kill a potential target cell, NK cells have to distinguish between small differences in the expression of ligands that report on the viral infection or transformation of the target. NK killing requires tight coupling to the target cell and extensive NK cell polarization. Here we discuss, often in contrast to the second cytolytic immune cell type, cytotoxic T cells, how NK cell polarization is shaped by three constraints of their activation. First, NK cell have to respond to cytokines: Different priming cytokines yield dramatically divergent NK cell polarization. Second, NK cells have to distinguish small differences in ligand expression: NK cell polarization is tentative, likely to allow discriminatory recognition close to the NK cell activation threshold. A critical contributor to the tentative nature of NK cell polarization may be poorly developed spatiotemporal organization of NK cell signaling. Third, NK cells have to kill effectively: NK cell polarization is transient, allowing for efficient killing by sequential interactions of a single NK cell with numerous target cells.

  20. 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

  1. Genetic Requirements for the Transformation of Human Cells

    DTIC Science & Technology

    2003-07-01

    thus progression of the cell cycle to S phase. The complex of cyclin E and CDK2 is responsible for this hyper-phosphorylation of Rb. Apoptosis studies...expressing E1AACR2 and Ha-RasV12 are senescent. In his experiments testing complementation of ElA mutants to re-establish apoptosis , Andy Samuelson...also based upon results in apoptosis studies where c-myc and cyclin E expression could rescue drug-induced apoptosis in IMR90 human fibroblasts (A

  2. E-cadherin is required for centrosome and spindle orientation in Drosophila male germline stem cells.

    PubMed

    Inaba, Mayu; Yuan, Hebao; Salzmann, Viktoria; Fuller, Margaret T; Yamashita, Yukiko M

    2010-08-31

    Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.

  3. Requirement of soluble factors produced by bone marrow stromal cells on the growth of novel established human myeloma cell line.

    PubMed

    Aikawa, Shingo; Hatta, Yoshihiro; Tanaka, Megumi; Kaneita, Yoshitaka; Yasukawa, Kiyotaka; Sawada, Umihiko; Horie, Takashi; Tsuboi, Isao; Aizawa, Shin

    2003-03-01

    The growth of myeloma cells is believed to be mediated by functional interactions between tumor cells and the marrow environment involving the action of several cytokines. We report on the establishment and characterization of a new human myeloma cell line (TAB1) that can be long-term maintained in the presence of conditioned medium of bone marrow stromal cells (BMCM) and a BMCM independent variant, C2-2. Both cell lines have plasma cell morphology and express plasma cell antigens (CD38, PCA-1 and immunoglobulin kappa light chain). In the absence of BMCM, TAB1 cells undergoing apoptosis were observed. Among the adherent molecules tested, these cells expressed VLA-4, ICAM-1 and H-CAM, but not VLA-5, suggesting that these were mostly immature plasmacytes. Introduction with exogenous IL-6 and/or GM-CSF, which were detected in BMCM, partially supported the proliferation of TAB1 cells. Treatment with anti-IL-6 antibody partially inhibited the proliferation of TAB1 cells cultured with BMCM. These findings strongly suggest that TAB1 required at least two or more factors on their growth in vitro; IL-6 was one of the factors necessary for cell growth. Further studies are required to clarify the precise molecules which support TAB1 cell growth in combination with IL-6, however, TAB1 and its variant C2-2 cells may offer an attractive model to unravel novel molecular mechanisms involved in bone marrow stroma-dependent growth of myeloma cells.

  4. 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.

  5. 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

  6. 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

  7. 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.

  8. Dual requirement for Pax6 in retinal progenitor cells

    PubMed Central

    Elgart, Michael; Marquardt, Till; Remizova, Lena; Yaron, Orly; Xie, Qing; Cvekl, Ales; Ashery-Padan, Ruth

    2014-01-01

    Throughout the developing central nervous system, pre-patterning of the ventricular zone into discrete neural progenitor domains is one of the predominant strategies used to produce neuronal diversity in a spatially coordinated manner. In the retina, neurogenesis proceeds in an intricate chronological and spatial sequence, yet it remains unclear whether retinal progenitor cells (RPCs) display intrinsic heterogeneity at any given time point. Here, we performed a detailed study of RPC fate upon temporally and spatially confined inactivation of Pax6. Timed genetic removal of Pax6 appeared to unmask a cryptic divergence of RPCs into qualitatively divergent progenitor pools. In the more peripheral RPCs under normal circumstances, Pax6 seemed to prevent premature activation of a photoreceptor-differentiation pathway by suppressing expression of the transcription factor Crx. More centrally, Pax6 contributed to the execution of the comprehensive potential of RPCs: Pax6 ablation resulted in the exclusive generation of amacrine interneurons. Together, these data suggest an intricate dual role for Pax6 in retinal neurogenesis, while pointing to the cryptic divergence of RPCs into distinct progenitor pools. PMID:19004853

  9. 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

  10. Germ Cells Are Not Required to Establish the Female Pathway in Mouse Fetal Gonads

    PubMed Central

    Maatouk, Danielle M.; Mork, Lindsey; Hinson, Ashley; Kobayashi, Akio; McMahon, Andrew P.; Capel, Blanche

    2012-01-01

    The fetal gonad is composed of a mixture of somatic cell lineages and germ cells. The fate of the gonad, male or female, is determined by a population of somatic cells that differentiate into Sertoli or granulosa cells and direct testis or ovary development. It is well established that germ cells are not required for the establishment or maintenance of Sertoli cells or testis cords in the male gonad. However, in the agametic ovary, follicles do not form suggesting that germ cells may influence granulosa cell development. Prior investigations of ovaries in which pre-meiotic germ cells were ablated during fetal life reported no histological changes during stages prior to birth. However, whether granulosa cells underwent normal molecular differentiation was not investigated. In cases where germ cell loss occurred secondary to other mutations, transdifferentiation of granulosa cells towards a Sertoli cell fate was observed, raising questions about whether germ cells play an active role in establishing or maintaining the fate of granulosa cells. We developed a group of molecular markers associated with ovarian development, and show here that the loss of pre-meiotic germ cells does not disrupt the somatic ovarian differentiation program during fetal life, or cause transdifferentiation as defined by expression of Sertoli markers. Since we do not find defects in the ovarian somatic program, the subsequent failure to form follicles at perinatal stages is likely attributable to the absence of germ cells rather than to defects in the somatic cells. PMID:23091613

  11. Murine thymic NK cells are distinct from ILC1s and have unique transcription factor requirements.

    PubMed

    Gabrielli, Sara; Sun, Mengxi; Bell, April; Zook, Erin C; de Pooter, Renee F; Zamai, Loris; Kee, Barbara L

    2017-03-09

    Group 1 innate lymphoid cells include natural killer (NK) cells and ILC1s, which mediate the response to intracellular pathogens. Thymic NK (tNK) cells were described with hybrid features of immature NK cells and ILC1 but whether these cells are related to NK cells or ILC1 has not been fully investigated. We report that murine tNK cells expressed the NK-cell associated transcription factor EOMES and developed independent of the essential ILC1 factor TBET, confirming their placement within the NK lineage. Moreover, tNK cells resemble NK cells rather than ILC1 in their requirements for the E protein transcription factor inhibitor ID2. We provide further insight into the mechanisms governing tNK-cell development by showing that the transcription factor ETS1 prevented tNK cell acquisition of the conventional NK-cell maturation markers CD11b and KLRG1. Our data reveal few ILC1 in the thymus and clarify the identity and developmental requirements of tNK cells. This article is protected by copyright. All rights reserved.

  12. Classical dendritic cells are required for dietary antigen-mediated peripheral regulatory T cell and tolerance induction

    PubMed Central

    Esterházy, Daria; Loschko, Jakob; London, Mariya; Jove, Veronica; Oliveira, Thiago Y.; Mucida, Daniel

    2016-01-01

    Oral tolerance prevents pathological inflammatory responses towards innocuous foreign antigens via peripheral regulatory T cells (pTreg cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure to instruct naïve CD4+ T cells to differentiate into pTreg cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs are dispensable, while classical dendritic cells (cDCs) are critical for pTreg cell induction and oral tolerance. CD11b− cDCs from the gut-draining lymph nodes efficiently induced pTreg cells, and conversely, loss of IRF8-dependent CD11b− cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in pTreg cell induction and their redundancy during oral tolerance development. PMID:27019226

  13. Memory CD4+ T cells are required for optimal NK cell effector functions against the opportunistic fungal pathogen Pneumocystis murina.

    PubMed

    Kelly, Michelle N; Zheng, Mingquan; Ruan, Sanbao; Kolls, Jay; D'Souza, Alain; Shellito, Judd E

    2013-01-01

    Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4(+) T cell-depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4(+) T cell-intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4(+) T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4(+) T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4(+) T cells and NK cells in the host response to an opportunistic fungal pathogen.

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

    PubMed

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

    2015-09-08

    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.

  15. CD22 is required for formation of memory B cell precursors within germinal centers

    PubMed Central

    Chappell, Craig P.; Draves, Kevin E.

    2017-01-01

    CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens. PMID:28346517

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

    DTIC Science & Technology

    2010-02-01

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

  17. Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation.

    PubMed

    Lee, Peishan; Zhu, Zilu; Hachmann, Janna; Nojima, Takuya; Kitamura, Daisuke; Salvesen, Guy; Rickert, Robert C

    2017-02-01

    During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.

  18. 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...

  19. 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.

  20. Multiple conductances cooperatively regulate spontaneous bursting in mouse olfactory bulb external tufted cells.

    PubMed

    Liu, Shaolin; Shipley, Michael T

    2008-02-13

    External tufted (ET) cells are juxtaglomerular neurons that spontaneously generate bursts of action potentials, which persist when fast synaptic transmission is blocked. The intrinsic mechanism of this autonomous bursting is unknown. We identified a set of voltage-dependent conductances that cooperatively regulate spontaneous bursting: hyperpolarization-activated inward current (I(h)), persistent Na+ current (I(NaP)), low-voltage-activated calcium current (I(L/T)) mediated by T- and/or L-type Ca2+ channels, and large-conductance Ca2+-dependent K+ current (I(BK)). I(h) is important in setting membrane potential and depolarizes the cell toward the threshold of I(NaP) and I(T/L), which are essential to generate the depolarizing envelope that is crowned by a burst of action potentials. Action potentials depolarize the membrane and induce Ca2+ influx via high-voltage-activated Ca2+ channels (I(HVA)). The combined depolarization and increased intracellular Ca2+ activates I(BK), which terminates the burst by hyperpolarizing the membrane. Hyperpolarization activates I(h) and the cycle is regenerated. A novel finding is the role of L-type Ca2+ channels in autonomous ET cells bursting. A second novel feature is the role of BK channels, which regulate burst duration. I(L) and I(BK) may go hand-in-hand, the slow inactivation of I(L) requiring I(BK)-dependent hyperpolarization to deactivate inward conductances and terminate the burst. ET cells receive monosynaptic olfactory nerve input and drive the major inhibitory interneurons of the glomerular circuit. Modulation of the conductances identified here can regulate burst frequency, duration, and spikes per burst in ET cells and thus significantly shape the impact of glomerular circuits on mitral and tufted cells, the output channels of the olfactory bulb.

  1. 40 CFR Table 8 to Subpart IIIii of... - Requirements for Cell Room Monitoring Program

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... CATEGORIES (CONTINUED) National Emission Standards for Hazardous Air Pollutants: Mercury Emissions From 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...

  2. 40 CFR Table 8 to Subpart IIIii of... - Requirements for Cell Room Monitoring Program

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... CATEGORIES (CONTINUED) National Emission Standards for Hazardous Air Pollutants: Mercury Emissions From 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...

  3. 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...

  4. Embryonic origin of adult stem cells required for tissue homeostasis and regeneration.

    PubMed

    Davies, Erin L; Lei, Kai; Seidel, Christopher W; Kroesen, Amanda E; McKinney, Sean A; Guo, Longhua; Robb, Sofia Mc; Ross, Eric J; Gotting, Kirsten; Alvarado, Alejandro Sánchez

    2017-01-10

    Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration.

  5. Mouse granulated metrial gland cells require contact with stromal cells to maintain viability

    PubMed Central

    STEWART, I. J.

    2000-01-01

    Granulated metrial gland (GMG) cells differentiate in the uterine wall in pregnancy in mice but the mechanisms which control their differentiation and maintenance are unknown. In vivo, GMG cells share an intimate association with fibroblast-like stromal cells. The importance of this association has been assessed by examining the effects of withdrawal of stromal cell contact on GMG cell maintenance in vitro. When single cell suspensions of cells were prepared from mouse metrial glands there was a steady decline in numbers with days of culture but usually some remained at 7 d of culture. The ability of metrial gland cells to kill Wehi 164 target cells in 51Cr-release cytotoxicity assays was retained by cells cultured for at least 3 d. When explants of metrial gland were maintained in culture to allow GMG cell migration onto the culture flask, the attached GMG cells were lost by 1 d later. Overall, these results suggest that a juxtacrine regulatory mechanism maintains GMG cells. The rapid loss of unsupported GMG cells in culture has major implications in the design of assays to examine GMG cell function. PMID:11117633

  6. National Ingition Facility subsystem design requirements pockels cell subsystem SSDR 1.3.3

    SciTech Connect

    Rhodes, M.

    1996-10-31

    This Subsystem Design Requirement document is a development specification that establishes the performance, design, development, and test requirements for the Pockels cell subsystem (WBS 1.3.3) of the NIF Laser System (WBS 1.3). The NIF is a multi-pass, 192-beam, high-power, neodymium-glass laser that meets requirements set forth in the NIF SDR 002 (Laser System). 5 figs., 1 tab.

  7. 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.

  8. 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.

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

    PubMed Central

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

    2016-01-01

    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

  10. 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.

  11. Peripheral T Cell Survival Requires Continual Ligation of the T Cell Receptor to Major Histocompatibility Complex–Encoded Molecules

    PubMed Central

    Kirberg, Jörg; Berns, Anton; Boehmer, Harald von

    1997-01-01

    In the thymus, T cells are selected according to their T cell receptor (TCR) specificity. After positive selection, mature cells are exported from primary lymphoid organs to seed the secondary lymphoid tissue. An important question is whether survival of mature T cells is an intrinsic property or requires continuous survival signals, i.e., engagement of the TCR by major histocompatibility complex (MHC) molecules in the periphery, perhaps in a similar way as occurring during thymic positive selection. To address this issue we used recombination-activating gene (Rag)-deficient H-2b mice expressing a transgenic TCR restricted by I-Ed class II MHC molecules. After engraftment with Rag−/− H-2d fetal thymi, CD4+8− peripheral T cells emerged. These cells were isolated and transferred into immunodeficient hosts of H-2b or H-2d haplotype, some of the latter being common cytokine receptor γ chain deficient to exclude rejection of H-2b donor cells by host natural killer cells. Our results show that in the absence, but not in the presence, of selecting MHC molecules, peripheral mature T cells are short lived and disappear within 7 wk, indicating that continuous contact of the TCR with selecting MHC molecules is required for survival of T cells. PMID:9334366

  12. Invariant NKT cells require autophagy to coordinate proliferation and survival signals during differentiation.

    PubMed

    Pei, Bo; Zhao, Meng; Miller, Brian C; Véla, Jose Luis; Bruinsma, Monique W; Virgin, Herbert W; Kronenberg, Mitchell

    2015-06-15

    Autophagy regulates cell differentiation, proliferation, and survival in multiple cell types, including cells of the immune system. In this study, we examined the effects of a disruption of autophagy on the differentiation of invariant NKT (iNKT) cells. Using mice with a T lymphocyte-specific deletion of Atg5 or Atg7, two members of the macroautophagic pathway, we observed a profound decrease in the iNKT cell population. The deficit is cell-autonomous, and it acts predominantly to reduce the number of mature cells, as well as the function of peripheral iNKT cells. In the absence of autophagy, there is reduced progression of iNKT cells in the thymus through the cell cycle, as well as increased apoptosis of these cells. Importantly, the reduction in Th1-biased iNKT cells is most pronounced, leading to a selective reduction in iNKT cell-derived IFN-γ. Our findings highlight the unique metabolic and genetic requirements for the differentiation of iNKT cells.

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

    PubMed

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

    2015-02-15

    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.

  14. 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

  15. Calpain 2 Is Required for the Invasion of Glioblastoma Cells in the Zebrafish Brain Microenvironment

    PubMed Central

    Lal, Sangeet; La Du, Jane; Tanguay, Robert L.; Greenwood, Jeffrey A.

    2012-01-01

    Glioblastoma is an aggressive primary brain tumor with a 5-year survival rate of less than 5%. The ability of glioblastoma cells to invade surrounding brain tissue presents the primary challenge for the success of focal therapeutic approaches. We previously reported that the calcium-activated protease calpain 2 is critical for glioblastoma cell invasion in vitro. Here, we show that expression of calpain 2 is required for the dispersal of glioblastoma cells in a living brain microenvironment. Knockdown of calpain 2 resulted in a 2.9-fold decrease in the invasion of human glioblastoma cells in zebrafish brain. Control cells diffusely migrated up to 450 μm from the site of injection, whereas knockdown cells remained confined in clusters. The invasion study was repeated in organotypic mouse brain tissues, and calpain 2 knockdown cells demonstrated a 2.3-fold lower area of dispersal compared with control cells. In zebrafish brain, glioblastoma cells appeared to migrate in part along the blood vessels of the host. Furthermore, angiogenesis was detected in 27% of zebrafish injected with control cells, whereas only 12.5% of fish receiving knockdown cells showed the formation of new vessels, suggesting a role for calpain 2 in tumor cell angiogenesis. Consistent with the progression of glioblastoma in humans, transplanted tumor cells were not observed to metastasize outside the brain of zebrafish. This study demonstrates that calpain 2 expression is required for the dispersal of glioblastoma cells within the dynamic microenvironment of the brain, identifying zebrafish as a valuable orthotopic system for studying glioblastoma cell invasion. PMID:22183788

  16. The Drosophila toucan (toc) gene is required in germline cells for the somatic cell patterning during oogenesis.

    PubMed

    Grammont, M; Dastugue, B; Couderc, J L

    1997-12-01

    We have characterized a new gene, called toucan, that is expressed and required in germline cells to promote proper differentiation of the somatic follicle cells. toucan mutant ovaries are defective in (i) the enclosure of newly formed germline cysts by the follicle cells, (ii) the formation of interfollicular stalks, (iii) the migration of the follicle cells over the oocyte and (iv) the formation of the eggshell. Overexpression of a toucan cDNA in the germline leads to the production of longer interfollicular stalks than wild-type ovaries, a phenotype that is the exact opposite of the toucan mutant phenotype. This observation shows that the formation of the interfollicular stalks depends not only on interactions among the somatic cells but also requires a germline signal. Moreover, dominant interactions have been observed between toucan and certain alleles of the daughterless, Notch and Delta genes, each of which is required in the somatic cells for the formation of egg chambers. toucan encodes for a large protein with a coiled-coil domain but has no other homology with known proteins. We propose that toucan participates in the production or localization of a germline-specific signal(s) that is required for the patterning of the follicular epithelium.

  17. Successful correction of murine sickle cell disease with reduced stem cell requirements reinforced by fractionated marrow infusions.

    PubMed

    Felfly, Hady; Trudel, Marie

    2010-02-01

    Minimal criteria requirements of stem cell replacement, conditioning regimen and modalities of infusion essential for cure of sickle cell disease (SCD) by bone marrow(BM)/stem cell transplantation or gene therapy must be established prior to clinical trials. The threshold of normal BM/stem cells for therapeutic correction of this red blood cell disorder was evaluated in the SAD murine SCD model from peripheral donor white blood cells. From 11 groups of stable chimeric SAD mice (5-92%) analyzed over approximately 2 years, mice with approximately 16% normal donor stem cells showed improvement of haematological and erythroid responses. Mice in the 26% chimeric group and above demonstrated substantial amelioration of organ pathologies with generalized decreased iron deposits, fibrosis and reached normal lifespan. Subsequently, the minimal myelosuppression concurrently with number and timing of infusions and number of BM cells was determined to reach therapeutic threshold in SAD mice. Higher myelosuppression (2 Gy vs. 1 Gy) and cell number in single infusion led to increased chimerism. Importantly, administration of three-equivalent cell subdoses within 28 h of mild myelosuppression resulted in 100% recipient engraftment at therapeutic levels. These studies established the long-term therapeutic chimeric threshold of normal white blood cells at approximately 26% and determined the minimal fractionated BM/stem cell doses concomitant with mild myelosuppression for significant correction of SCD in SAD mice.

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

    PubMed Central

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

    2009-01-01

    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 (TReg) cells play a critical role in tumor-specific CD8+ T-cell tolerance after transplantation. Removal of TReg 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+ TReg cells in recipients after transplantation, and depletion of TReg 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

  19. 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.

  20. Membranes replace irradiated blast cells as growth requirement for leukemic blast progenitors in suspension culture

    SciTech Connect

    Nara, N.; McCulloch, E.A.

    1985-11-01

    The blast cells of acute myeloblastic leukemia (AML) may be considered as a renewal population, maintained by blast stem cells capable of both self-renewal and the generation of progeny with reduced or absent proliferative potential. This growth requires that two conditions be met: first, the cultures must contain growth factors in media conditioned either by phytohemagglutinin (PHA)-stimulated mononuclear leukocytes (PHA-LCM), or by cells of the continuous bladder carcinoma line HTB9 (HTB9-CM). Second, the cell density must be maintained at 10(6) blasts/ml; this may be achieved by adding irradiated cells to smaller numbers of intact blasts. The authors are concerned with the mechanism of the feeding function. They present evidence that (a) cell-cell contact is required. (b) Blasts are heterogeneous in respect to their capacity to support growth. (c) Fractions containing membranes from blast cells will substitute for intact cells in promoting the generation of new blast progenitors in culture. (d) This membrane function may be specific for AML blasts, since membranes from blasts of lymphoblastic leukemia or normal marrow cells were inactive.

  1. 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

  2. Bällchen is required for self-renewal of germline stem cells in Drosophila melanogaster.

    PubMed

    Herzig, Bettina; Yakulov, Toma A; Klinge, Kathrin; Günesdogan, Ufuk; Jäckle, Herbert; Herzig, Alf

    2014-05-29

    Self-renewing stem cells are pools of undifferentiated cells, which are maintained in cellular niche environments by distinct tissue-specific signalling pathways. In Drosophila melanogaster, female germline stem cells (GSCs) are maintained in a somatic niche of the gonads by BMP signalling. Here we report a novel function of the Drosophila kinase Bällchen (BALL), showing that its cell autonomous role is to maintain the self-renewing capacity of female GSCs independent of BMP signalling. ball mutant GSCs are eliminated from the niche and subsequently differentiate into mature eggs, indicating that BALL is largely dispensable for differentiation. Similar to female GSCs, BALL is required to maintain self-renewal of male GSCs, suggesting a tissue independent requirement of BALL for self-renewal of germline stem cells.

  3. Pharyngeal Satellite Cells Undergo Myogenesis Under Basal Conditions and Are Required for Pharyngeal Muscle Maintenance.

    PubMed

    Randolph, Matthew E; Phillips, Brittany L; Choo, Hyo-Jung; Vest, Katherine E; Vera, Yandery; Pavlath, Grace K

    2015-12-01

    The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging.

  4. Cell polarization is required for ricin sensitivity in a Caco-2 cell line selected for ricin resistance.

    PubMed Central

    Jackman, M R; Ellis, J A; Gray, S R; Shurety, W; Luzio, J P

    1999-01-01

    It has been proposed that killing of mammalian cells by ricin requires efficient endocytic delivery to the trans-Golgi network (TGN) prior to retrograde transport to the endoplasmic reticulum and entry to the cytosol. In polarized epithelial cells, an efficient membrane-traffic pathway to the TGN is present from the basolateral but not the apical plasma-membrane domain. Thus one can hypothesize that a ricin-resistant phenotype might be demonstrated by polarized cells that fail to differentiate and thus fail to develop an efficient membrane-traffic pathway from the basolateral plasma membrane to the TGN. We have isolated and studied a ricin-resistant Caco-2 cell clone (Caco-2-RCAr clone 2) which, when grown on plastic, was deficient in differentiation, measured by the development of polarized-cell-surface marker enzymes. The deficiency in differentiation was partially reversed, and ricin sensitivity was restored, when the cells were grown on filter supports. Our data provide the first evidence of a ricin-resistant cell line where resistance is due to the lack of development of polarized cell surfaces. The observed ricin resistance is consistent with the requirement that ricin is delivered to the TGN before its A chain enters the cytosol to mediate cell killing. PMID:10393089

  5. Nonspecific cytotoxic cells in fish (Ictalurus punctatus). V. Metabolic requirements of lysis.

    PubMed

    Carlson, R L; Evans, D L; Graves, S S

    1985-01-01

    The mechanisms of lysis of nonspecific cytotoxic cells (NCC) from the channel catfish (Ictalurus punctatus) were studied by determining the effects of various inhibitors of cellular metabolism on cytolysis of NC-37 human lymphoma target cells. Inhibition of NCC-mediated lysis by dinitrophenol (DNP) and sodium azide (NaN3) indicated a requirement for cellular energy metabolism. Cytochalasin B, an inhibitor of microfilaments, and monensin, an inhibitor of cellular secretion, both prevented lysis by NCC. Three microtubule inhibitors, vinblastine sulfate, colchicine, and demecolcine, all inhibited target cell lysis. Two divalent cation chelators, EDTA and EGTA, blocked NCC activity. Elimination of both Ca2+ and Mg2+ by EDTA prevented target cell binding and killing. Selective removal of Ca2+ by EGTA prevented killing but did not block target cell binding. These results indicated that nonspecific cytotoxicity in fish is an active process which requires cell movement and an intact secretory apparatus. The mechanisms of cytolysis by NCC were found (except for the requirement of microtubules) to be analogous to those of mammalian NK cells. Combined with morphological studies, these data strongly suggest that a phylogenetic relationship exists between these effector cells.

  6. 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.

  7. Tumour metastasis as an adaptation of tumour cells to fulfil their phosphorus requirements.

    PubMed

    de Carvalho, Carla C C R; Caramujo, Maria José

    2012-05-01

    Inorganic phosphate (Pi) is a vital component of nucleotides, membrane phospholipids, and phosphorylated intermediates in cellular signalling. The Growth Rate Hypothesis (GRH) states that fast growing organisms should be richer in phosphorus (relatively low C:P and N:P cell content) than slow developing organisms as a result of high ribosome biogenesis. Cells that proliferate rapidly, such as cancer cells, require a high amount of ribosomes and other P-rich RNA components that are necessary to manufacture proteins. The GRH hypothesis may be applied to cancer predicting that tumour cells are richer in phosphorus than the surrounding tissue, and that they resort to metastasis in order to meet their nutrient demands. Considering that the cells most P-deprived should be located in the inner parts of the tumour we propose that changes in the membrane of these cells favour the detachment of the more peripheral cells.

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

    PubMed

    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

    2016-02-02

    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.

  9. 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

  10. L1-mediated colon cancer cell metastasis does not require changes in EMT and cancer stem cell markers.

    PubMed

    Gavert, Nancy; Vivanti, Alessia; Hazin, John; Brabletz, Thomas; Ben-Ze'ev, Avri

    2011-01-01

    Aberrant activation of Wnt/β-catenin signaling is common in most sporadic and inherited colorectal cancer (CRC) cells leading to elevated β-catenin/TCF transactivation. We previously identified the neural cell adhesion molecule L1 as a target gene of β-catenin/TCF in CRC cells. Forced expression of L1 confers increased cell motility, invasion, and tumorigenesis, and the induction of human CRC cell metastasis to the liver. In human CRC tissue, L1 is exclusively localized at the invasive front of such tumors in a subpopulation of cells displaying nuclear β-catenin. We determined whether L1 expression confers metastatic capacities by inducing an epithelial to mesenchymal transition (EMT) and whether L1 cosegregates with cancer stem cell (CSC) markers. We found that changes in L1 levels do not affect the organization or expression of E-cadherin in cell lines, or in invading CRC tissue cells, and no changes in other epithelial or mesenchymal markers were detected after L1 transfection. The introduction of major EMT regulators (Slug and Twist) into CRC cell lines reduced the levels of E-cadherin and induced fibronectin and vimentin, but unlike L1, Slug and Twist expression was insufficient for conferring metastasis. In CRC cells L1 did not specifically cosegregate with CSC markers including CD133, CD44, and EpCAM. L1-mediated metastasis required NF-κB signaling in cells harboring either high or low levels of endogenous E-cadherin. The results suggest that L1-mediated metastasis of CRC cells does not require changes in EMT and CSC markers and operates by activating NF-κβ signaling.

  11. ELT-5 and ELT-6 are required continuously to regulate epidermal seam cell differentiation and cell fusion in C. elegans.

    PubMed

    Koh, K; Rothman, J H

    2001-08-01

    The C. elegans epidermis is a simple epithelium comprised of three major cell types, the seam, syncytial and P cells. While specification of all major epidermal cells is known to require the ELT-1 GATA transcription factor, little is known about how the individual epidermal cell types are specified. We report that elt-5 and -6, adjacent genes encoding GATA factors, are essential for the development of the lateral epidermal cells, the seam cells. Inhibition of elt-5 and -6 function by RNA-mediated interference results in penetrant late embryonic and early larval lethality. Seam cells in affected animals do not differentiate properly: the alae, seam-specific cuticular structures, are generally absent and expression of several seam-specific markers is blocked. In addition, elt-3, which encodes another GATA factor normally expressed in non-seam epidermis, is often ectopically expressed in the seam cells of affected animals, demonstrating that ELT-5 and -6 repress elt-3 expression in wild-type seam cells. Seam cells in affected animals often undergo inappropriate fusion with the epidermal syncytia. Interference of elt-5 and -6 function during larval development can cause fusion of all seam cells with the surrounding syncytia and pronounced defects in molting. elt-5 and -6 are both expressed in seam cells and many other cells, and are apparently functionally interchangeable. Their expression is controlled by separable tissue-specific regulatory elements and the apportionment of monocistronic versus dicistronic transcription of both genes appears to be subject to cell-type-specific regulation. Collectively, these findings indicate that elt-5 and -6 function continuously throughout C. elegans development to regulate seam cell differentiation and cell fusion.

  12. 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.

  13. Short circuit testing of a nickel-hydrogen cell for compliance with range safety requirements

    SciTech Connect

    Tracinski, W.A.; Applewhite, A.Z.

    1997-12-01

    Short circuit testing was performed on a single stack, Independent Pressure Vessel (IPV) aerospace Nickel-Hydrogen cell with axial terminals for compliance with range safety requirements. The cell contained two brazed ceramic seals, was 3 1/2 inches in diameter, and had a nameplate rating of 85.5 Ah. The majority of the energy was released in the first ten minutes with peak terminal temperature reaching 192 degrees Celsius. No breaching of the cell was evident and the cell returned to normal open circuit voltage within fifteen minutes of the load being removed.

  14. Th17 cells give rise to Th1 cells that are required for the pathogenesis of colitis.

    PubMed

    Harbour, Stacey N; Maynard, Craig L; Zindl, Carlene L; Schoeb, Trenton R; Weaver, Casey T

    2015-06-02

    Th17 cells reactive to the enteric microbiota are central to the pathogenesis of certain types of inflammatory bowel disease. However, Th17 cells display substantial developmental plasticity, such that some progeny of Th17 cell precursors retain a predominantly IL-17A(+) phenotype, whereas others extinguish IL-17 expression and acquire expression of IFN-γ, giving rise to "Th1-like" cells. It remains unclear what role these subsets play in inflammatory bowel disease. Using a Th17 transfer model of colitis, we found that IFN-γ-deficient Th17 cells retained an IL-17A(+) phenotype and were unable to induce colitis in recipients. Development of disease required the transition of a subset of Th17 precursors to Th1-like cells and was contingent on the expression of both Stat4 and T-bet, but not the IL-12 or IFN-γ receptors. Moreover, Th17 cells could provide "help" for the development of pathogenic Th1 cells from naïve precursors. These results indicate that Th17 cells are potent mediators of colitis pathogenesis by dual mechanisms: by directly transitioning to Th1-like cells and by supporting the development of classic Th1 cells.

  15. Germline self-renewal requires cyst stem cells and stat regulates niche adhesion in Drosophila testes.

    PubMed

    Leatherman, Judith L; Dinardo, Stephen

    2010-08-01

    Adults maintain tissue-specific stem cells through niche signals. A model for niche function is the Drosophila melanogaster testis, where a small cluster of cells called the hub produce locally available signals that allow only adjacent cells to self-renew. We show here that the principal signalling pathway implicated in this niche, chemokine activation of STAT, does not primarily regulate self-renewal of germline stem cells (GSCs), but rather governs GSC adhesion to hub cells. In fact, GSC renewal does not require hub cell contact, as GSCs can be renewed solely by contact with the second resident stem cell population, somatic cyst stem cells (CySCs), and this involves BMP signalling. These data suggest a modified paradigm whereby the hub cells function as architects of the stem cell environment, drawing into proximity cellular components necessary for niche function. Self-renewal functions are shared by the hub cells and the CySCs. This work also reconciles key differences in GSC renewal between Drosophila testis and ovary niches, and highlights how a niche can coordinate the production of distinct lineages by having one stem cell type rely on a second.

  16. 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.

  17. Th9 cell development requires a BATF-regulated transcriptional network

    PubMed Central

    Jabeen, Rukhsana; Goswami, Ritobrata; Awe, Olufolakemi; Kulkarni, Aishwarya; Nguyen, Evelyn T.; Attenasio, Andrea; Walsh, Daniel; Olson, Matthew R.; Kim, Myung H.; Tepper, Robert S.; Sun, Jie; Kim, Chang H.; Taparowsky, Elizabeth J.; Zhou, Baohua; Kaplan, Mark H.

    2013-01-01

    T helper 9 (Th9) cells are specialized for the production of IL-9, promote allergic inflammation in mice, and are associated with allergic disease in humans. It has not been determined whether Th9 cells express a characteristic transcriptional signature. In this study, we performed microarray analysis to identify genes enriched in Th9 cells compared with other Th subsets. This analysis defined a transcriptional regulatory network required for the expression of a subset of Th9-enriched genes. The activator protein 1 (AP1) family transcription factor BATF (B cell, activating transcription factor–like) was among the genes enriched in Th9 cells and was required for the expression of IL-9 and other Th9-associated genes in both human and mouse T cells. The expression of BATF was increased in Th9 cultures derived from atopic infants compared with Th9 cultures from control infants. T cells deficient in BATF expression had a diminished capacity to promote allergic inflammation compared with wild-type controls. Moreover, mouse Th9 cells ectopically expressing BATF were more efficient at promoting allergic inflammation than control transduced cells. These data indicate that BATF is a central regulator of the Th9 phenotype and contributes to the development of allergic inflammation. PMID:24216482

  18. Th9 cell development requires a BATF-regulated transcriptional network.

    PubMed

    Jabeen, Rukhsana; Goswami, Ritobrata; Awe, Olufolakemi; Kulkarni, Aishwarya; Nguyen, Evelyn T; Attenasio, Andrea; Walsh, Daniel; Olson, Matthew R; Kim, Myung H; Tepper, Robert S; Sun, Jie; Kim, Chang H; Taparowsky, Elizabeth J; Zhou, Baohua; Kaplan, Mark H

    2013-11-01

    T helper 9 (Th9) cells are specialized for the production of IL-9, promote allergic inflammation in mice, and are associated with allergic disease in humans. It has not been determined whether Th9 cells express a characteristic transcriptional signature. In this study, we performed microarray analysis to identify genes enriched in Th9 cells compared with other Th subsets. This analysis defined a transcriptional regulatory network required for the expression of a subset of Th9-enriched genes. The activator protein 1 (AP1) family transcription factor BATF (B cell, activating transcription factor–like) was among the genes enriched in Th9 cells and was required for the expression of IL-9 and other Th9-associated genes in both human and mouse T cells. The expression of BATF was increased in Th9 cultures derived from atopic infants compared with Th9 cultures from control infants. T cells deficient in BATF expression had a diminished capacity to promote allergic inflammation compared with wild-type controls. Moreover, mouse Th9 cells ectopically expressing BATF were more efficient at promoting allergic inflammation than control transduced cells. These data indicate that BATF is a central regulator of the Th9 phenotype and contributes to the development of allergic inflammation.

  19. Nuclear cathepsin L activity is required for cell cycle progression of colorectal carcinoma cells.

    PubMed

    Tamhane, Tripti; Lllukkumbura, Rukshala; Lu, Shiying; Maelandsmo, Gunhild M; Haugen, Mads H; Brix, Klaudia

    2016-03-01

    Prominent tasks of cysteine cathepsins involve endo-lysosomal proteolysis and turnover of extracellular matrix constituents or plasma membrane proteins for maintenance of intestinal homeostasis. Here we report on enhanced levels and altered subcellular localization of distinct cysteine cathepsins in adenocarcinoma tissue in comparison to adjacent normal colon. Immunofluorescence and immunoblotting investigations revealed the presence of cathepsin L in the nuclear compartment in addition to its expected endo-lysosomal localization in colorectal carcinoma cells. Cathepsin L was represented as the full-length protein in the nuclei of HCT116 cells from which stefin B, a potent cathepsin L inhibitor, was absent. Fluorescence activated cell sorting analyses with synchronized cell cultures revealed deceleration of cell cycle progression of HCT116 cells upon inhibition of cathepsin L activity, while expression of cathepsin L-enhanced green fluorescent protein chimeras accelerated S-phase entry. We conclude that the activity of cathepsin L is high in the nucleus of colorectal carcinoma cells because of lacking stefin B inhibitory activity. Furthermore, we hypothesize that nuclear cathepsin L accelerates cell cycle progression of HCT116 cells thereby supporting the notion that cysteine cathepsins may play significant roles in carcinogenesis due to deregulated trafficking.

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

    PubMed Central

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

    2015-01-01

    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

  1. 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.

  2. Efficient Hepatitis Delta Virus RNA Replication in Avian Cells Requires a Permissive Factor(s) from Mammalian Cells

    PubMed Central

    Liu, Yu-Tsueng; Brazas, Rob; Ganem, Don

    2001-01-01

    Hepatitis delta virus (HDV) is a highly pathogenic human RNA virus whose genome is structurally related to those of plant viroids. Although its spread from cell to cell requires helper functions supplied by hepatitis B virus (HBV), intracellular HDV RNA replication can proceed in the absence of HBV proteins. As HDV encodes no RNA-dependent RNA polymerase, the identity of the (presumably cellular) enzyme responsible for this reaction remains unknown. Here we show that, in contrast to mammalian cells, avian cells do not support efficient HDV RNA replication and that this defect cannot be rescued by provision of HDV gene products in trans. Contrary to earlier assertions, this defect is not due to enhanced apoptosis triggered in avian cells by HDV. Fusion of avian cells to mammalian cells rescues HDV replication in avian nuclei, indicating that the nonpermissive phenotype of avian cells is not due to the presence of dominantly acting inhibitors of replication. Rather, avian cells lack one or more essential permissive factors present in mammalian cells. These results set the stage for the identification of such factors and also explain the failure of earlier efforts to transmit HDV infection to avian hosts harboring indigenous hepadnaviruses. PMID:11462021

  3. 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.

  4. 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.

  5. 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.

  6. JNK controls the onset of mitosis in planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling.

    PubMed

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

    2014-06-01

    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.

  7. 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

  8. Mouse B-Type Lamins Are Required for Proper Organogenesis But Not by Embryonic Stem Cells

    PubMed Central

    Kim, Youngjo; Sharov, Alexei A.; McDole, Katie; Cheng, Melody; Hao, Haiping; Fan, Chen-Ming; Gaiano, Nicholas; Ko, Minoru S. H.; Zheng, Yixian

    2012-01-01

    B-type lamins, the major components of the nuclear lamina, are believed to be essential for cell proliferation and survival. We found that mouse embryonic stem cells (ESCs) do not need any lamins for self-renewal and pluripotency. Although genome-wide lamin-B binding profiles correlate with reduced gene expression, such binding is not directly required for gene silencing in ESCs or trophectoderm cells. However, B-type lamins are required for proper organogenesis. Defects in spindle orientation in neural progenitor cells and migration of neurons probably cause brain disorganizations found in lamin-B null mice. Thus, our studies not only disprove several prevailing views of lamin-Bs but also establish a foundation for redefining the function of the nuclear lamina in the context of tissue building and homeostasis. PMID:22116031

  9. 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.

  10. Different growth factor requirements for human Th2 cells may reflect in vivo induced anergy.

    PubMed Central

    Van Reijsen, F C; Wijburg, O L; Gebhardt, M; Van Ieperen-Van Dijk, A G; Betz, S; Poellabauer, E M; Thepen, T; Bruijnzeel-Koomen, C A; Mudde, G C

    1994-01-01

    We previously reported the isolation of allergen-specific Th2 lines and clones from atopy patch test (APT) sites of atopic dermatitis (AD) patients. Upon stimulation with allergen or anti-CD3+ phorbol myristate acetate (PMA) IL-4 was released with or without IL-5, while no (or extremely low concentrations of) IL-2 and interferon-gamma (IFN-gamma) were detectable. A high IL-4/IFN-gamma ratio facilitates production of allergen-specific IgE, of which high levels are observed in AD patients. Here we show that the above mentioned Th2 cells are notably different from murine Th2 cells. Not IL-4, which is the autocrine acting growth factor for murine Th2 cells, but IL-2 was needed for proliferation of these human APT-derived Th2 lines and clones. Of significance, unless exogenous IL-2 was added, no proliferative response to allergen, presented by Epstein-Barr virus-transformed B (EBV-B) cells, non-T cells or IgE-bearing Langerhans cells (LC), occurred. Lack of proliferation and IL-2 production after full T cell receptor (TCR) triggering is a characteristic first described for in vitro anergized T cells. However, like the clones we describe in this study, anergic T cells may retain production of cytokines other than IL-2. A further resemblance between anergic T cells and the human Th2 clones reported here is that IL-4 can enhance IL-2-driven proliferation, but is not capable of inducing T cell growth by itself. The absence of IL-4-driven proliferation differentiates human Th2 cells from murine Th2 cells. Both produce IL-4 when stimulated in a cognate fashion, but only murine Th2 cells will proliferate. We conclude that the presently reported human Th2 cells are different from murine Th2 cells, in that they need other T cells to produce IL-2 required for their expansion. Moreover, the Th2 cells phenotypically resemble anergic T cells. As yet, however, we have no clue as to whether these features account for the current Th2 cells only or for human Th2 cells in general. We

  11. Cyclin D3 is selectively required for proliferative expansion of germinal center B cells.

    PubMed

    Cato, Matthew H; Chintalapati, Suresh K; Yau, Irene W; Omori, Sidne A; Rickert, Robert C

    2011-01-01

    The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.

  12. 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.

  13. 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.

  14. Efficient Norovirus and Reovirus Replication in the Mouse Intestine Requires Microfold (M) Cells

    PubMed Central

    Gonzalez-Hernandez, Mariam B.; Liu, Thomas; Payne, Hilary C.; Stencel-Baerenwald, Jennifer E.; Ikizler, Mine; Yagita, Hideo; Dermody, Terence S.; Williams, Ifor R.

    2014-01-01

    ABSTRACT Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. M cells have also been suggested as a portal for pathogen entry into the host. While virus particles have been observed in M cells, it is not known whether viruses use M cells to initiate a productive infection. Noroviruses (NoVs) are single-stranded RNA viruses that infect host organisms via the fecal-oral route. Murine NoV (MNV) infects intestinal macrophages and dendritic cells and provides a tractable experimental system for understanding how an enteric virus overcomes the intestinal epithelial barrier to infect underlying target cells. We found that replication of two divergent MNV strains was reduced in mice depleted of M cells. Reoviruses are double-stranded RNA viruses that infect hosts via respiratory or enteric routes. In contrast to MNV, reovirus infects enterocytes in the intestine. Despite differences in cell tropism, reovirus infection was also reduced in M cell-depleted mice. These data demonstrate that M cells are required for the pathogenesis of two unrelated enteric viruses that replicate in different cell types within the intestine. IMPORTANCE To successfully infect their hosts, pathogens that infect via the gastrointestinal tract must overcome the multilayered system of host defenses. Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. Virus particles have been observed within M cells. However, it is not known whether viruses use M cells to initiate a productive infection. To address this question, we use MNV and reovirus, two enteric viruses that replicate in different cell types in the intestine, intestinal epithelial cells for reovirus and intestinal mononuclear phagocytes for MNV. Interestingly, MNV- and reovirus-infected mice depleted of M

  15. 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

  16. Ethanolamine requirement of mammary epithelial cells is due to reduced activity of base exchange enzyme

    SciTech Connect

    Kano-Sueoka, T.; King, D.M.

    1987-05-01

    Epithelial cells and some of their transformed derivatives require ethanolamine (Etn) to proliferate normally in defined culture medium. The amount of cellular phosphatidylethanolamine (PtdEtn) is considerably reduced when these cells are cultured without Etn. Using Etn-responsive and -nonresponsive rat mammary carcinoma cell lines, the biochemical mechanism of Etn-responsiveness of investigated. The incorporation of (/sup 3/H)serine into phosphatidylserine (PtdSer) and PtdEtn in Etn-responsive cells was 60 and 37%, respectively, of those in Etn-nonresponsive cells. There was no significant difference between the two cell types in the activities of enzymes involved in PtdEtn synthesis via CDP-Etn. The activity of PtdSer decarboxylase was also very similar in these two cell types. When these cells were cultured in the presence of (/sup 32/P)PtdEtn, the rate of accumulation of (/sup 32/P)-labeled PtdSer from the radioactive PtdEtn was considerably reduced in Etn-responsive cells as compared to Etn-nonresponsive cells. Whereas there was no significant difference in the accumulation of the labeled PtdSer from (/sup 32/P)phosphatidylcholine. These results demonstrate that the Etn-responsiveness is due to a limited ability to synthesize PtdSer resulting from a limited base exchange activity utilizing PtdEtn.

  17. Gammadelta T cells in EAE: early trafficking events and cytokine requirements.

    PubMed

    Wohler, Jillian E; Smith, Sherry S; Zinn, Kurt R; Bullard, Dan C; Barnum, Scott R

    2009-06-01

    We have previously shown that gammadelta T cells traffic to the CNS during EAE with concurrently increased expression of beta(2)-integrins and production of IFN-gamma and TNF-alpha. To extend these studies, we transferred bioluminescent gammadelta T cells to WT mice and followed their movement through the acute stages of disease. We found that gammadelta T cells rapidly migrated to the site of myelin oligodendrocyte glycoprotein peptide injection and underwent massive expansion. Within 6 days after EAE induction, bioluminescent gammadelta T cells were found in the spinal cord and brain, peaking in number between days 10 and 12 and then rapidly declining by day 15. Reconstitution of gammadelta T cell(-/-) mice with gammadelta T cells derived from beta(2)-integrin-deficient mice (CD11a, -b or -c) demonstrated that gammadelta T-cell trafficking to the CNS during EAE is independent of this family of adhesion molecules. We also examined the role of gammadelta T-cell-produced IFN-gamma and TNF-alpha in EAE and found that production of both cytokines by gammadelta T cells was required for full development of EAE. These results indicate that gammadelta T cells are critical for the development of EAE and suggest a therapeutic target in demyelinating disease.

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

    PubMed Central

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

    2015-01-01

    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

  19. Lgr4 is required for Paneth cell differentiation and maintenance of intestinal stem cells ex vivo.

    PubMed

    Mustata, Roxana C; Van Loy, Tom; Lefort, Anne; Libert, Frédérick; Strollo, Sandra; Vassart, Gilbert; Garcia, Marie-Isabelle

    2011-06-01

    Gene inactivation of the orphan G protein-coupled receptor LGR4, a paralogue of the epithelial-stem-cell marker LGR5, results in a 50% decrease in epithelial cell proliferation and an 80% reduction in terminal differentiation of Paneth cells in postnatal mouse intestinal crypts. When cultured ex vivo, LGR4-deficient crypts or progenitors, but not LGR5-deficient progenitors, die rapidly with marked downregulation of stem-cell markers and Wnt target genes, including Lgr5. Partial rescue of this phenotype is achieved by addition of LiCl to the culture medium, but not Wnt agonists. Our results identify LGR4 as a permissive factor in the Wnt pathway in the intestine and, as such, as a potential target for intestinal cancer therapy.

  20. Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance.

    PubMed

    Esterházy, Daria; Loschko, Jakob; London, Mariya; Jove, Veronica; Oliveira, Thiago Y; Mucida, Daniel

    2016-05-01

    Oral tolerance prevents pathological inflammatory responses to innocuous foreign antigens by peripheral regulatory T cells (pT(reg) cells). However, whether a particular subset of antigen-presenting cells (APCs) is required during dietary antigen exposure for the 'instruction' of naive CD4(+) T cells to differentiate into pT(reg) cells has not been defined. Using myeloid lineage-specific APC depletion in mice, we found that monocyte-derived APCs were dispensable, while classical dendritic cells (cDCs) were critical, for pT(reg) cell induction and oral tolerance. CD11b(-) cDCs from the gut-draining lymph nodes efficiently induced pT(reg) cells and, conversely, loss of transcription factor IRF8-dependent CD11b(-) cDCs impaired their polarization, although oral tolerance remained intact. These data reveal the hierarchy of cDC subsets in the induction of pT(reg) cells and their redundancy during the development of oral tolerance.

  1. Tissue-tissue interaction-triggered calcium elevation is required for cell polarization during Xenopus gastrulation.

    PubMed

    Shindo, Asako; Hara, Yusuke; Yamamoto, Takamasa S; Ohkura, Masamichi; Nakai, Junichi; Ueno, Naoto

    2010-02-02

    The establishment of cell polarity is crucial for embryonic cells to acquire their proper morphologies and functions, because cell alignment and intracellular events are coordinated in tissues during embryogenesis according to the cell polarity. Although much is known about the molecules involved in cell polarization, the direct trigger of the process remains largely obscure. We previously demonstrated that the tissue boundary between the chordamesoderm and lateral mesoderm of Xenopus laevis is important for chordamesodermal cell polarity. Here, we examined the intracellular calcium dynamics during boundary formation between two different tissues. In a combination culture of nodal-induced chordamesodermal explants and a heterogeneous tissue, such as ectoderm or lateral mesoderm, the chordamesodermal cells near the boundary frequently displayed intracellular calcium elevation; this frequency was significantly less when homogeneous explants were used. Inhibition of the intracellular calcium elevation blocked cell polarization in the chordamesodermal explants. We also observed frequent calcium waves near the boundary of the dorsal marginal zone (DMZ) dissected from an early gastrula-stage embryo. Optical sectioning revealed that where heterogeneous explants touched, the chordamesodermal surface formed a wedge with the narrow end tucked under the heterogeneous explant. No such configuration was seen between homogeneous explants. When physical force was exerted against a chordamesodermal explant with a glass needle at an angle similar to that created in the explant, or migrating chordamesodermal cells crawled beneath a silicone block, intracellular calcium elevation was frequent and cell polarization was induced. Finally, we demonstrated that a purinergic receptor, which is implicated in mechano-sensing, is required for such frequent calcium elevation in chordamesoderm and for cell polarization. This study raises the possibility that tissue-tissue interaction generates

  2. Hyaluronan Is Required for Generation of Hematopoietic Cells during Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Schraufstatter, Ingrid U.; Serobyan, Naira; Loring, Jeanne; Khaldoyanidi, Sophia K.

    2010-01-01

    Hyaluronan (HA) is an important component of the microenvironment in bone marrow, but its role in regulation of the development of hematopoietic cells is not well understood. To address the role of HA in regulation of human embryonic stem cell (hESC) differentiation into the hematopoietic lineage, we screened for genes encoding components of the HA pathway. Using gene arrays, we found that HA synthases and HA receptors are expressed in both undifferentiated and differentiating hESCs. Enzymatic degradation of HA resulted in decreased numbers of hematopoietic progenitors and lower numbers of CD45+ cells generated in HA-deprived embryoid bodies (EBs). In addition, deprivation of HA resulted in the inhibition of generation of CD31+ cells, stromal fibroblast-like cells and contracting myocytes in EBs. RT-PCR and immunocytochemistry revealed that HA deprivation did not influence the dynamics of OCT4 expression, but decreased the expression of BRY, an early mesoderm marker, and BMP2, a later mesoderm marker in differentiating EBs. In addition, the endoderm markers α-FP and SOX17 were decreased, whereas the expression of the ectoderm markers GFAP and FGF5 was higher in HA-deprived cultures. Our findings indicate that endogenously produced HA contributes to the network that regulates the differentiation of hESC and the generation of mesodermal lineage in general and hematopoietic cells specifically. PMID:20861924

  3. The pleiotrophin-ALK axis is required for tumorigenicity of glioblastoma stem cells.

    PubMed

    Koyama-Nasu, R; Haruta, R; Nasu-Nishimura, Y; Taniue, K; Katou, Y; Shirahige, K; Todo, T; Ino, Y; Mukasa, A; Saito, N; Matsui, M; Takahashi, R; Hoshino-Okubo, A; Sugano, H; Manabe, E; Funato, K; Akiyama, T

    2014-04-24

    Increasing evidence suggests that brain tumors arise from the transformation of neural stem/precursor/progenitor cells. Much current research on human brain tumors is focused on the stem-like properties of glioblastoma. Here we show that anaplastic lymphoma kinase (ALK) and its ligand pleiotrophin are required for the self-renewal and tumorigenicity of glioblastoma stem cells (GSCs). Furthermore, we demonstrate that pleiotrophin is transactivated directly by SOX2, a transcription factor essential for the maintenance of both neural stem cells and GSCs. We speculate that the pleiotrophin-ALK axis may be a promising target for the therapy of glioblastoma.

  4. The C. elegans TIA-1/TIAR homolog TIAR-1 is required to induce germ cell apoptosis.

    PubMed

    Silva-García, Carlos Giovanni; Estela Navarro, Rosa

    2013-10-01

    In Caenorhabditis elegans, physiological germ cell apoptosis eliminates more than half of the cells in the hermaphrodite gonad to support gamete quality and germline homeostasis by a still unidentified mechanism. External factors can also affect germ cell apoptosis. The BH3-only protein EGL-1 induces germ cell apoptosis when animals are exposed to pathogens or agents that produce DNA damage. DNA damage-induced apoptosis also requires the nematode p53 homolog CEP-1. Previously, we found that heat shock, oxidative, and osmotic stresses induce germ cell apoptosis through an EGL-1 and CEP-1 independent mechanism that requires the MAPKK pathway. However, we observed that starvation increases germ cell apoptosis by an unknown pathway. Searching for proteins that participate in stress-induced apoptosis, we found the RNA-binding protein TIAR-1 (a homolog of the mammalian TIA-1/TIAR family of proteins). Here, we show that TIAR-1 in C. elegans is required to induce apoptosis in the germline under several conditions. We also show that TIAR-1 acts downstream of CED-9 (a BCL2 homolog) to induce apoptosis under stress conditions, and apparently does not seem to regulate ced-4 or ced-3 mRNAs accumulation directly. TIAR-1 is expressed ubiquitously in the cytoplasm of the soma as well as the germline, where it sometimes associates with P granules. We show that animals lacking TIAR-1 expression are temperature sensitive sterile due to oogenesis and spermatogenesis defects. Our work shows that TIAR-1 is required for proper germline function and demonstrates that this protein is important to induce germ cell apoptosis under several conditions.

  5. Merkel Cell Polyomavirus Small T Antigen Promotes Pro-Glycolytic Metabolic Perturbations Required for Transformation

    PubMed Central

    Keibler, Mark A.; Park, Donglim Esther; Molla, Vadim; Cheng, Jingwei; Stephanopoulos, Gregory

    2016-01-01

    Merkel cell polyomavirus (MCPyV) is an etiological agent of Merkel cell carcinoma (MCC), a highly aggressive skin cancer. The MCPyV small tumor antigen (ST) is required for maintenance of MCC and can transform normal cells. To gain insight into cellular perturbations induced by MCPyV ST, we performed transcriptome analysis of normal human fibroblasts with inducible expression of ST. MCPyV ST dynamically alters the cellular transcriptome with increased levels of glycolytic genes, including the monocarboxylate lactate transporter SLC16A1 (MCT1). Extracellular flux analysis revealed increased lactate export reflecting elevated aerobic glycolysis in ST expressing cells. Inhibition of MCT1 activity suppressed the growth of MCC cell lines and impaired MCPyV-dependent transformation of IMR90 cells. Both NF-κB and MYC have been shown to regulate MCT1 expression. While MYC was required for MCT1 induction, MCPyV-induced MCT1 levels decreased following knockdown of the NF-κB subunit RelA, supporting a synergistic activity between MCPyV and MYC in regulating MCT1 levels. Several MCC lines had high levels of MYCL and MYCN but not MYC. Increased levels of MYCL was more effective than MYC or MYCN in increasing extracellular acidification in MCC cells. Our results demonstrate the effects of MCPyV ST on the cellular transcriptome and reveal that transformation is dependent, at least in part, on elevated aerobic glycolysis. PMID:27880818

  6. 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

  7. eor-1 and eor-2 are required for cell-specific apoptotic death in C. elegans.

    PubMed

    Hoeppner, Daniel J; Spector, Mona S; Ratliff, Thomas M; Kinchen, Jason M; Granat, Susan; Lin, Shih-Chieh; Bhusri, Satjit S; Conradt, Barbara; Herman, Michael A; Hengartner, Michael O

    2004-10-01

    Programmed cell death occurs in every multicellular organism and in diverse cell types yet the genetic controls that define which cells will live and which will die remain poorly understood. During development of the nematode Caenorhabditis elegans, the coordinated activity of four gene products, EGL-1, CED-9, CED-4 and CED-3, results in the death of essentially all cells fated to die. To identify novel upstream components of the cell death pathway, we performed a genetic screen for mutations that abolish the death of the hermaphrodite-specific neurons (HSNs), a homologous pair of cells required for egg-laying in the hermaphrodite. We identified and cloned the genes, eor-1 and eor-2, which are required to specify the fate of cell death in male HSNs. In addition to defects in HSN death, mutation of either gene leads to defects in coordinated movement, neuronal migration, male tail development, and viability; all consistent with abnormal neuronal differentiation. eor-1 encodes a putative transcription factor related to the human oncogene PLZF. eor-2 encodes a novel but conserved protein. We propose that eor-1 and eor-2 function together throughout the nervous system to promote terminal differentiation of neurons and function specifically in male HSNs to promote apoptotic death of the HSNs.

  8. RNASET2 is required for ROS propagation during oxidative stress-mediated cell death

    PubMed Central

    Caputa, G; Zhao, S; Criado, A E G; Ory, D S; Duncan, J G; Schaffer, J E

    2016-01-01

    RNASET2 is a ubiquitously expressed acidic ribonuclease that has been implicated in diverse pathophysiological processes including tumorigeneis, vitiligo, asthenozoospermia, and neurodegeneration. Prior studies indicate that RNASET2 is induced in response to oxidative stress and that overexpression of RNASET2 sensitizes cells to reactive oxygen species (ROS)-induced cell death through a mechanism that is independent of catalytic activity. Herein, we report a loss-of-function genetic screen that identified RNASET2 as an essential gene for lipotoxic cell death. Haploinsufficiency of RNASET2 confers increased antioxidant capacity and generalized resistance to oxidative stress-mediated cell death in cultured cells. This function is critically dependent on catalytic activity. Furthermore, knockdown of RNASET2 in the Drosophila fat body confers increased survival in the setting of oxidative stress inducers. Together, these findings demonstrate that RNASET2 regulates antioxidant tone and is required for physiological ROS responses. PMID:26206090

  9. 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

    2015-05-11

    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.

  10. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    PubMed

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  11. Compatibility of the movement protein and the coat protein of cucumoviruses is required for cell-to-cell movement.

    PubMed

    Salánki, Katalin; Gellért, Akos; Huppert, Emese; Náray-Szabó, Gábor; Balázs, Ervin

    2004-04-01

    For the cell-to-cell movement of cucumoviruses both the movement protein (MP) and the coat protein (CP) are required. These are not reversibly exchangeable between Cucumber mosaic virus (CMV) and Tomato aspermy virus (TAV). The MP of CMV is able to function with the TAV CP (chimera RT), but TAV MP is unable to promote the cell-to-cell movement in the presence of CMV CP (chimera TR). To gain further insight into the non-infectious nature of the TR recombinant, RNA 3 chimeras were constructed with recombinant MPs and CPs. The chimeric MP and one of the CP recombinants were infectious. The other recombinant CP enabled virus movement only after the introduction of two point mutations (Glu-->Lys and Lys-->Arg at aa 62 and 65, respectively). The mutations served to correct the CP surface electrostatic potential that was altered by the recombination. The infectivity of the TR virus on different test plants was restored by replacing the sequence encoding the C-terminal 29 aa of the MP with the corresponding sequence of the CMV MP gene or by exchanging the sequence encoding the C-terminal 15 aa of the CP with the same region of TAV. The analysis of the recombinant clones suggests a requirement for compatibility between the C-terminal 29 aa of the MP and the C-terminal two-thirds of the CP for cell-to-cell movement of cucumoviruses.

  12. The actin gene ACT1 is required for phagocytosis, motility, and cell separation of Tetrahymena thermophila.

    PubMed

    Williams, Norman E; Tsao, Che-Chia; Bowen, Josephine; Hehman, Gery L; Williams, Ruth J; Frankel, Joseph

    2006-03-01

    A previously identified Tetrahymena thermophila actin gene (C. G. Cupples and R. E. Pearlman, Proc. Natl. Acad. Sci. USA 83:5160-5164, 1986), here called ACT1, was disrupted by insertion of a neo3 cassette. Cells in which all expressed copies of this gene were disrupted exhibited intermittent and extremely slow motility and severely curtailed phagocytic uptake. Transformation of these cells with inducible genetic constructs that contained a normal ACT1 gene restored motility. Use of an epitope-tagged construct permitted visualization of Act1p in the isolated axonemes of these rescued cells. In ACT1Delta mutant cells, ultrastructural abnormalities of outer doublet microtubules were present in some of the axonemes. Nonetheless, these cells were still able to assemble cilia after deciliation. The nearly paralyzed ACT1Delta cells completed cleavage furrowing normally, but the presumptive daughter cells often failed to separate from one another and later became reintegrated. Clonal analysis revealed that the cell cycle length of the ACT1Delta cells was approximately double that of wild-type controls. Clones could nonetheless be maintained for up to 15 successive fissions, suggesting that the ACT1 gene is not essential for cell viability or growth. Examination of the cell cortex with monoclonal antibodies revealed that whereas elongation of ciliary rows and formation of oral structures were normal, the ciliary rows of reintegrated daughter cells became laterally displaced and sometimes rejoined indiscriminately across the former division furrow. We conclude that Act1p is required in Tetrahymena thermophila primarily for normal ciliary motility and for phagocytosis and secondarily for the final separation of daughter cells.

  13. 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...

  14. FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration.

    PubMed

    Zhou, Lan; Jiang, Sufang; Fu, Qiang; Smith, Kelly; Tu, Kailing; Li, Hua; Zhao, Yuhua

    2016-05-01

    Both fatty acid synthase (FASN) and ErbB2 have been shown to promote breast cancer cell migration. However, the underlying molecular mechanism remains poorly understood and there is no reported evidence that directly links glycolysis to breast cancer cell migration. In this study, we investigated the role of FASN, ErbB2-mediated glycolysis in breast cancer cell migration. First, we compared lactate dehydrogenase A (LDHA) protein levels, glycolysis and cell migration between FASN, ErbB2-overexpressing SK-BR-3 cells and FASN, ErbB2-low-expressing MCF7 cells. Then, SK-BR-3 cells were treated with cerulenin (Cer), an inhibitor of FASN, and ErbB2, LDHA protein levels, glycolysis, and cell migration were detected. Next, we transiently transfected ErbB2 plasmid into MCF7 cells and detected FASN, LDHA protein levels, glycolysis and cell migration. Heregulin-β1 (HRG-β1) is an activator of ErbB2 and 2-deoxyglucose (2-DG) and oxamate (OX) are inhibitors of glycolysis. MCF7 cells were treated with HRG-β1 alone, HRG-β1 plus 2-DG, OX or cerulenin and glycolysis, and cell migration were measured. We found that FASN, ErbB2-high-expressing SK-BR-3 cells displayed higher levels of glycolysis and migration than FASN, ErbB2-low-expressing MCF7 cells. Inhibition of FASN by cerulenin impaired glycolysis and migration in SK-BR-3 cells. Transient overexpression of ErbB2 in MCF7 cells promotes glycolysis and migration. Moreover, 2-deoxyglucose (2-DG), oxamate (OX), or cerulenin partially reverses heregulin-β1 (HRG-β1)-induced glycolysis and migration in MCF7 cells. In conclusion, this study demonstrates that FASN, ErbB2-mediated glycolysis is required for breast cancer cell migration. These novel findings indicate that targeting FASN, ErbB2-mediated glycolysis may be a new approach to reverse breast cancer cell migration.

  15. Scrib is required for epithelial cell identity and prevents epithelial to mesenchymal transition in the mouse.

    PubMed

    Yamben, Idella F; Rachel, Rivka A; Shatadal, Shalini; Copeland, Neal G; Jenkins, Nancy A; Warming, Soren; Griep, Anne E

    2013-12-01

    The integrity and function of epithelial tissues depend on the establishment and maintenance of defining characteristics of epithelial cells, cell-cell adhesion and cell polarity. Disruption of these characteristics can lead to the loss of epithelial identity through a process called epithelial to mesenchymal transition (EMT), which can contribute to pathological conditions such as tissue fibrosis and invasive cancer. In invertebrates, the epithelial polarity gene scrib plays a critical role in establishing and maintaining cell adhesion and polarity. In this study we asked if the mouse homolog, Scrib, is required for establishment and/or maintenance of epithelial identity in vivo. To do so, we conditionally deleted Scrib in the head ectoderm tissue that gives rise to both the ocular lens and the corneal epithelium. Deletion of Scrib in the lens resulted in a change in epithelial cell shape from cuboidal to flattened and elongated. Early in the process, the cell adhesion protein, E-cadherin, and apical polarity protein, ZO-1, were downregulated and the myofibroblast protein, αSMA, was upregulated, suggesting EMT was occurring in the Scrib deficient lenses. Correlating temporally with the upregulation of αSMA, Smad3 and Smad4, TGFβ signaling intermediates, accumulated in the nucleus and Snail, a TGFβ target and transcriptional repressor of the gene encoding E-cadherin, was upregulated. Pax6, a lens epithelial transcription factor required to maintain lens epithelial cell identity also was downregulated. Loss of Scrib in the corneal epithelium also led to molecular changes consistent with EMT, suggesting that the effect of Scrib deficiency was not unique to the lens. Together, these data indicate that mammalian Scrib is required to maintain epithelial identity and that loss of Scrib can culminate in EMT, mediated, at least in part, through TGFβ signaling.

  16. 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.

  17. Notch signaling is required for normal prostatic epithelial cell proliferation and differentiation.

    PubMed

    Wang, Xi-De; Leow, Ching Ching; Zha, Jiping; Tang, Zhijun; Modrusan, Zora; Radtke, Freddy; Aguet, Michel; de Sauvage, Frederic J; Gao, Wei-Qiang

    2006-02-01

    Notch pathway is crucial for stem/progenitor cell maintenance, growth and differentiation in a variety of tissues. Using a transgenic cell ablation approach, we found in our previous study that cells expressing Notch1 are crucial for prostate early development and re-growth. Here, we further define the role of Notch signaling in regulating prostatic epithelial cell growth and differentiation using biochemical and genetic approaches in ex vivo or in vivo systems. Treatment of developing prostate grown in culture with inhibitors of gamma-secretase/presenilin, which is required for Notch cleavage and activation, caused a robust increase in proliferation of epithelial cells co-expressing cytokeratin 8 and 14, lack of luminal/basal layer segregation and dramatically reduced branching morphogenesis. Using conditional Notch1 gene deletion mouse models, we found that inactivation of Notch1 signaling resulted in profound prostatic alterations, including increased tufting, bridging and enhanced epithelial proliferation. Cells within these lesions co-expressed both luminal and basal cell markers, a feature of prostatic epithelial cells in predifferentiation developmental stages. Microarray analysis revealed that the gene expression in a number of genetic networks was altered following Notch1 gene deletion in prostate. Furthermore, expression of Notch1 and its effector Hey-1 gene in human prostate adenocarcinomas were found significantly down-regulated compared to normal control tissues. Taken together, these data suggest that Notch signaling is critical for normal cell proliferation and differentiation in the prostate, and deregulation of this pathway may facilitate prostatic tumorigenesis.

  18. 14-3-3ε Is Required for Germ Cell Migration in Drosophila

    PubMed Central

    Tsigkari, K. Kirki; Acevedo, Summer F.; Skoulakis, Efthimios M. C.

    2012-01-01

    Although 14-3-3 proteins participate in multiple biological processes, isoform-specific specialized functions, as well as functional redundancy are emerging with tissue and developmental stage-specificity. Accordingly, the two 14-3-3ε proteins in Drosophila exhibit functional specificity and redundancy. Homozygotes for loss of function alleles of D14-3-3ε contain significantly fewer germ line cells (pole cells) in their gonads, a phenotype not shared by mutants in the other 14-3-3 gene leo. We show that although D14-3-3ε is enriched within pole cells it is required in mesodermal somatic gonad precursor cells which guide pole cells in their migration through the mesoderm and coalesce with them to form the embryonic gonad. Loss of D14-3-3ε results in defective pole cell migration, reduced pole cell number. We present evidence that D14-3-3ε loss results in reduction or loss of the transcription factor Zfh-1, one of the main regulatory molecules of the pole cell migration, from the somatic gonad precursor cells. PMID:22666326

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

    PubMed Central

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

    2015-01-01

    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

  20. 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.

  1. Pineal Photoreceptor Cells Are Required for Maintaining the Circadian Rhythms of Behavioral Visual Sensitivity in Zebrafish

    PubMed Central

    Li, Xinle; Montgomery, Jake; Cheng, Wesley; Noh, Jung Hyun; Hyde, David R.; Li, Lei

    2012-01-01

    In non-mammalian vertebrates, the pineal gland functions as the central pacemaker that regulates the circadian rhythms of animal behavior and physiology. We generated a transgenic zebrafish line [Tg(Gnat2:gal4-VP16/UAS:nfsB-mCherry)] in which the E. coli nitroreductase is expressed in pineal photoreceptor cells. In developing embryos and young adults, the transgene is expressed in both retinal and pineal photoreceptor cells. During aging, the expression of the transgene in retinal photoreceptor cells gradually diminishes. By 8 months of age, the Gnat2 promoter-driven nitroreductase is no longer expressed in retinal photoreceptor cells, but its expression in pineal photoreceptor cells persists. This provides a tool for selective ablation of pineal photoreceptor cells, i.e., by treatments with metronidazole. In the absence of pineal photoreceptor cells, the behavioral visual sensitivity of the fish remains unchanged; however, the circadian rhythms of rod and cone sensitivity are diminished. Brief light exposures restore the circadian rhythms of behavioral visual sensitivity. Together, the data suggest that retinal photoreceptor cells respond to environmental cues and are capable of entraining the circadian rhythms of visual sensitivity; however, they are insufficient for maintaining the rhythms. Cellular signals from the pineal photoreceptor cells may be required for maintaining the circadian rhythms of visual sensitivity. PMID:22815753

  2. Phospholipase D-mTOR requirement for the Warburg effect in human cancer cells.

    PubMed

    Toschi, Alfredo; Lee, Evan; Thompson, Sebastian; Gadir, Noga; Yellen, Paige; Drain, C Michael; Ohh, Michael; Foster, David A

    2010-12-18

    A characteristic of cancer cells is the generation of lactate from glucose in spite of adequate oxygen for oxidative phosphorylation. This property - known as the "Warburg effect" or aerobic glycolysis - contrasts with anaerobic glycolysis, which is triggered in hypoxic normal cells. The Warburg effect is thought to provide a means for cancer cells to survive under conditions where oxygen is limited and to generate metabolites necessary for cell growth. The shift from oxidative phosphorylation to glycolysis in response to hypoxia is mediated by the production of hypoxia-inducible factor (HIF) - a transcription factor family that stimulates the expression of proteins involved in glucose uptake and glycolysis. We reported previously that elevated phospholipase D (PLD) activity in renal and breast cancer cells is required for the expression of the α subunits of HIF1 and HIF2. We report here that the aerobic glycolysis observed in human breast and renal cancer cells is dependent on the elevated PLD activity. Intriguingly, the effect of PLD on the Warburg phenotype was dependent on the mammalian target of rapamycin complex 1 (mTORC1) in the breast cancer cells and on mTORC2 in the renal cancer cells. These data indicate that elevated PLD-mTOR signaling, which is common in human cancer cells, is critical for the metabolic shift to aerobic glycolysis.

  3. 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

  4. γδ T cells in Experimental Autoimmune Encephalomyelitis: Early Trafficking Events and Cytokine Requirements

    PubMed Central

    Wohler, Jillian E.; Smith, Sherry S.; Zinn, Kurt R.; Bullard, Dan C.; Barnum, Scott R.

    2010-01-01

    We have previously shown that γδ T cells traffic to the CNS during EAE with concurrent increased expression of β2-integrins and production of IFN-γ and TNF-α. To extend these studies, we transferred bioluminescent γδ T cells to wild type mice and followed their movement through the acute stages of disease. We found that γδ T cells rapidly migrated to the site of myelin oligodendrocyte glycoprotein (MOG) peptide injection and underwent massive expansion. Within six days after EAE induction, bioluminescent γδ T cells were found in the spinal cord and brain, peaking in number between days ten and twelve and then rapidly declining by day fifteen. Reconstitution of γδ T cell−/− mice with γδ T cells derived from β2-integrin-deficient mice (CD11a, -b or -c) demonstrated that γδ T cell trafficking to the CNS during EAE is independent of this family of adhesion molecules. We also examined the role of γδ T cell-produced IFN-γ and TNF-α in EAE and found that production of both cytokines by γδ T cells was required for full development of EAE. These results indicate that γδ T cells are critical for the development of EAE and suggest a therapeutic target in demyelinating disease. PMID:19384874

  5. The effect of erythrocyte antigen structure on requirement for T cells*

    PubMed Central

    Byrd, W.; Feldmann, Marc; Palmer, J.

    1974-01-01

    The induction in mice of a humoral immune response to intact sheep erythrocytes, both in vivo and in vitro, requires participation of thymus-derived (T) lymphocytes. In an in vitro system, spleen cells from both neonatally thymectomized and adult thymectomized irradiated bone marrow protected mice were successfully immunized, using washed sonicated sheep erythrocyte membrane fragments as antigen. This obviation of the requirement of T lymphocytes in the immune response, coupled with previous work on macrophage independence, indicates that sonicated membrane fragments were capable of directly immunizing bone marrow-derived (B) lymphocytes in vitro. These results further confirm the signal importance of antigenic structure in determining the cellular requirements for an immunological response; whereas antigens of particulate or monomeric form require the presence of both T cells and macrophages, polymeric antigens of intermediate size such as polymerized flagellin and sonicated sheep erythrocyte membranes require neither of these accessory cells. The results caution against the use of erythrocytes as models of thymus-dependent antigens. The data further suggest that reports of late antibody responses of relatively normal magnitude in thymectomized animals given larger doses of heterologous erythrocytes may have been due to direct immunization of B lymphocytes by degraded erythrocyte antigen. PMID:4138234

  6. Murine Polyomavirus Cell Surface Receptors Activate Distinct Signaling Pathways Required for Infection

    PubMed Central

    O’Hara, Samantha D.

    2016-01-01

    ABSTRACT Virus binding to the cell surface triggers an array of host responses, including activation of specific signaling pathways that facilitate steps in virus entry. Using mouse polyomavirus (MuPyV), we identified host signaling pathways activated upon virus binding to mouse embryonic fibroblasts (MEFs). Pathways activated by MuPyV included the phosphatidylinositol 3-kinase (PI3K), FAK/SRC, and mitogen-activated protein kinase (MAPK) pathways. Gangliosides and α4-integrin are required receptors for MuPyV infection. MuPyV binding to both gangliosides and the α4-integrin receptors was required for activation of the PI3K pathway; however, either receptor interaction alone was sufficient for activation of the MAPK pathway. Using small-molecule inhibitors, we confirmed that the PI3K and FAK/SRC pathways were required for MuPyV infection, while the MAPK pathway was dispensable. Mechanistically, the PI3K pathway was required for MuPyV endocytosis, while the FAK/SRC pathway enabled trafficking of MuPyV along microtubules. Thus, MuPyV interactions with specific cell surface receptors facilitate activation of signaling pathways required for virus entry and trafficking. Understanding how different viruses manipulate cell signaling pathways through interactions with host receptors could lead to the identification of new therapeutic targets for viral infection. PMID:27803182

  7. MiR-24 Is Required for Hematopoietic Differentiation of Mouse Embryonic Stem Cells

    PubMed Central

    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

  8. Growth rate and cell size modulate the synthesis of, and requirement for, G1-phase cyclins at start.

    PubMed

    Schneider, Brandt L; Zhang, Jian; Markwardt, J; Tokiwa, George; Volpe, Tom; Honey, Sangeet; Futcher, Bruce

    2004-12-01

    In Saccharomyces cerevisiae, commitment to cell cycle progression occurs at Start. Progression past Start requires cell growth and protein synthesis, a minimum cell size, and G(1)-phase cyclins. We examined the relationships among these factors. Rapidly growing cells expressed, and required, dramatically more Cln protein than did slowly growing cells. To clarify the role of cell size, we expressed defined amounts of CLN mRNA in cells of different sizes. When Cln was expressed at nearly physiological levels, a critical threshold of Cln expression was required for cell cycle progression, and this critical threshold varied with both cell size and growth rate: as cells grew larger, they needed less CLN mRNA, but as cells grew faster, they needed more Cln protein. At least in part, large cells had a reduced requirement for CLN mRNA because large cells generated more Cln protein per unit of mRNA than did small cells. When Cln was overexpressed, it was capable of promoting Start rapidly, regardless of cell size or growth rate. In summary, the amount of Cln required for Start depends dramatically on both cell size and growth rate. Large cells generate more Cln1 or Cln2 protein for a given amount of CLN mRNA, suggesting the existence of a novel posttranscriptional size control mechanism.

  9. 2B4-SAP signaling is required for the priming of naive CD8(+) T cells by antigen-expressing B cells and B lymphoma cells.

    PubMed

    Huang, Yu-Hsuan; Tsai, Kevin; Tan, Sara Y; Kang, Sohyeong; Ford, Mandy L; Harder, Kenneth W; Priatel, John J

    2017-01-01

    Mutations in SH2D1A gene that encodes SAP (SLAM-associated protein) result in X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency disease defined by exquisite sensitivity to the B-lymphotropic Epstein-Barr virus (EBV) and B cell lymphomas. However, the precise mechanism of how the loss of SAP function contributes to extreme vulnerability to EBV and the development of B cell lymphomas remains unclear. Here, we investigate the hypothesis that SAP is critical for CD8(+) T cell immune surveillance of antigen (Ag)-expressing B cells or B lymphoma cells under conditions of defined T cell receptor (TCR) signaling. Sh2d1a(-)(/)(-) CD8(+) T cells exhibited greatly diminished proliferation relative to wild type when Ag-presenting-B cells or -B lymphoma cells served as the primary Ag-presenting cell (APC). By contrast, Sh2d1a(-)(/)(-) CD8(+) T cells responded equivalently to wild-type CD8(+) T cells when B cell-depleted splenocytes, melanoma cells or breast carcinoma cells performed Ag presentation. Through application of signaling lymphocyte activation molecule (SLAM) family receptor blocking antibodies or SLAM family receptor-deficient CD8(+) T cells and APCs, we found that CD48 engagement on the B cell surface by 2B4 is crucial for initiating SAP-dependent signaling required for the Ag-driven CD8(+) T cell proliferation and differentiation. Altogether, a pivotal role for SAP in promoting the expansion and differentiation of B cell-primed viral-specific naive CD8(+) T cells may explain the selective immune deficiency of XLP patients to EBV and B cell lymphomas.

  10. Embryonic origin of adult stem cells required for tissue homeostasis and regeneration

    PubMed Central

    Davies, Erin L; Lei, Kai; Seidel, Christopher W; Kroesen, Amanda E; McKinney, Sean A; Guo, Longhua; Robb, Sofia MC; Ross, Eric J; Gotting, Kirsten; Alvarado, Alejandro Sánchez

    2017-01-01

    Planarian neoblasts are pluripotent, adult somatic stem cells and lineage-primed progenitors that are required for the production and maintenance of all differentiated cell types, including the germline. Neoblasts, originally defined as undifferentiated cells residing in the adult parenchyma, are frequently compared to embryonic stem cells yet their developmental origin remains obscure. We investigated the provenance of neoblasts during Schmidtea mediterranea embryogenesis, and report that neoblasts arise from an anarchic, cycling piwi-1+ population wholly responsible for production of all temporary and definitive organs during embryogenesis. Early embryonic piwi-1+ cells are molecularly and functionally distinct from neoblasts: they express unique cohorts of early embryo enriched transcripts and behave differently than neoblasts in cell transplantation assays. Neoblast lineages arise as organogenesis begins and are required for construction of all major organ systems during embryogenesis. These subpopulations are continuously generated during adulthood, where they act as agents of tissue homeostasis and regeneration. DOI: http://dx.doi.org/10.7554/eLife.21052.001 PMID:28072387

  11. 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

  12. Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo.

    PubMed

    Maki, Nobuyasu; Suetsugu-Maki, Rinako; Sano, Shozo; Nakamura, Kenta; Nishimura, Osamu; Tarui, Hiroshi; Del Rio-Tsonis, Katia; Ohsumi, Keita; Agata, Kiyokazu; Tsonis, Panagiotis A

    2010-09-01

    The ability to reprogram in vivo a somatic cell after differentiation is quite limited. One of the most impressive examples of such a process is transdifferentiation of pigmented epithelial cells (PECs) to lens cells during lens regeneration in newts. However, very little is known of the molecular events that allow newt cells to transdifferentiate. Histone B4 is an oocyte-type linker histone that replaces the somatic-type linker histone H1 during reprogramming mediated by somatic cell nuclear transfer (SCNT). We found that B4 is expressed and required during transdifferentiation of PECs. Knocking down of B4 decreased proliferation and increased apoptosis, which resulted in considerable smaller lens. Furthermore, B4 knockdown altered gene expression of key genes of lens differentiation and nearly abolished expression of gamma-crystallin. These data are the first to show expression of oocyte-type linker histone in somatic cells and its requirement in newt lens transdifferentiation and suggest that transdifferentiation in newts might share common strategies with reprogramming after SCNT.

  13. 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

    2015-06-26

    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.

  14. 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

  15. Malignant Precursor Cells Pre-Exist in Human Breast DCIS and Require Autophagy for Survival

    PubMed Central

    Espina, Virginia; Mariani, Brian D.; Gallagher, Rosa I.; Tran, Khoa; Banks, Stacey; Wiedemann, Joy; Huryk, Heather; Mueller, Claudius; Adamo, Luana; Deng, Jianghong; Petricoin, Emanuel F.; Pastore, Lucia; Zaman, Syed; Menezes, Geetha; Mize, James; Johal, Jasbir; Edmiston, Kirsten; Liotta, Lance A.

    2010-01-01

    Background While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. Methodology and Principal Findings We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from

  16. NOTCH1 is required for regeneration of Clara cells during repair of airway injury.

    PubMed

    Xing, Yiming; Li, Aimin; Borok, Zea; Li, Changgong; Minoo, Parviz

    2012-05-01

    The airways of the mammalian lung are lined with highly specialized epithelial cell types that are the targets of airborne toxicants and injury. Notch signaling plays an important role in the ontogeny of airway epithelial cells, but its contributions to recruitment, expansion or differentiation of resident progenitor/stem cells, and repair and re-establishment of the normal composition of airway epithelium following injury have not been addressed. In this study, the role of a specific Notch receptor, Notch1, was investigated by targeted inactivation in the embryonic lung epithelium using the epithelial-specific Gata5-Cre driver line. Notch1-deficient mice are viable without discernible defects in pulmonary epithelial cell-fate determination and differentiation. However, in an experimental model of airway injury, activity of Notch1 is found to be required for normal repair of the airway epithelium. Absence of Notch1 reduced the ability of a population of cells distinguished by expression of PGP9.5, otherwise a marker of pulmonary neuroendocrine cells, which appears to serve as a reservoir for regeneration of Clara cells. Hairy/enhancer of split-5 (Hes5) and paired-box-containing gene 6 (Pax6) were found to be downstream targets of Notch1. Both Hes5 and Pax6 expressions were significantly increased in association with Clara cell regeneration in wild-type lungs. Ablation of Notch1 reduced Hes5 and Pax6 and inhibited airway epithelial repair. Thus, although dispensable in developmental ontogeny of airway epithelial cells, normal activity of Notch1 is required for repair of the airway epithelium. The signaling pathway by which Notch1 regulates the repair process includes stimulation of Hes5 and Pax6 gene expression.

  17. Development of promyelocytic leukemia zinc finger-expressing innate CD4 T cells requires stronger T-cell receptor signals than conventional CD4 T cells.

    PubMed

    Qiao, Yu; Zhu, Lingqiao; Sofi, Hanief; Lapinski, Philip E; Horai, Reiko; Mueller, Kristen; Stritesky, Gretta L; He, Xi; Teh, Hung-Sia; Wiest, David L; Kappes, Dietmar J; King, Philip D; Hogquist, Kristin A; Schwartzberg, Pamela L; Sant'Angelo, Derek B; Chang, Cheong-Hee

    2012-10-02

    MHC class II-expressing thymocytes and thymic epithelial cells can mediate CD4 T-cell selection resulting in functionally distinct thymocyte-selected CD4 (T-CD4) and epithelial-selected CD4 (E-CD4) T cells, respectively. However, little is known about how T-cell receptor (TCR) signaling influences the development of these two CD4 T-cell subsets. To study TCR signaling for T-CD4 T-cell development, we used a GFP reporter system of Nur77 in which GFP intensity directly correlates with TCR signaling strength. T-CD4 T cells expressed higher levels of GFP than E-CD4 T cells, suggesting that T-CD4 T cells received stronger TCR signaling than E-CD4 T cells during selection. Elimination of Ras GTPase-activating protein enhanced E-CD4 but decreased T-CD4 T-cell selection efficiency, suggesting a shift to negative selection. Conversely, the absence of IL-2-inducible T-cell kinase that causes poor E-CD4 T-cell selection due to insufficient TCR signaling improved T-CD4 T-cell generation, consistent with rescue from negative selection. Strong TCR signaling during T-CD4 T-cell development correlates with the expression of the transcription factor promyelocytic leukemia zinc finger protein. However, although modulation of the signaling strength affected the efficiency of T-CD4 T-cell development during positive and negative selection, the signaling strength is not as important for the effector function of T-CD4 T cells. These findings indicate that innate T-CD4 T cells, together with invariant natural killer T cells and γδ T cells, receive strong TCR signals during their development and that signaling requirements for the development and the effector functions are distinct.

  18. Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI)

    PubMed Central

    Diner, Elie J.; Beck, Christina M.; Webb, Julia S.; Low, David A.; Hayes, Christopher S.

    2012-01-01

    Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI+ inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI+ cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK+ cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells. PMID:22333533

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

    PubMed Central

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

    2009-01-01

    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 demonstrate 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 NK cells. Lung metastasis required CCR4+ Tregs which directly killed NK cells utilizing 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

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

    PubMed

    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

    2013-02-21

    It is widely appreciated that T cells increase glycolytic flux during activation, but the role of mitochondrial flux is unclear. Here, we have shown that mitochondrial metabolism in the absence of glucose metabolism is 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, but 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 the production of complex III ROS.

  1. Cell contacts are required for induction by cortisol of glutamine synthetase gene transcription in the retina.

    PubMed Central

    Vardimon, L; Fox, L L; Degenstein, L; Moscona, A A

    1988-01-01

    In embryonic neural retina the enzyme glutamine synthetase [GS; L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2] is a glia-specific differentiation marker inducible with cortisol. We show that cortisol elicits GS mRNA accumulation by stimulating transcription of the GS gene and that this stimulation requires cell contacts: in dissociated and separated retina cells GS gene transcription was not induced; when the separated cells were reassembled into multicellular aggregates, restoring cell contacts, accumulation of GS mRNA was again inducible. In cells dissociated from retina tissue that had been preinduced with cortisol, GS gene transcription rapidly declined, despite continued hormone availability. In the separated cells transcription of the histone H3.3 gene and accumulation of carbonic anhydrase II mRNA were unaffected; therefore, cell separation selectively precluded induction of the GS gene. These findings provide direct evidence for the regulatory role of cell contacts in hormonal control of gene transcription. Images PMID:2901094

  2. Protein kinase C theta is required for efficient induction of IL-10-secreting T cells

    PubMed Central

    Burton, Bronwen R.

    2017-01-01

    Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance. PMID:28158245

  3. Histone demethylase Jmjd3 is required for the development of subsets of retinal bipolar cells.

    PubMed

    Iida, Atsumi; Iwagawa, Toshiro; Kuribayashi, Hiroshi; Satoh, Shinya; Mochizuki, Yujin; Baba, Yukihiro; Nakauchi, Hiromitsu; Furukawa, Takahisa; Koseki, Haruhiko; Murakami, Akira; Watanabe, Sumiko

    2014-03-11

    Di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) is an important gene repression mechanism. H3K27me2/3-specific demethylase, Jmjd3, was expressed in the inner nuclear layer during late retinal development. In contrast, H3K27 methyltransferase, Ezh2, was highly expressed in the embryonic retina but its expression decreased rapidly after birth. Jmjd3 loss of function in the developing retina resulted in failed differentiation of PKC-positive bipolar cell subsets (rod-ON-BP) and reduced transcription factor Bhlhb4 expression, which is critical for the differentiation of rod-ON-BP cells. Overexpression of Bhlhb4, but not of other BP cell-related genes, such as transcription factors Neurod and Chx10, in Jmjd3-knockdown retina rescued loss of PKC-positive BP cells. Populations of other retinal cell subsets were not significantly affected. In addition, proliferation activity and apoptotic cell number during retinal development were not affected by the loss of Jmjd3. Levels of histone H3 trimethyl Lys27 (H3K27me3) in the Bhlhb4 locus were lower in Islet-1-positive BP cells and amacrine cells than in the Islet-1-negative cell fraction. The Islet-1-negative cell fraction consisted mainly of photoreceptors, suggestive of lineage-specific demethylation of H3K27me3 in the Bhlhb4 locus. We propose that lineage-specific H3K27me3 demethylation of critical gene loci by spatiotemporal-specific Jmjd3 expression is required for appropriate maturation of retinal cells.

  4. 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

  5. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells.

    PubMed

    Ishiai, M; Kurosaki, M; Pappu, R; Okawa, K; Ronko, I; Fu, C; Shibata, M; Iwamatsu, A; Chan, A C; Kurosaki, T

    1999-01-01

    Signaling through the B cell receptor (BCR) is essential for B cell function and development. Despite the key role of Syk in BCR signaling, little is known about the mechanism by which Syk transmits downstream effectors. BLNK (B cell LiNKer protein), a substrate for Syk, is now shown to be essential in activating phospholipase C (PLC)gamma 2 and JNK. The BCR-induced PLC gamma 2 activation, but not the JNK activation, was restored by introduction of PLC gamma 2 membrane-associated form into BLNK-deficient B cells. As JNK activation requires both Rac1 and PLC gamma 2, our results suggest that BLNK regulates the Rac1-JNK pathway, in addition to modulating PLC gamma 2 localization.

  6. Requirement for tyrosine phosphorylation in lipopolysaccharide-induced murine B-cell proliferation.

    PubMed Central

    Dearden-Badet, M T; Revillard, J P

    1993-01-01

    Bacterial lipopolysaccharide (LPS) induces a strong B-cell proliferative response with subsequent differentiation, through a complex signal transduction pathway. This process is known to be mediated through protein kinase C (PKC) translocation without Ca2+ mobilization. Here, we show that B-cell proliferative responses induced by five different LPS preparations, as well as by F(ab')2 anti-IgM antibodies, are inhibited by the tyrosine kinase inhibitors, genistein and herbimycin A. In contrast, B-cell proliferation induced by the combination of phorbol 12-myristate 13-acetate (PMA) plus ionomycin was not influenced by treatment with either herbimycin A or genistein. These data indicate that tyrosine phosphorylation is required to initiate B-cell proliferation by LPS. PMID:8307617

  7. Prox1 is required for granule cell maturation and intermediate progenitor maintenance during brain neurogenesis.

    PubMed

    Lavado, Alfonso; Lagutin, Oleg V; Chow, Lionel M L; Baker, Suzanne J; Oliver, Guillermo

    2010-08-17

    The dentate gyrus has an important role in learning and memory, and adult neurogenesis in the subgranular zone of the dentate gyrus may play a role in the acquisition of new memories. The homeobox gene Prox1 is expressed in the dentate gyrus during embryonic development and adult neurogenesis. Here we show that Prox1 is necessary for the maturation of granule cells in the dentate gyrus during development and for the maintenance of intermediate progenitors during adult neurogenesis. We also demonstrate that Prox1-expressing intermediate progenitors are required for adult neural stem cell self-maintenance in the subgranular zone; thus, we have identified a previously unknown non-cell autonomous regulatory feedback mechanism that controls adult neurogenesis in this region of the mammalian brain. Finally, we show that the ectopic expression of Prox1 induces premature differentiation of neural stem cells.

  8. Identification of a novel cyclin required for the intrinsic apoptosis pathway in lymphoid cells.

    PubMed

    Roig, M B; Roset, R; Ortet, L; Balsiger, N A; Anfosso, A; Cabellos, L; Garrido, M; Alameda, F; Brady, H J M; Gil-Gómez, G

    2009-02-01

    We have identified an early step common to pathways activated by different forms of intrinsic apoptosis stimuli. It requires de novo synthesis of a novel cyclin, cyclin O, that forms active complexes primarily with Cdk2 upon apoptosis induction in lymphoid cells. Cyclin O expression precedes glucocorticoid and gamma-radiation-induced apoptosis in vivo in mouse thymus and spleen, and its overexpression induces caspase-dependent apoptosis in cultured cells. Knocking down the endogenous expression of cyclin O by shRNA leads to the inhibition of glucocorticoid and DNA damage-induced apoptosis due to a failure in the activation of apical caspases while leaving CD95 death receptor-mediated apoptosis intact. Our data demonstrate that apoptosis induction in lymphoid cells is one of the physiological roles of cyclin O and it does not act by perturbing a normal cellular process such as the cell cycle, the DNA damage checkpoints or transcriptional response to glucocorticoids.

  9. 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.

  10. humpty dumpty is required for developmental DNA amplification and cell proliferation in Drosophila.

    PubMed

    Bandura, Jennifer L; Beall, Eileen L; Bell, Maren; Silver, Hannah R; Botchan, Michael R; Calvi, Brian R

    2005-04-26

    The full complement of proteins required for the proper regulation of genome duplication are yet to be described. We employ a genetic DNA-replication model system based on developmental amplification of Drosophila eggshell (chorion) genes [1]. Hypomorphic mutations in essential DNA replication genes result in a distinct thin-eggshell phenotype owing to reduced amplification [2]. Here, we molecularly identify the gene, which we have named humpty dumpty (hd), corresponding to the thin-eggshell mutant fs(3)272-9 [3]. We confirm that hd is essential for DNA amplification in the ovary and show that it also is required for cell proliferation during development. Mosaic analysis of hd mutant cells during development and RNAi in Kc cells reveal that depletion of Hd protein results in severe defects in genomic replication and DNA damage. Most Hd protein is found in nuclear foci, and some may traverse the nuclear envelope. Consistent with a role in DNA replication, expression of Hd protein peaks during late G1 and S phase, and it responds to the E2F1/Dp transcription factor. Hd protein sequence is conserved from plants to humans, and published microarrays indicate that expression of its putative human ortholog also peaks at G1/S [4]. Our data suggest that hd defines a new gene family likely required for cell proliferation in all multicellular eukaryotes.

  11. Cell cycle progression in Caulobacter requires a nucleoid-associated protein with high AT sequence recognition

    PubMed Central

    Ricci, Dante P.; Melfi, Michael D.; Lasker, Keren; Dill, David L.; McAdams, Harley H.; Shapiro, Lucy

    2016-01-01

    Faithful cell cycle progression in the dimorphic bacterium Caulobacter crescentus requires spatiotemporal regulation of gene expression and cell pole differentiation. We discovered an essential DNA-associated protein, GapR, that is required for Caulobacter growth and asymmetric division. GapR interacts with adenine and thymine (AT)-rich chromosomal loci, associates with the promoter regions of cell cycle-regulated genes, and shares hundreds of recognition sites in common with known master regulators of cell cycle-dependent gene expression. GapR target loci are especially enriched in binding sites for the transcription factors GcrA and CtrA and overlap with nearly all of the binding sites for MucR1, a regulator that controls the establishment of swarmer cell fate. Despite constitutive synthesis, GapR accumulates preferentially in the swarmer compartment of the predivisional cell. Homologs of GapR, which are ubiquitous among the α-proteobacteria and are encoded on multiple bacteriophage genomes, also accumulate in the predivisional cell swarmer compartment when expressed in Caulobacter. The Escherichia coli nucleoid-associated protein H-NS, like GapR, selectively associates with AT-rich DNA, yet it does not localize preferentially to the swarmer compartment when expressed exogenously in Caulobacter, suggesting that recognition of AT-rich DNA is not sufficient for the asymmetric accumulation of GapR. Further, GapR does not silence the expression of H-NS target genes when expressed in E. coli, suggesting that GapR and H-NS have distinct functions. We propose that Caulobacter has co-opted a nucleoid-associated protein with high AT recognition to serve as a mediator of cell cycle progression. PMID:27647925

  12. Fatty acid oxidation is required for the respiration and proliferation of malignant glioma cells

    PubMed Central

    Lin, Hua; Patel, Shaan; Affleck, Valerie S.; Wilson, Ian; Turnbull, Douglass M.; Joshi, Abhijit R.; Maxwell, Ross

    2017-01-01

    Background. Glioma is the most common form of primary malignant brain tumor in adults, with approximately 4 cases per 100 000 people each year. Gliomas, like many tumors, are thought to primarily metabolize glucose for energy production; however, the reliance upon glycolysis has recently been called into question. In this study, we aimed to identify the metabolic fuel requirements of human glioma cells. Methods. We used database searches and tissue culture resources to evaluate genotype and protein expression, tracked oxygen consumption rates to study metabolic responses to various substrates, performed histochemical techniques and fluorescence-activated cell sorting-based mitotic profiling to study cellular proliferation rates, and employed an animal model of malignant glioma to evaluate a new therapeutic intervention. Results. We observed the presence of enzymes required for fatty acid oxidation within human glioma tissues. In addition, we demonstrated that this metabolic pathway is a major contributor to aerobic respiration in primary-cultured cells isolated from human glioma and grown under serum-free conditions. Moreover, inhibiting fatty acid oxidation reduces proliferative activity in these primary-cultured cells and prolongs survival in a syngeneic mouse model of malignant glioma. Conclusions. Fatty acid oxidation enzymes are present and active within glioma tissues. Targeting this metabolic pathway reduces energy production and cellular proliferation in glioma cells. The drug etomoxir may provide therapeutic benefit to patients with malignant glioma. In addition, the expression of fatty acid oxidation enzymes may provide prognostic indicators for clinical practice. PMID:27365097

  13. p38α MAPK is required for arsenic-induced cell transformation.

    PubMed

    Kim, Hong-Gyum; Shi, Chengcheng; Bode, Ann M; Dong, Zigang

    2016-05-01

    Arsenic exposure has been reported to cause neoplastic transformation through the activation of PcG proteins. In the present study, we show that activation of p38α mitogen-activated protein kinase (MAPK) is required for arsenic-induced neoplastic transformation. Exposure of cells to 0.5 μM arsenic increased CRE and c-Fos promoter activities that were accompanied by increases in p38α MAPK and CREB phosphorylation and expression levels concurrently with AP-1 activation. Introduction of short hairpin (sh) RNA-p38α into BALB/c 3T3 cells markedly suppressed arsenic-induced colony formation compared with wildtype cells. CREB phosphorylation and AP-1 activation were decreased in p38α knockdown cells after arsenic treatment. Arsenic-induced AP-1 activation, measured as c-Fos and CRE promoter activities, and CREB phosphorylation were attenuated by p38 inhibition in BALB/c 3T3 cells. Thus, p38α MAPK activation is required for arsenic-induced neoplastic transformation mediated through CREB phosphorylation and AP-1 activation.

  14. TGFβ/Activin signalling is required for ribosome biogenesis and cell growth in Drosophila salivary glands.

    PubMed

    Martins, Torcato; Eusebio, Nadia; Correia, Andreia; Marinho, Joana; Casares, Fernando; Pereira, Paulo S

    2017-01-01

    Signalling by TGFβ superfamily factors plays an important role in tissue growth and cell proliferation. In Drosophila, the activity of the TGFβ/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. In this study, we show that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFβ/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, our results show that TGFβ/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis.

  15. TGFβ/Activin signalling is required for ribosome biogenesis and cell growth in Drosophila salivary glands

    PubMed Central

    Eusebio, Nadia; Correia, Andreia; Marinho, Joana; Casares, Fernando

    2017-01-01

    Signalling by TGFβ superfamily factors plays an important role in tissue growth and cell proliferation. In Drosophila, the activity of the TGFβ/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. In this study, we show that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFβ/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, our results show that TGFβ/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis. PMID:28123053

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

    PubMed Central

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

    2016-01-01

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

  17. TGF-β Effects on Prostate Cancer Cell Migration and Invasion Require FosB

    PubMed Central

    Barrett, Cachétne S.X.; Millena, Ana C.; Khan, Shafiq A.

    2017-01-01

    were also significantly decreased after FosB knockdown in PC3 cells. CONCLUSION Our data suggest that FosB is required for migration and invasion in prostate cancer cells. We also conclude that TGF-β1 effect on prostate cancer cell migration and invasion may be mediated through the induction of FosB. PMID:27604827

  18. Physiological levels of reactive oxygen species are required to maintain genomic stability in stem cells.

    PubMed

    Li, Tao-Sheng; Marbán, Eduardo

    2010-07-01

    Stem cell cytogenetic abnormalities constitute a roadblock to regenerative therapies. We investigated the possibility that reactive oxygen species (ROSs) influence genomic stability in cardiac and embryonic stem cells. Karyotypic abnormalities in primary human cardiac stem cells were suppressed by culture in physiological (5%) oxygen, but addition of antioxidants to the medium unexpectedly increased aneuploidy. Intracellular ROS levels were moderately decreased in physiological oxygen, but dramatically decreased by the addition of high-dose antioxidants. Quantification of DNA damage in cardiac stem cells and in human embryonic stem cells revealed a biphasic dose-dependence: antioxidants suppressed DNA damage at low concentrations, but potentiated such damage at higher concentrations. High-dose antioxidants decreased cellular levels of ATM (ataxia-telangiectasia mutated) and other DNA repair enzymes, providing a potential mechanistic basis for the observed effects. These results indicate that physiological levels of intracellular ROS are required to activate the DNA repair pathway for maintaining genomic stability in stem cells. The concept of an "oxidative optimum" for genomic stability has broad implications for stem cell biology and carcinogenesis.

  19. Type I interferon is selectively required by dendritic cells for immune rejection of tumors.

    PubMed

    Diamond, Mark S; Kinder, Michelle; Matsushita, Hirokazu; Mashayekhi, Mona; Dunn, Gavin P; Archambault, Jessica M; Lee, Hsiaoju; Arthur, Cora D; White, J Michael; Kalinke, Ulrich; Murphy, Kenneth M; Schreiber, Robert D

    2011-09-26

    Cancer immunoediting is the process whereby the immune system suppresses neoplastic growth and shapes tumor immunogenicity. We previously reported that type I interferon (IFN-α/β) plays a central role in this process and that hematopoietic cells represent critical targets of type I IFN's actions. However, the specific cells affected by IFN-α/β and the functional processes that type I IFN induces remain undefined. Herein, we show that type I IFN is required to initiate the antitumor response and that its actions are temporally distinct from IFN-γ during cancer immunoediting. Using mixed bone marrow chimeric mice, we demonstrate that type I IFN sensitivity selectively within the innate immune compartment is essential for tumor-specific T cell priming and tumor elimination. We further show that mice lacking IFNAR1 (IFN-α/β receptor 1) in dendritic cells (DCs; Itgax-Cre(+)Ifnar1(f/f) mice) cannot reject highly immunogenic tumor cells and that CD8α(+) DCs from these mice display defects in antigen cross-presentation to CD8(+) T cells. In contrast, mice depleted of NK cells or mice that lack IFNAR1 in granulocytes and macrophage populations reject these tumors normally. Thus, DCs and specifically CD8α(+) DCs are functionally relevant targets of endogenous type I IFN during lymphocyte-mediated tumor rejection.

  20. 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.

  1. 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.

  2. Enterogenous bacterial glycolipids are required for the generation of natural killer T cells mediated liver injury

    PubMed Central

    Wei, Yingfeng; Zeng, Benhua; Chen, Jianing; Cui, Guangying; Lu, Chong; Wu, Wei; Yang, Jiezuan; Wei, Hong; Xue, Rufeng; Bai, Li; Chen, Zhi; Li, Lanjuan; Iwabuchi, Kazuya; Uede, Toshimitsu; Van Kaer, Luc; Diao, Hongyan

    2016-01-01

    Glycolipids are potent activator of natural killer T (NKT) cells. The relationship between NKT cells and intestinal bacterial glycolipids in liver disorders remained unclear. We found that, in sharp contrast to specific pathogen-free (SPF) mice, germ-free (GF) mice are resistant to Concanavalin A (ConA)-induced liver injury. ConA treatment failed to trigger the activation of hepatic NKT cells in GF mice. These defects correlated with the sharply reduced levels of CD1d-presented glycolipid antigens in ConA-treated GF mice compared with SPF counterparts. Nevertheless, CD1d expression was similar between these two kinds of mice. The absence of intestinal bacteria did not affect the incidence of αGalCer-induced liver injury in GF mice. Importantly, we found the intestinal bacteria contain glycolipids which can be presented by CD1d and recognized by NKT cells. Furthermore, supplement of killed intestinal bacteria was able to restore ConA-mediated NKT cell activation and liver injury in GF mice. Our results suggest that glycolipid antigens derived from intestinal commensal bacteria are important hepatic NKT cell agonist and these antigens are required for the activation of NKT cells during ConA-induced liver injury. These finding provide a mechanistic explanation for the capacity of intestinal microflora to control liver inflammation. PMID:27821872

  3. Tumor suppressor gene Rb is required for self-renewal of spermatogonial stem cells in mice.

    PubMed

    Hu, Yueh-Chiang; de Rooij, Dirk G; Page, David C

    2013-07-30

    The retinoblastoma tumor suppressor gene Rb is essential for maintaining the quiescence and for regulating the differentiation of somatic stem cells. Inactivation of Rb in somatic stem cells typically leads to their overexpansion, often followed by increased apoptosis, defective terminal differentiation, and tumor formation. However, Rb's roles in germ-line stem cells have not been explored. We conditionally disrupted the Rb gene in mouse germ cells in vivo and discovered unanticipated consequences for GFRa1-protein-expressing A(single) (GFRa1(+) A(s)) spermatogonia, the major source of male germ-line stem cells. Rb-deficient GFRa1(+) A(s) spermatogonia were present at normal density in testes 5 d after birth, but they lacked the capacity for self-renewal, resulting in germ cell depletion by 2 mo of age. Rb deficiency did not affect the proliferative activity of GFRa1(+) A(s) spermatogonia, but their progeny were exclusively transit-amplifying progenitor spermatogonia and did not include GFRa1(+) A(s) spermatogonia. In addition, Rb deficiency caused prolonged proliferation of progenitor spermatogonia, transiently enlarging this population. Despite these defects, Rb deficiency did not block terminal differentiation into functional sperm; offspring were readily obtained from young males whose germ cell pool was not yet depleted. We conclude that Rb is required for self-renewal of germ-line stem cells, but contrary to its critical roles in somatic stem cells, it is dispensable for their proliferative activity and terminal differentiation. Thus, this study identifies an unexpected function for Rb in maintaining the stem cell pool in the male germ line.

  4. Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis

    PubMed Central

    Félix, Marie-Anne

    2016-01-01

    Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change—less than 30%—in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR) binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression. PMID:27588814

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

    PubMed

    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.

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

    PubMed Central

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

    2014-01-01

    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 ~226x 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

  7. 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

  8. Invasive Cell Fate Requires G1 Cell-Cycle Arrest and Histone Deacetylase-Mediated Changes in Gene Expression.

    PubMed

    Matus, David Q; Lohmer, Lauren L; Kelley, Laura C; Schindler, Adam J; Kohrman, Abraham Q; Barkoulas, Michalis; Zhang, Wan; Chi, Qiuyi; Sherwood, David R

    2015-10-26

    Despite critical roles in development and cancer, the mechanisms that specify invasive cellular behavior are poorly understood. Through a screen of transcription factors in Caenorhabditis elegans, we identified G1 cell-cycle arrest as a precisely regulated requirement of the anchor cell (AC) invasion program. We show that the nuclear receptor nhr-67/tlx directs the AC into G1 arrest in part through regulation of the cyclin-dependent kinase inhibitor cki-1. Loss of nhr-67 resulted in non-invasive, mitotic ACs that failed to express matrix metalloproteinases or actin regulators and lack invadopodia, F-actin-rich membrane protrusions that facilitate invasion. We further show that G1 arrest is necessary for the histone deacetylase HDA-1, a key regulator of differentiation, to promote pro-invasive gene expression and invadopodia formation. Together, these results suggest that invasive cell fate requires G1 arrest and that strategies targeting both G1-arrested and actively cycling cells may be needed to halt metastatic cancer.

  9. Dll1- and Dll4-mediated Notch signaling is required for homeostasis of intestinal stem cells

    PubMed Central

    Pellegrinet, Luca; Rodilla, Veronica; Liu, Zhenyi; Chen, Shuang; Koch, Ute; Espinosa, Lluis; Kaestner, Klaus H.; Kopan, Raphael; Lewis, Julian; Radtke, Freddy

    2011-01-01

    Background & Aims Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors, due to their conversion into post-mitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SC), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiological ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4). Methods Trasgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ERT2). Results Notch1 signaling was found to be activated in intestinal SC. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into post-mitotic goblet cells, concomitant with loss of SC (Olfm4+, Lgr5+ and Ascl2+). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut. Conclusions Notch signaling in SC and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SC. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice. PMID:21238454

  10. TIA-1 or TIAR is required for DT40 cell viability.

    PubMed

    Le Guiner, Caroline; Gesnel, Marie-Claude; Breathnach, Richard

    2003-03-21

    TIA-1 and TIAR are a pair of related RNA-binding proteins which have been implicated in apoptosis. We show that chicken DT40 cells with both tia-1 alleles and one tiar allele disrupted (tia-1(-/-)tiar(-/+) cells) are viable. However, their growth and survival in medium containing low serum levels is significantly reduced compared with DT40 cells. The remaining intact tiar allele in tia-1(-/-)tiar(-/+) cells can only be disrupted if TIA-1 expression is first restored to the cells by transfection of a TIA-1 expression vector. We conclude that DT40 cells require either TIA-1 or TIAR for viability. TIA-1 overexpression in tia-1(-/-)tiar(-/+) cells leads to a radical drop in TIAR levels, by inducing efficient splicing of two tiar alternative exons carrying in-frame stop codons. In wild-type DT40 cells, tiar transcripts including these exons can also be detected. These transcripts increase significantly in abundance in cycloheximide-treated cells, suggesting that splicing of the exons exposes mRNAs to nonsense-mediated mRNA decay. TIA-1 or TIAR depletion leads to a marked drop in splicing of the exons. The human tiar gene contains a corresponding pair of TIA-1-inducible alternative exons, and we show that there is very high sequence conservation between chickens and humans of the exon pair and parts of the flanking introns. The TIA-1/TIAR responsiveness of these alternative tiar exons is likely to be of physiological importance for controlling TIAR levels.

  11. β-Catenin is required for the tumorigenic behavior of triple-negative breast cancer cells.

    PubMed

    Xu, Jinhua; Prosperi, Jenifer R; Choudhury, Noura; Olopade, Olufunmilayo I; Goss, Kathleen H

    2015-01-01

    Our previous data illustrated that activation of the canonical Wnt signaling pathway was enriched in triple-negative breast cancer and associated with reduced overall survival in all patients. To determine whether Wnt signaling may be a promising therapeutic target for triple-negative breast cancer, we investigated whether β-catenin was necessary for tumorigenic behaviors in vivo and in vitro. β-catenin expression level was significantly reduced in two human triple-negative breast cancer cell lines, MDA-MB-231 and HCC38, using lentiviral delivery of β-catenin-specific small hairpin RNAs (shRNAs). Upon implantation of the cells in the mammary fat pad of immunocompromised mice, we found that β-catenin shRNA HCC38 cells formed markedly smaller tumors than control cells and grew much more slowly. In in vitro assays, β-catenin silencing significantly reduced the percentage of Aldefluor-positive cells, a read-out of the stem-like cell population, as well as the expression of stem cell-related target genes including Bmi-1 and c-Myc. β-catenin-knockdown cells were also significantly impaired in their ability to migrate in wound-filling assays and form anchorage-independent colonies in soft agar. β-catenin-knockdown cells were more sensitive to chemotherapeutic agents doxorubicin and cisplatin. Collectively, these data suggest that β-catenin is required for triple-negative breast cancer development by controlling numerous tumor-associated properties, such as migration, stemness, anchorage-independent growth and chemosensitivity.

  12. 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.

  13. Protective cellular retroviral immunity requires both CD4+ and CD8+ immune T cells.

    PubMed Central

    Hom, R C; Finberg, R W; Mullaney, S; Ruprecht, R M

    1991-01-01

    We have found previously that postexposure chemoprophylaxis with 3'-azido-3'-deoxythymidine (also known as zidovudine or AZT) in combination with recombinant human alpha A/D interferon fully protected mice exposed to a lethal dose of Rauscher murine leukemia virus (RLV) against viremia and disease. After cessation of therapy, over 90% of these mice were able to resist rechallenge with live RLV, thus demonstrating an acquired immunity. Adoptive cell transfer of 4 x 10(7) cells from immunized mice fully protected naive recipients from viremia and splenomegaly after RLV challenge. However, when these immune T cells were fractionated into CD4+ and CD8+ subpopulations, only partial protection was found when 4 x 10(7) T cells of either subset were given. Full protection against RLV challenge was seen again when the T-cell subsets from immunized mice were recombined and transferred at the same number into naive mice. We conclude that cellular immunity alone is protective and that both CD4+ and CD8+ cell types are required for conferring full protection against live virus challenge. Images PMID:1898666

  14. 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.

  15. The vacuole/lysosome is required for cell-cycle progression

    PubMed Central

    Jin, Yui; Weisman, Lois S

    2015-01-01

    Organelles are distributed to daughter cells, via inheritance pathways. However, it is unclear whether there are mechanisms beyond inheritance, which ensure that organelles are present in all cells. Here we present the unexpected finding that the yeast vacuole plays a positive essential role in initiation of the cell-cycle. When inheritance fails, a new vacuole is generated. We show that this occurs prior to the next cell-cycle, and gain insight into this alternative pathway. Moreover, we find that a combination of a defect in inheritance with an acute block in the vacuole biogenesis results in the loss of a functional vacuole and a specific arrest of cells in early G1 phase. Furthermore, this role for the vacuole in cell-cycle progression requires an intact TORC1-SCH9 pathway that can only signal from a mature vacuole. These mechanisms may serve as a checkpoint for the presence of the vacuole/lysosome. DOI: http://dx.doi.org/10.7554/eLife.08160.001 PMID:26322385

  16. 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.

  17. Cell cycle progression requires the CDC-48UFD-1/NPL-4 complex for efficient DNA replication.

    PubMed

    Mouysset, Julien; Deichsel, Alexandra; Moser, Sandra; Hoege, Carsten; Hyman, Anthony A; Gartner, Anton; Hoppe, Thorsten

    2008-09-02

    Since cdc48 mutants were isolated by the first genetic screens for cell division cycle (cdc) mutants in yeast, the requirement of the chaperone-like ATPase Cdc48/p97 during cell division has remained unclear. Here, we discover an unanticipated function for Caenorhabditis elegans CDC-48 in DNA replication linked to cell cycle control. Our analysis of the CDC-48(UFD-1/NPL-4) complex identified a general role in S phase progression of mitotic cells essential for embryonic cell division and germline development of adult worms. These developmental defects result from activation of the DNA replication checkpoint caused by replication stress. Similar to loss of replication licensing factors, DNA content is strongly reduced in worms depleted for CDC-48, UFD-1, and NPL-4. In addition, these worms show decreased DNA synthesis and hypersensitivity toward replication blocking agents. Our findings identified a role for CDC-48(UFD-1/NPL-4) in DNA replication, which is important for cell cycle progression and genome stability.

  18. 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.

  19. 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

  20. AP4 is required for mitogen- and c-MYC-induced cell cycle progression

    PubMed Central

    Jackstadt, Rene; Hermeking, Heiko

    2014-01-01

    AP4 represents a c-MYC-inducible bHLH-LZ transcription factor, which displays elevated expression in many types of tumors. We found that serum-starved AP4-deficient mouse embryo fibroblasts (MEFs) were unable to resume proliferation and showed a delayed S-phase entry after restimulation. Furthermore, they accumulated as tetraploid cells due to a cytokinesis defect. In addition, AP4 was required for c-MYC-induced cell cycle re-entry. AP4-deficient MEFs displayed decreased expression of CDK2 (cyclin-dependent kinase 2), which we characterized as a conserved and direct AP4 target. Activation of an AP4 estrogen receptor fusion protein (AP4-ER) enhanced proliferation of human diploid fibroblasts in a CDK2-dependent manner. However, in contrast to c-MYC-ER, AP4-ER activation was not sufficient to induce cell cycle re-entry or apoptosis in serum-starved MEFs. AP4-deficiency was accompanied by increased spontaneous and c-MYC-induced DNA damage in MEFs. Furthermore, c-MYC-induced apoptosis was decreased in AP4-deficient MEFs, suggesting that induction of apoptosis by c-MYC is linked to its ability to activate AP4 and thereby cell cycle progression. Taken together, these results indicate that AP4 is a central mediator and coordinator of cell cycle progression in response to mitogenic signals and c-MYC activation. Therefore, inhibition of AP4 function may represent a therapeutic approach to block tumor cell proliferation. PMID:25261373

  1. 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

  2. Barx2 is expressed in satellite cells and is required for normal muscle growth and regeneration.

    PubMed

    Meech, Robyn; Gonzalez, Katie N; Barro, Marietta; Gromova, Anastasia; Zhuang, Lizhe; Hulin, Julie-Ann; Makarenkova, Helen P

    2012-02-01

    Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell-dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell-derived myoblasts cultured from Barx2(-/-) mice show decreased proliferation and ability to differentiate relative to those from wild-type or Barx2(+/-) mice. Barx2(-/-) myoblasts show reduced expression of the differentiation-associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation.

  3. Hydrogen Monitoring Requirements in the Global Technical Regulation on Hydrogen and Fuel Cell Vehicles: Preprint

    SciTech Connect

    Buttner, William; Rivkin, Carl; Burgess, Robert; Hartmann, Kevin; Bubar, Max; Post, Matthew; Boon-Brett, Lois; Weidner, Eveline; Moretto, Pietro

    2016-07-01

    The United Nations Global Technical Regulation (GTR) Number 13 (Global Technical Regulation on Hydrogen and Fuel Cell Vehicles) is the defining document regulating safety requirements in hydrogen vehicles, and in particular fuel cell electric vehicles (FCEV). GTR Number 13 has been formally implemented and will serve as the basis for the national regulatory standards for FCEV safety in North America (Canada, United States), Japan, Korea, and the European Union. The GTR defines safety requirement for these vehicles, including specifications on the allowable hydrogen levels in vehicle enclosures during in-use and post-crash conditions and on the allowable hydrogen emissions levels in vehicle exhaust during certain modes of normal operation. However, in order to be incorporated into national regulations, that is, in order to be binding, methods to verify compliance to the specific requirements must exist. In a collaborative program, the Sensor Laboratories at the National Renewable Energy Laboratory in the United States and the Joint Research Centre, Institute for Energy and Transport in the Netherlands have been evaluating and developing analytical methods that can be used to verify compliance to the hydrogen release requirement as specified in the GTR.

  4. Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis

    PubMed Central

    Albini, Sonia; Coutinho Toto, Paula; Dall’Agnese, Alessandra; Malecova, Barbora; Cenciarelli, Carlo; Felsani, Armando; Caruso, Maurizia; Bultman, Scott J; Puri, Pier Lorenzo

    2015-01-01

    Although the two catalytic subunits of the SWI/SNF chromatin-remodeling complex—Brahma (Brm) and Brg1—are almost invariably co-expressed, their mutually exclusive incorporation into distinct SWI/SNF complexes predicts that Brg1- and Brm-based SWI/SNF complexes execute specific functions. Here, we show that Brg1 and Brm have distinct functions at discrete stages of muscle differentiation. While Brg1 is required for the activation of muscle gene transcription at early stages of differentiation, Brm is required for Ccnd1 repression and cell cycle arrest prior to the activation of muscle genes. Ccnd1 knockdown rescues the ability to exit the cell cycle in Brm-deficient myoblasts, but does not recover terminal differentiation, revealing a previously unrecognized role of Brm in the activation of late muscle gene expression independent from the control of cell cycle. Consistently, Brm null mice displayed impaired muscle regeneration after injury, with aberrant proliferation of satellite cells and delayed formation of new myofibers. These data reveal stage-specific roles of Brm during skeletal myogenesis, via formation of repressive and activatory SWI/SNF complexes. PMID:26136374

  5. Left-right asymmetry in the chick embryo requires core planar cell polarity protein Vangl2

    PubMed Central

    Zhang, Ying; Levin, Michael

    2009-01-01

    Consistent left-right patterning is a fascinating and biomedically important problem. In the chick embryo, it is not known how cells determine their position (left or right) relative to the primitive streak, which is required for subsequent asymmetric gene expression cascades. We show that the subcellular localization of Vangl2, a core planar cell polarity (PCP) protein, is consistently polarized, giving cells in the blastoderm a vector pointing toward the primitive streak. Moreover, morpholino-mediated loss-of-function of Vangl2 by electroporation into chicks at very early stages randomizes the normally left-sided expression of Sonic hedgehog. Strikingly, Vangl2 morpholinos also induce a de-synchronization of asymmetric gene expression within the left and right domains of Hensen’s node. These data reveal the existence of polarized planar cell polarity protein localization in gastrulating chick and demonstrate that the PCP pathway is functionally required for normal asymmetry in the chick upstream of Sonic hedgehog. These data suggest a new and widely-applicable class of models for the spread and coordination of left-right patterning information in the embryonic blastoderm. PMID:19621439

  6. The atypical cadherin Flamingo is required for sensory axon advance beyond intermediate target cells.

    PubMed

    Steinel, Martin C; Whitington, Paul M

    2009-03-15

    The Drosophila atypical cadherin Flamingo plays key roles in a number of developmental processes. We have used the sensory nervous system of the Drosophila embryo to shed light on the mechanism by which Flamingo regulates axon growth. flamingo loss of function mutants display a highly penetrant sensory axon stall phenotype. The location of these axon stalls is stereotypic and corresponds to the position of intermediate target cells, with which sensory axons associate during normal development. This suggests that Flamingo mediates an interaction between the sensory neuron growth cones and these intermediate targets, which is required for continued axon advance. Mutant rescue experiments show that Flamingo expression is required only in sensory neurons for normal axon growth. The flamingo mutant phenotype can be partially rescued by expressing a Flamingo construct lacking most of the extracellular domain, suggesting that regulation of sensory axon advance by Flamingo does not absolutely depend upon a homophilic Flamingo-Flamingo interaction or its ability to mediate cell-cell adhesion. Loss of function mutants for a number of key genes that act together with Flamingo in the planar cell polarity pathway do not display the highly penetrant stalling phenotype seen in flamingo mutants.

  7. 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.

  8. Oxidation of alpha-ketoglutarate is required for reductive carboxylation in cancer cells with mitochondrial defects

    PubMed Central

    Mullen, Andrew R.; Hu, Zeping; Shi, Xiaolei; Jiang, Lei; Boroughs, Lindsey K.; Kovacs, Zoltan; Boriack, Richard; Rakheja, Dinesh; Sullivan, Lucas B.; Linehan, W. Marston; Chandel, Navdeep S.; DeBerardinis, Ralph J.

    2014-01-01

    Summary Mammalian cells generate citrate by decarboxylating pyruvate in the mitochondria to supply the tricarboxylic acid (TCA) cycle. In contrast, hypoxia and other impairments of mitochondrial function induce an alternative pathway that produces citrate by reductively carboxylating α-ketoglutarate (AKG) via NADPH-dependent isocitrate dehydrogenase (IDH). It is unknown how cells generate reducing equivalents necessary to supply reductive carboxylation in the setting of mitochondrial impairment. Here we identified shared metabolic features in cells using reductive carboxylation. Paradoxically, reductive carboxylation was accompanied by concomitant AKG oxidation in the TCA cycle. Inhibiting AKG oxidation decreased reducing equivalent availability and suppressed reductive carboxylation. Interrupting transfer of reducing equivalents from NADH to NADPH by nicotinamide nucleotide transhydrogenase increased NADH abundance and decreased NADPH abundance while suppressing reductive carboxylation. The data demonstrate that reductive carboxylation requires bidirectional AKG metabolism along oxidative and reductive pathways, with the oxidative pathway producing reducing equivalents used to operate IDH in reverse. PMID:24857658

  9. An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.

    PubMed

    Whitney, John C; Quentin, Dennis; Sawai, Shin; LeRoux, Michele; Harding, Brittany N; Ledvina, Hannah E; Tran, Bao Q; Robinson, Howard; Goo, Young Ah; Goodlett, David R; Raunser, Stefan; Mougous, Joseph D

    2015-10-22

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.

  10. 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.

  11. Requirement for three signals in B cell responses. II. Analysis of antigen- and Ia-restricted T helper cell-B cell interaction

    PubMed Central

    1982-01-01

    We have recently reported that resting B cells must receive at least three different signals in a T helper cell (TH)-dependent as well as in a lipopolysaccharide (LPS)-induced B cell response (3), i.e., a specific TH signal (that can be bypassed by LPS), a nonspecific TH signal (mediated by Ia or antigen-nonspecific B cell helper factor), and an antigen (hapten) signal. In a system using male (H-Y) antigen- specific cloned TH of C57BL/6 origin and male (or female) B cells, we now confirm and extend these findings by demonstrating that H-Y- specific TH must see both H-Y and Ia determinants on the B cells (and not only on macrophages) to provide the first specific TH signal required for a plaque-forming cell (PFC) response. This signal was interfered with by a monoclonal anti-I-Ab antibody at the B cell level, was not mediated by detectable soluble factors (in contrast to the nonspecific signal also provided by the TH), and could be bypassed by LPS, in which case anti-I-Ab antibody had no effect. However, although the H-Y-specific TH induced a polyclonal PFC response (B cell differentiation) in the apparent absence of an antigen seen by the B cells, significant clonal expansion of PFC precursors occurred only when the B cells also recognized an antigen (hapten). PMID:6980255

  12. 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

  13. Akt Requires Glucose Metabolism to Suppress Puma Expression and Prevent Apoptosis of Leukemic T Cells*

    PubMed Central

    Coloff, Jonathan L.; Mason, Emily F.; Altman, Brian J.; Gerriets, Valerie A.; Liu, Tingyu; Nichols, Amanda N.; Zhao, Yuxing; Wofford, Jessica A.; Jacobs, Sarah R.; Ilkayeva, Olga; Garrison, Sean P.; Zambetti, Gerard P.; Rathmell, Jeffrey C.

    2011-01-01

    The PI3K/Akt pathway is activated in stimulated cells and in many cancers to promote glucose metabolism and prevent cell death. Although inhibition of Akt-mediated cell survival may provide a means to eliminate cancer cells, this survival pathway remains incompletely understood. In particular, unlike anti-apoptotic Bcl-2 family proteins that prevent apoptosis independent of glucose, Akt requires glucose metabolism to inhibit cell death. This glucose dependence may occur in part through metabolic regulation of pro-apoptotic Bcl-2 family proteins. Here, we show that activated Akt relies on glycolysis to inhibit induction of Puma, which was uniquely sensitive to metabolic status among pro-apoptotic Bcl-2 family members and was rapidly up-regulated in glucose-deficient conditions. Importantly, preventing Puma expression was critical for Akt-mediated cell survival, as Puma deficiency protected cells from glucose deprivation and Akt could not readily block Puma-mediated apoptosis. In contrast, the pro-apoptotic Bcl-2 family protein Bim was induced normally even when constitutively active Akt was expressed, yet Akt could provide protection from Bim cytotoxicity. Up-regulation of Puma appeared mediated by decreased availability of mitochondrial metabolites rather than glycolysis itself, as alternative mitochondrial fuels could suppress Puma induction and apoptosis upon glucose deprivation. Metabolic regulation of Puma was mediated through combined p53-dependent transcriptional induction and control of Puma protein stability, with Puma degraded in nutrient-replete conditions and long lived in nutrient deficiency. Together, these data identify a key role for Bcl-2 family proteins in Akt-mediated cell survival that may be critical in normal immunity and in cancer through Akt-dependent stimulation of glycolysis to suppress Puma expression. PMID:21159778

  14. Protein Kinase A Activity and Anchoring Are Required for Ovarian Cancer Cell Migration and Invasion

    PubMed Central

    McKenzie, Andrew J.; Campbell, Shirley L.; Howe, Alan K.

    2011-01-01

    Epithelial ovarian cancer (EOC) is the deadliest of the gynecological malignancies, due in part to its clinically occult metastasis. Therefore, understanding the mechanisms governing EOC dissemination and invasion may provide new targets for antimetastatic therapies or new methods for detection of metastatic disease. The cAMP-dependent protein kinase (PKA) is often dysregulated in EOC. Furthermore, PKA activity and subcellular localization by A-kinase anchoring proteins (AKAPs) are important regulators of cytoskeletal dynamics and cell migration. Thus, we sought to study the role of PKA and AKAP function in both EOC cell migration and invasion. Using the plasma membrane-directed PKA biosensor, pmAKAR3, and an improved migration/invasion assay, we show that PKA is activated at the leading edge of migrating SKOV-3 EOC cells, and that inhibition of PKA activity blocks SKOV-3 cell migration. Furthermore, we show that while the PKA activity within the leading edge of these cells is mediated by anchoring of type-II regulatory PKA subunits (RII), inhibition of anchoring of either RI or RII PKA subunits blocks cell migration. Importantly, we also show – for the first time – that PKA activity is up-regulated at the leading edge of SKOV-3 cells during invasion of a three-dimensional extracellular matrix and, as seen for migration, inhibition of either PKA activity or AKAP-mediated PKA anchoring blocks matrix invasion. These data are the first to demonstrate that the invasion of extracellular matrix by cancer cells elicits activation of PKA within the invasive leading edge and that both PKA activity and anchoring are required for matrix invasion. These observations suggest a role for PKA and AKAP activity in EOC metastasis. PMID:22028904

  15. 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

  16. Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

    PubMed

    Eshraghi, Aria; Dixon, Shandee D; Tamilselvam, Batcha; Kim, Emily Jin-Kyung; Gargi, Amandeep; Kulik, Julia C; Damoiseaux, Robert; Blanke, Steven R; Bradley, Kenneth A

    2014-07-01

    Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

  17. NK cells require antigen-specific memory CD4+ T cells to mediate superior effector functions during HSV-2 recall responses in vitro.

    PubMed

    Chen, Branson; Lee, Amanda J; Chew, Marianne V; Ashkar, Ali A

    2016-12-14

    Natural killer (NK) cells have an important role in mounting protective innate responses against genital herpes simplex virus type 2 (HSV-2) infections. However their role as effectors in adaptive immune responses against HSV-2 is unclear. Here, we demonstrate that NK cells from C57BL/6 mice in an ex vivo splenocyte culture produce significantly more interferon γ (IFN-γ) upon re-exposure to HSV-2 antigens in a mouse model of genital HSV-2 immunization. We find that naïve NK cells do not require any prior stimulation or priming to be activated to produce IFN-γ. Our results demonstrate that HSV-2-experienced CD4(+) T cells have a crucial role in coordinating NK cell activation and that their presence during HSV-2 antigen presentation is required to activate NK cells in this model of secondary immune response. We also examined the requirement of cell-to-cell contacts for both CD4(+) T cells and NK cells. NK cells are dependent on direct interactions with other HSV-2-experienced splenocytes, and CD4(+) T cells need to be in close proximity to NK cells to activate them. This study revealed that NK cells do not exhibit any memory toward HSV-2 antigens and, in fact, require specific interactions with HSV-2-experienced CD4(+) T cells to produce IFN-γ.

  18. [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.

  19. 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.

  20. Epithelial cell integrin β1 is required for developmental angiogenesis in the pituitary gland

    PubMed Central

    Scully, Kathleen M.; Skowronska-Krawczyk, Dorota; Krawczyk, Michal; Merkurjev, Daria; Taylor, Havilah; Livolsi, Antonia; Tollkuhn, Jessica; Stan, Radu V.; Rosenfeld, Michael G.

    2016-01-01

    As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non–cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development. PMID:27810956

  1. Ethylene signaling pathway and MAPK cascades are required for AAL toxin-induced programmed cell death.

    PubMed

    Mase, Keisuke; Mizuno, Takahito; Ishihama, Nobuaki; Fujii, Takayuki; Mori, Hitoshi; Kodama, Motoichiro; Yoshioka, Hirofumi

    2012-08-01

    Programmed cell death (PCD), known as hypersensitive response cell death, has an important role in plant defense response. The signaling pathway of PCD remains unknown. We employed AAL toxin and Nicotiana umbratica to analysis plant PCD. AAL toxin is a pathogenicity factor of the necrotrophic pathogen Alternaria alternata f. sp. lycopersici. N. umbratica is sensitive to AAL toxin, susceptible to pathogens, and effective in Tobacco rattle virus-based virus-induced gene silencing (VIGS). VIGS analyses indicated that AAL toxin-triggered cell death (ACD) is dependent upon the mitogen-activated protein (MAP) kinase kinase MEK2, which is upstream of both salicylic acid-induced protein kinase (SIPK) and wound-induced protein kinase (WIPK) responsible for ethylene (ET) synthesis. ET treatment of MEK2-silenced N. umbratica re-established ACD. In SIPK- and WIPK-silenced N. umbratica, ACD was compromised and ET accumulation was not observed. However, in contrast to the case of MEK2-silenced plants, ET treatment did not induce cell death in SIPK- and WIPK-silenced plants. This work showed that ET-dependent pathway and MAP kinase cascades are required in ACD. Our results suggested that MEK2-SIPK/WIPK cascades have roles in ET biosynthesis; however, SIPK and WIPK have other roles in ET signaling or another pathway leading to cell death by AAL toxin.

  2. 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

  3. Epithelial cell integrin β1 is required for developmental angiogenesis in the pituitary gland.

    PubMed

    Scully, Kathleen M; Skowronska-Krawczyk, Dorota; Krawczyk, Michal; Merkurjev, Daria; Taylor, Havilah; Livolsi, Antonia; Tollkuhn, Jessica; Stan, Radu V; Rosenfeld, Michael G

    2016-11-22

    As a key component of the vertebrate neuroendocrine system, the pituitary gland relies on the progressive and coordinated development of distinct hormone-producing cell types and an invading vascular network. The molecular mechanisms that drive formation of the pituitary vasculature, which is necessary for regulated synthesis and secretion of hormones that maintain homeostasis, metabolism, and endocrine function, remain poorly understood. Here, we report that expression of integrin β1 in embryonic pituitary epithelial cells is required for angiogenesis in the developing mouse pituitary gland. Deletion of pituitary epithelial integrin β1 before the onset of angiogenesis resulted in failure of invading endothelial cells to recruit pericytes efficiently, whereas deletion later in embryogenesis led to decreased vascular density and lumen formation. In both cases, lack of epithelial integrin β1 was associated with a complete absence of vasculature in the pituitary gland at birth. Within pituitary epithelial cells, integrin β1 directs a large transcriptional program that includes components of the extracellular matrix and associated signaling factors that are linked to the observed non-cell-autonomous effects on angiogenesis. We conclude that epithelial integrin β1 functions as a critical and canonical regulator of developmental angiogenesis in the pituitary gland, thus providing insight into the long-standing systems biology conundrum of how vascular invasion is coordinated with tissue development.

  4. 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.

  5. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.

    PubMed

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw; Syljuåsen, Randi G; Lundin, Cecilia; Bartek, Jiri; Helleday, Thomas

    2005-02-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

  6. Aurora B kinase is required for cell cycle progression in silkworm.

    PubMed

    Gang, Xiaoxu; Qian, Wenliang; Zhang, Tianlei; Yang, Xinxin; Xia, Qingyou; Cheng, Daojun

    2017-01-30

    Aurora B kinase, a member of serine/threonine kinase family, is the catalytic subunit of the chromosomal passenger complex and is essential for chromosome alignment, chromosome segregation, and cytokinesis during mitosis. Here, we cloned the full-length cDNA sequence of silkworm Aurora B (BmAurB) gene and predicted that BmAurB protein contains a conserved S_TKc domain. Phylogenetic analysis between BmAurB and other Aurora kinases indicates that Aurora kinases may have evolved after separation between mammalian and insect, and prior to radiation of either mammalian or insects. RT-PCR examination revealed that the expression of the BmAurB gene was high in mitotic cycling gonads, moderate in mitotic cycling brain, and undetectable in endocycling silk gland during silkworm larval development. RNAi or inhibitor-mediated inhibition of the BmAurB gene in silkworm ovary-derived BmN4-SID1 cells disrupted cell cycle progression during mitosis and induced an accumulation of polyploid cells, cell cycle arrest at G2/M phase, chromosome misalignment, chromosome bridge, and bi-nucleation. Taken together, our results suggest that the BmAurB gene is required for cell cycle progression in silkworm.

  7. 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.

  8. Nrf2 is required to maintain the self-renewal of glioma stem cells

    PubMed Central

    2013-01-01

    Background Glioblastomas are deadly cancers that display a functional cellular hierarchy maintained by self-renewing glioma stem cells (GSCs). Self-renewal is a complex biological process necessary for maintaining the glioma stem cells. Nuclear factor rythroid 2-related factor 2(Nrf2) plays a significant role in protecting cells from endogenous and exogenous stresses. Nrf2 is a key nuclear transcription factor that regulates antioxidant response element (ARE)-containing genes. Previous studies have demonstrated the significant role of Nrf2 in the proliferation of glioblastoma, and in their resistance to radioactive therapies. We examined the effect of knocking down Nrf2 in GSCs. Methods Nrf2 expression was down-regulated by shRNA transinfected with lentivirus. Expression levels of Nestin, Nrf2, BMI-1, Sox2 and Cyclin E were assessed by western blotting, quantitative polymerase chain reaction (qPCR) and immunohistochemistry analysis. The capacity for self-renewal in vitro was assessed by genesis of colonies. The capacity for self-renewal in vivo was analyzed by tumor genesis of xenografts in nude mice. Results Knockdown of Nrf2 inhibited the proliferation of GSCs, and significantly reduced the expression of BMI-1, Sox2 and CyclinE. Knocking down of Nrf2 changed the cell cycle distribution of GSCs by causing an uncharacteristic increase in the proportion of cells in the G2 phase and a decrease in the proportion of cells in the S phase of the cell cycle. Conclusions Nrf2 is required to maintain the self-renewal of GSCs, and its down-regulation can attenuate the self-renewal of GSCs significantly. PMID:23937621

  9. Requirement of lipid II biosynthesis for cell division in cell wall-less Wolbachia, endobacteria of arthropods and filarial nematodes.

    PubMed

    Vollmer, Jennifer; Schiefer, Andrea; Schneider, Tanja; Jülicher, Karen; Johnston, Kelly L; Taylor, Mark J; Sahl, Hans-Georg; Hoerauf, Achim; Pfarr, Kenneth

    2013-04-01

    Obligate Wolbachia endobacteria have a reduced genome and retained genes are hypothesized to be crucial for survival. Although intracellular bacteria do not need a stress-bearing peptidoglycan cell wall, Wolbachia encode proteins necessary to synthesize the peptidoglycan precursor lipid II. The activity of the enzymes catalyzing the last two steps of this pathway was previously shown, and Wolbachia are sensitive to inhibition of lipid II synthesis. A puzzling characteristic of Wolbachia is the lack of genes for l-amino acid racemases essential for lipid II synthesis. Transcription analysis showed the expression of a possible alternative racemase metC, and recombinant Wolbachia MetC indeed had racemase activity that may substitute for the absent l-Ala racemase. However, enzymes needed to form mature peptidoglycan are absent and the function of Wolbachia lipid II is unknown. Inhibition of lipid II biosynthesis resulted in enlargement of Wolbachia cells and redistribution of Wolbachia peptidoglycan-associated lipoprotein, demonstrating that lipid II is required for coordinated cell division and may interact with the lipoprotein. We conclude that lipid II is essential for Wolbachia cell division and that this function is potentially conserved in the Gram-negative bacteria.

  10. 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

  11. PI4KIIIα is required for cortical integrity and cell polarity during Drosophila oogenesis.

    PubMed

    Tan, Julie; Oh, Karen; Burgess, Jason; Hipfner, David R; Brill, Julie A

    2014-03-01

    Phosphoinositides regulate myriad cellular processes, acting as potent signaling molecules in conserved signaling pathways and as organelle gatekeepers that recruit effector proteins to membranes. Phosphoinositide-generating enzymes have been studied extensively in yeast and cultured cells, yet their roles in animal development are not well understood. Here, we analyze Drosophila melanogaster phosphatidylinositol 4-kinase IIIα (PI4KIIIα) during oogenesis. We demonstrate that PI4KIIIα is required for production of plasma membrane PtdIns4P and PtdIns(4,5)P2 and is crucial for actin organization, membrane trafficking and cell polarity. Female germ cells mutant for PI4KIIIα exhibit defects in cortical integrity associated with failure to recruit the cytoskeletal-membrane crosslinker Moesin and the exocyst subunit Sec5. These effects reflect a unique requirement for PI4KIIIα, as egg chambers from flies mutant for either of the other Drosophila PI4Ks, fwd or PI4KII, show Golgi but not plasma membrane phenotypes. Thus, PI4KIIIα is a vital regulator of a functionally distinct pool of PtdIns4P that is essential for PtdIns(4,5)P2-dependent processes in Drosophila development.

  12. Phosphatidylserine is polarized and required for proper Cdc42 localization and for development of cell polarity.

    PubMed

    Fairn, Gregory D; Hermansson, Martin; Somerharju, Pentti; Grinstein, Sergio

    2011-10-02

    Polarity is key to the function of eukaryotic cells. On the establishment of a polarity axis, cells can vectorially target secretion, generating an asymmetric distribution of plasma membrane proteins. From Saccharomyces cerevisiae to mammals, the small GTPase Cdc42 is a pivotal regulator of polarity. We used a fluorescent probe to visualize the distribution of phosphatidylserine in live S. cerevisiae. Remarkably, phosphatidylserine was polarized in the plasma membrane, accumulating in bud necks, the bud cortex and the tips of mating projections. Polarization required vectorial delivery of phosphatidylserine-containing secretory vesicles, and phosphatidylserine was largely excluded from endocytic vesicles, contributing to its polarized retention. Mutants lacking phosphatidylserine synthase had impaired polarization of the Cdc42 complex, leading to a delay in bud emergence, and defective mating. The addition of lysophosphatidylserine resulted in resynthesis and polarization of phosphatidylserine, as well as repolarization of Cdc42. The results indicate that phosphatidylserine--and presumably its polarization--are required for optimal Cdc42 targeting and activation during cell division and mating.

  13. Goblet Cell Hyperplasia Requires High Bicarbonate Transport To Support Mucin Release

    PubMed Central

    Gorrieri, Giulia; Scudieri, Paolo; Caci, Emanuela; Schiavon, Marco; Tomati, Valeria; Sirci, Francesco; Napolitano, Francesco; Carrella, Diego; Gianotti, Ambra; Musante, Ilaria; Favia, Maria; Casavola, Valeria; Guerra, Lorenzo; Rea, Federico; Ravazzolo, Roberto; Di Bernardo, Diego; Galietta, Luis J. V.

    2016-01-01

    Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus. PMID:27786259

  14. 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...

  15. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 31 2012-07-01 2012-07-01 false Fuel economy label-special requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  16. 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...

  17. 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...

  18. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 30 2014-07-01 2014-07-01 false Fuel economy label-special requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  19. 40 CFR 600.304-12 - Fuel economy label-special requirements for hydrogen fuel cell vehicles.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 31 2013-07-01 2013-07-01 false Fuel economy label-special requirements for hydrogen fuel cell vehicles. 600.304-12 Section 600.304-12 Protection of Environment... fuel cell vehicles. Fuel economy labels for hydrogen fuel cell vehicles must meet the...

  20. 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...

  1. 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...

  2. 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

    Yang, Jian; Wang, Wei; Ooi, Jolene; Campos, Lia S; Lu, Liming; Liu, Pentao

    2015-05-01

    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.

  3. AKT induces erythroid-cell maturation of JAK2-deficient fetal liver progenitor cells and is required for Epo regulation of erythroid-cell differentiation.

    PubMed

    Ghaffari, Saghi; Kitidis, Claire; Zhao, Wei; Marinkovic, Dragan; Fleming, Mark D; Luo, Biao; Marszalek, Joseph; Lodish, Harvey F

    2006-03-01

    AKT serine threonine kinase of the protein kinase B (PKB) family plays essential roles in cell survival, growth, metabolism, and differentiation. In the erythroid system, AKT is known to be rapidly phosphorylated and activated in response to erythropoietin (Epo) engagement of Epo receptor (EpoR) and to sustain survival signals in cultured erythroid cells. Here we demonstrate that activated AKT complements EpoR signaling and supports erythroid-cell differentiation in wild-type and JAK2-deficient fetal liver cells. We show that erythroid maturation of AKT-transduced cells is not solely dependent on AKT-induced cell survival or proliferation signals, suggesting that AKT transduces also a differentiation-specific signal downstream of EpoR in erythroid cells. Down-regulation of expression of AKT kinase by RNA interference, or AKT activity by expression of dominant negative forms, inhibits significantly fetal liver-derived erythroid-cell colony formation and gene expression, demonstrating that AKT is required for Epo regulation of erythroid-cell maturation.

  4. Proper Control of Caulobacter crescentus Cell Surface Adhesion Requires the General Protein Chaperone DnaK

    PubMed Central

    Eaton, Daniel S.; Crosson, Sean

    2016-01-01

    ABSTRACT Growth in a surface-attached bacterial community, or biofilm, confers a number of advantages. However, as a biofilm matures, high-density growth imposes stresses on individual cells, and it can become less advantageous for progeny to remain in the community. Thus, bacteria employ a variety of mechanisms to control attachment to and dispersal from surfaces in response to the state of the environment. The freshwater oligotroph Caulobacter crescentus can elaborate a polysaccharide-rich polar organelle, known as the holdfast, which enables permanent surface attachment. Holdfast development is strongly inhibited by the small protein HfiA; mechanisms that control HfiA levels in the cell are not well understood. We have discovered a connection between the essential general protein chaperone, DnaK, and control of C. crescentus holdfast development. C. crescentus mutants partially or completely lacking the C-terminal substrate binding “lid” domain of DnaK exhibit enhanced bulk surface attachment. Partial or complete truncation of the DnaK lid domain increases the probability that any single cell will develop a holdfast by 3- to 10-fold. These results are consistent with the observation that steady-state levels of an HfiA fusion protein are significantly diminished in strains that lack the entire lid domain of DnaK. While dispensable for growth, the lid domain of C. crescentus DnaK is required for proper chaperone function, as evidenced by observed dysregulation of HfiA and holdfast development in strains expressing lidless DnaK mutants. We conclude that DnaK is an important molecular determinant of HfiA stability and surface adhesion control. IMPORTANCE Regulatory control of cell adhesion ensures that bacterial cells can transition between free-living and surface-attached states. We define a role for the essential protein chaperone, DnaK, in the control of Caulobacter crescentus cell adhesion. C. crescentus surface adhesion is mediated by an envelope

  5. 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

  6. Cis- and cell-type-dependent trans-requirements for Lassa virus-like particle production.

    PubMed

    Urata, Shuzo; Yasuda, Jiro

    2015-07-01

    Lassa virus (LASV) small zinc-finger protein (Z), which contains two L-domain motifs, plays a central role in virus budding. Here, we report that co-expression of glycoprotein (GPC) altered the requirements for cholesterol but not the L-domains and host factor, Tsg101, for Z-induced virus-like particle (VLP) production. In particular, the cholesterol requirement for VLP production was cell-type-dependent. In addition, GPC was found to be important for co-localization of Z with CD63, a late endosomal marker. We also found that the N-terminal region (aa 3-10) of Z was critical for its myristoylation and VLP production. These findings will contribute to our understanding of LASV assembly and budding.

  7. 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

  8. Mineral Nutrient Requirements of a Loblolly Pine (Pinus taeda) Cell Suspension Culture 1

    PubMed Central

    Teasdale, Robert D.; Dawson, Pamela A.; Woolhouse, Harold W.

    1986-01-01

    The mineral nutrient requirements of Pinus taeda cells were explored using quantitative cell culture growth measurements. An appraisal was thereby made of the critical features of a novel and successful medium which was developed specifically for this gymnosperm using chemical composition data for developing seeds, and characterized by generally high concentration of all micronutrients, high magnesium, and low calcium. The high magnesium concentration was found not to be detrimental and possibly beneficial whereas the calcium level bordered on a deficiency threshold. Within the microelements high iodide was found to be essential, as was a higher borate level than is present in media developed for angiosperms. High zinc concentrations were also beneficial, with normal levels permitting slower but nevertheless healthy growth. An improved medium was thereby formulated which was stress-free and exhibited broader genotype specificity. This new formulation has proved very successful in maintaining long-term growth of highly uniform and apparently meristematic suspension cultures of Pinus radiata. PMID:16665170

  9. Quantitative and Temporal Requirements Revealed for Zap-70 Catalytic Activity During T Cell Development

    PubMed Central

    Au-Yeung, Byron B.; Melichar, Heather J.; Ross, Jenny O.; Cheng, Debra A.; Zikherman, Julie; Shokat, Kevan M.; Robey, Ellen A.; Weiss, Arthur

    2014-01-01

    The catalytic activity of Zap-70 is crucial for T cell receptor (TCR) signaling, but the quantitative and temporal requirements for its function in thymocyte development are not known. Using a chemical-genetic system to selectively and reversibly inhibit Zap-70 catalytic activity in a model of synchronized thymic selection, we showed that CD4+CD8+ thymocytes integrate multiple, transient, Zap-70-dependent signals over more than 36 h to reach a cumulative threshold for positive selection, whereas one hour of signaling was sufficient for negative selection. Titration of Zap-70 activity resulted in graded reductions in positive and negative selection but did not decrease the cumulative TCR signals integrated by positively selected OT-I cells, revealing heterogeneity, even among CD4+CD8+ thymocytes expressing identical TCRs undergoing positive selection. PMID:24908390

  10. Notch1 is required for maintenance of the reservoir of adult hippocampal stem cells

    PubMed Central

    Ables, Jessica L.; DeCarolis, Nathan A.; Johnson, Madeleine A.; Rivera, Phillip D.; Gao, Zhengliang; Cooper, Don C.; Radtke, Freddy; Hsieh, Jenny; Eisch, Amelia J.

    2010-01-01

    Notch1 regulates neural stem cell (NSC) number during development, but its role in adult neurogenesis is unclear. We generated nestin-CreERT2/R26R-YFP/Notch1loxP/loxP (Notch1 iKO) mice to allow tamoxifen (TAM)-inducible elimination of Notch1 and concomitant expression of yellow fluorescent protein (YFP) in nestin-expressing Type-1 NSCs and their progeny in the adult hippocampal subgranular zone (SGZ). Consistent with previous research, YFP+ cells in all stages of neurogenesis were evident in the subgranular zone (SGZ) of wild type mice (WT; nestin-CreERT2/R26R-YFP/Notch1wt/wt) after tamoxifen (post-TAM), producing adult-generated YFP+ dentate gyrus neurons. Compared to WT littermates, Notch1 iKO mice had similar numbers of total SGZ YFP+ cells 13 and 30 days post-TAM but had significantly fewer SGZ YFP+ cells 60 and 90 days post-TAM. Significantly fewer YFP+ Type-1 NSCs and transiently-amplifying progenitors (TAPs) resulted in generation of fewer YFP+ granule neurons in Notch1 iKO mice. Strikingly, 30 days of running rescued this deficit, as the total YFP+ cell number in Notch iKO mice was equivalent to WT levels. This was even more notable given the persistent deficits in the Type-1 NSC and TAP reservoirs. Our data show that Notch1 signaling is required to maintain a reservoir of undifferentiated cells and ensure continuity of adult hippocampal neurogenesis, but that alternative Notch1- and Type-1 NSC-independent pathways compensate in response to physical activity. These data shed light on the complex relationship between Type-1 NSCs, adult neurogenesis, the neurogenic niche, and environmental stimuli. PMID:20685991

  11. Lung Angiogenesis Requires CD4+Forkhead Homeobox Protein-3+ Regulatory T Cells

    PubMed Central

    D’Alessio, Franco R.; Zhong, Qiong; Jenkins, John; Moldobaeva, Aigul

    2015-01-01

    Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (Treg) cells modulate tumor-induced neovascularization, we questioned whether this CD4+ lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4+ CD25+ forkhead homeobox protein-3 (Foxp3)+ cells was observed 3–5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3+ Treg cells can be depleted with diphtheria toxin (DT; Foxp3DTR), we unexpectedly found that Foxp3+ Treg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3gfp controls). Adoptive transfer studies using CD4+ CD25+ splenocytes from congenic CD45.1 mice into Foxp3+ Treg–depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3gfp controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-β, IL-10) cytokines showed Treg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in Treg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII]int, CD11C+) in Treg-deficient lungs compared with Treg-sufficient lungs. Treg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in Treg-depleted mice was the decline in the essential CXC chemokine, LIX. PMID:25275926

  12. Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface.

    PubMed Central

    Cheung, Joanne C; Reithmeier, Reinhart A F

    2004-01-01

    AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane. PMID:14640982

  13. ADP ribosylation factor 1 is required for synaptic vesicle budding in PC12 cells.

    PubMed

    Faúndez, V; Horng, J T; Kelly, R B

    1997-08-11

    Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

  14. The RhoGEF Pebble is required for cell shape changes during cell migration triggered by the Drosophila FGF receptor Heartless.

    PubMed

    Schumacher, Sabine; Gryzik, Tanja; Tannebaum, Sylvia; Müller, H-Arno J

    2004-06-01

    The FGF receptor Heartless (HTL) is required for mesodermal cell migration in the Drosophila gastrula. We show that mesoderm cells undergo different phases of specific cell shape changes during mesoderm migration. During the migratory phase, the cells adhere to the basal surface of the ectoderm and exhibit extensive protrusive activity. HTL is required for the protrusive activity of the mesoderm cells. Moreover, the early phenotype of htl mutants suggests that HTL is required for the adhesion of mesoderm cells to the ectoderm. In a genetic screen we identified pebble (pbl) as a novel gene required for mesoderm migration. pbl encodes a guanyl nucleotide exchange factor (GEF) for RHO1 and is known as an essential regulator of cytokinesis. We show that the function of PBL in cell migration is independent of the function of PBL in cytokinesis. Although RHO1 acts as a substrate for PBL in cytokinesis, compromising RHO1 function in the mesoderm does not block cell migration. These data suggest that the function of PBL in cell migration might be mediated through a pathway distinct from RHO1. This idea is supported by allele-specific differences in the expressivity of the cytokinesis and cell migration phenotypes of different pbl mutants. We show that PBL is autonomously required in the mesoderm for cell migration. Like HTL, PBL is required for early cell shape changes during mesoderm migration. Expression of a constitutively active form of HTL is unable to rescue the early cellular defects in pbl mutants, suggesting that PBL is required for the ability of HTL to trigger these cell shape changes. These results provide evidence for a novel function of the Rho-GEF PBL in HTL-dependent mesodermal cell migration.

  15. Cell Reprogramming Requires Silencing of a Core Subset of Polycomb Targets

    PubMed Central

    Fragola, Giulia; Cuomo, Alessandro; Blasimme, Alessandro; Gross, Fridolin; Signaroldi, Elena; Bucci, Gabriele; Sommer, Cesar; Pruneri, Giancarlo; Mazzarol, Giovanni; Bonaldi, Tiziana; Mostoslavsky, Gustavo; Casola, Stefano; Testa, Giuseppe

    2013-01-01

    Transcription factor (TF)–induced reprogramming of somatic cells into induced pluripotent stem cells (iPSC) is associated with genome-wide changes in chromatin modifications. Polycomb-mediated histone H3 lysine-27 trimethylation (H3K27me3) has been proposed as a defining mark that distinguishes the somatic from the iPSC epigenome. Here, we dissected the functional role of H3K27me3 in TF–induced reprogramming through the inactivation of the H3K27 methylase EZH2 at the onset of reprogramming. Our results demonstrate that surprisingly the establishment of functional iPSC proceeds despite global loss of H3K27me3. iPSC lacking EZH2 efficiently silenced the somatic transcriptome and differentiated into tissues derived from the three germ layers. Remarkably, the genome-wide analysis of H3K27me3 in Ezh2 mutant iPSC cells revealed the retention of this mark on a highly selected group of Polycomb targets enriched for developmental regulators controlling the expression of lineage specific genes. Erasure of H3K27me3 from these targets led to a striking impairment in TF–induced reprogramming. These results indicate that PRC2-mediated H3K27 trimethylation is required on a highly selective core of Polycomb targets whose repression enables TF–dependent cell reprogramming. PMID:23468641

  16. The Drosophila nuclear lamina protein otefin is required for germline stem cell survival.

    PubMed

    Barton, Lacy J; Pinto, Belinda S; Wallrath, Lori L; Geyer, Pamela K

    2013-06-24

    LEM domain (LEM-D) proteins are components of an extensive protein network that assembles beneath the inner nuclear envelope. Defects in LEM-D proteins cause tissue-restricted human diseases associated with altered stem cell homeostasis. Otefin (Ote) is a Drosophila LEM-D protein that is intrinsically required for female germline stem cell (GSC) maintenance. Previous studies linked Ote loss with transcriptional activation of the key differentiation gene bag-of-marbles (bam), leading to the model in which Ote tethers the bam gene to the nuclear periphery for gene silencing. Using genetic and phenotypic analyses of multiple ote(-/-) backgrounds, we obtained evidence that is inconsistent with this model. We show that bam repression is maintained in ote(-/-) GSCs and that germ cell loss persists in ote(-/-), bam(-/-) mutants, together demonstrating that GSC loss is independent of bam transcription. We show that the primary defect in ote(-/-) GSCs is a block of differentiation, which ultimately leads to germ cell death.

  17. Human Cytomegalovirus Carries a Cell-Derived Phospholipase A2 Required for Infectivity

    PubMed Central

    Allal, Cuider; Buisson-Brenac, Claire; Marion, Vincent; Claudel-Renard, Clotilde; Faraut, Thomas; Dal Monte, Paola; Streblow, Daniel; Record, Michel

    2004-01-01

    Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2α, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity. PMID:15220446

  18. The necessary length of carbon nanotubes required to optimize solar cells

    PubMed Central

    Vaezzadeh, Majid; Saeedi, Mohammad Reza; Barghi, Tirdad; Sadeghi, Mohammad Reza

    2007-01-01

    Background In recent years scientists have been trying both to increase the efficiency of solar cells, whilst at the same time reducing dimensions and costs. Increases in efficiency have been brought about by implanting carbon nanotubes onto the surface of solar cells in order to reduce the reflection of sunrays, as well as through the insertion of polymeric arrays into the intrinsic layer for charge separation. Results The experimental results show power rising linearly for intrinsic layer thicknesses between 0–50 nm. Wider thicknesses increase the possibility of recombination of electrons and holes, leading to perturbation of the linear behaviour of output power. This effect is studied and formulated as a function of thickness. Recognition of the critical intrinsic layer thickness can permit one to determine the length of carbon nanotube necessary for optimizing solar cells. Conclusion In this study the behaviour of output power as a function of intrinsic layer thicknesses has been described physically and also simulated. In addition, the implantation of carbon nanotubes into the intrinsic layer and the necessary nanotube length required to optimize solar cells have been suggested. PMID:17908295

  19. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy.

    PubMed

    Goh, Qingnian; Millay, Douglas P

    2017-02-10

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy.

  20. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy

    PubMed Central

    Goh, Qingnian; Millay, Douglas P

    2017-01-01

    Fusion of skeletal muscle stem/progenitor cells is required for proper development and regeneration, however the significance of this process during adult muscle hypertrophy has not been explored. In response to muscle overload after synergist ablation in mice, we show that myomaker, a muscle specific membrane protein essential for myoblast fusion, is activated mainly in muscle progenitors and not myofibers. We rendered muscle progenitors fusion-incompetent through genetic deletion of myomaker in muscle stem cells and observed a complete reduction of overload-induced hypertrophy. This blunted hypertrophic response was associated with a reduction in Akt and p70s6k signaling and protein synthesis, suggesting a link between myonuclear accretion and activation of pro-hypertrophic pathways. Furthermore, fusion-incompetent muscle exhibited increased fibrosis after muscle overload, indicating a protective role for normal stem cell activity in reducing myofiber strain associated with hypertrophy. These findings reveal an essential contribution of myomaker-mediated stem cell fusion during physiological adult muscle hypertrophy. DOI: http://dx.doi.org/10.7554/eLife.20007.001 PMID:28186492

  1. 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

  2. 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.

  3. Candida albicans RHO1 is required for cell viability in vitro and in vivo.

    PubMed

    Smith, Susan E; Csank, Csilla; Reyes, Guadalupe; Ghannoum, Mahmoud A; Berlin, Vivian

    2002-05-01

    In Saccharomyces cerevisiae, Rho1p plays an important role in cell wall integrity by regulating beta-1,3-glucan synthase, Pkc1p and the actin cytoskeleton. To determine the physiological role of Rho1p in the dimorphic fungus Candida albicans, the major human fungal pathogen, we constructed mutants that conditionally express Rho1p from the glucose-repressible phosphoenolpyruvate carboxykinase promoter (pPCK1). We examined the growth of these cells in a range of conditions. Depletion of Rho1p from yeast cells resulted in cell death, lysis, and aggregation. The Rho1p conditional mutant was inviable on 10% serum indicating that Rho1p was also required for hyphal viability. Furthermore, in a mouse model of systemic candidiasis, strains dependent on pPCK1-driven RHO1 expression failed to colonise the kidneys and establish disease, suggesting that the level of glucose in serum was sufficient to repress the pPCK1 and that Rho1p-depleted strains were inviable within the host. Therefore, Rho1p is essential for the viability of C. albicans in vitro and in vivo.

  4. Arginine methylation of SmB is required for Drosophila germ cell development.

    PubMed

    Anne, Joël

    2010-09-01

    Sm proteins constitute the common core of spliceosomal small nuclear ribonucleoproteins. Although Sm proteins are known to be methylated at specific arginine residues within the C-terminal arginine-glycine dipeptide (RG) repeats, the biological relevance of these modifications remains unknown. In this study, a tissue-specific function of arginine methylation of the SmB protein was identified in Drosophila. Analysis of the distribution of SmB during oogenesis revealed that this protein accumulates at the posterior pole of the oocyte, a cytoplasmic region containing the polar granules, which are necessary for the formation of primordial germ cells. The pole plasm localisation of SmB requires the methylation of arginine residues in its RG repeats by the Capsuléen-Valois methylosome complex. Functional studies showed that the methylation of these arginine residues is essential for distinct processes of the germline life cycle, including germ cell formation, migration and differentiation. In particular, the methylation of a subset of these arginine residues appears essential for the anchoring of the polar granules at the posterior cortex of the oocyte, whereas the methylation of another subset controls germ cell migration during embryogenesis. These results demonstrate a crucial role of arginine methylation in directing the subcellular localisation of SmB and that this modification contributes specifically to the establishment and development of germ cells.

  5. Histamine is required during neural stem cell proliferation to increase neuron differentiation.

    PubMed

    Rodríguez-Martínez, G; Velasco, I; García-López, G; Solís, K H; Flores-Herrera, H; Díaz, N F; Molina-Hernández, A

    2012-08-02

    Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 μM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

  6. Processing of microRNA primary transcripts requires heme in mammalian cells.

    PubMed

    Weitz, Sara H; Gong, Ming; Barr, Ian; Weiss, Shimon; Guo, Feng

    2014-02-04

    DiGeorge syndrome critical region gene 8 (DGCR8) is the RNA-binding partner protein of the nuclease Drosha. DGCR8 and Drosha recognize and cleave primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical microRNAs (miRNAs) in animals. We previously reported that human, frog, and starfish DGCR8 bind heme when expressed in Escherichia coli and that Fe(III) heme activates apoDGCR8 in reconstituted pri-miRNA processing assays. However, the physiological relevance of heme in miRNA maturation has not been clear. Here, we present a live-cell pri-miRNA processing assay that produces robust signals and faithfully indicates DGCR8 and Drosha activities. We demonstrate that all known heme-binding-deficient DGCR8 mutants are defective in pri-miRNA processing in HeLa cells. DGCR8 contains a previously uncharacterized heme-binding motif, "IPCL," that is also required for its activity. Heme availability and biosynthesis in HeLa cells positively affect pri-miRNA processing and production of mature miRNA. These results establish an essential function for heme in pri-miRNA processing in mammalian cells. Our study suggests that abnormal heme biosynthesis and degradation may contribute to diseases via miRNA-mediated gene regulation networks.

  7. 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.

  8. Venezuelan equine encephalitis virus entry mechanism requires late endosome formation and resists cell membrane cholesterol depletion

    SciTech Connect

    Kolokoltsov, Andrey A.; Fleming, Elisa H.; Davey, Robert A. . E-mail: radavey@utmb.edu

    2006-04-10

    Virus envelope proteins determine receptor utilization and host range. The choice of receptor not only permits specific targeting of cells that express it, but also directs the virus into specific endosomal trafficking pathways. Disrupting trafficking can result in loss of virus infectivity due to redirection of virions to non-productive pathways. Identification of the pathway or pathways used by a virus is, thus, important in understanding virus pathogenesis mechanisms and for developing new treatment strategies. Most of our understanding of alphavirus entry has focused on the Old World alphaviruses, such as Sindbis and Semliki Forest virus. In comparison, very little is known about the entry route taken by more pathogenic New World alphaviruses. Here, we use a novel contents mixing assay to identify the cellular requirements for entry of a New World alphavirus, Venezuelan equine encephalitis virus (VEEV). Expression of dominant negative forms of key endosomal trafficking genes shows that VEEV must access clathrin-dependent endocytic vesicles for membrane fusion to occur. Unexpectedly, the exit point is different from Old World alphaviruses that leave from early endosomes. Instead, VEEV also requires functional late endosomes. Furthermore, unlike the Old World viruses, VEEV entry is insensitive to cholesterol sequestration from cell membranes and may reflect a need to access an endocytic compartment that lacks cholesterol. This indicates fundamental differences in the entry route taken by VEEV compared to Old World alphaviruses.

  9. α6-Integrin is required for the adhesion and vasculogenic potential of hemangioma stem cells.

    PubMed

    Smadja, David M; Guerin, Coralie L; Boscolo, Elisa; Bieche, Ivan; Mulliken, John B; Bischoff, Joyce

    2014-03-01

    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. 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 HemSC in vitro and the vasculogenic and homing properties of HemSC in 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 i.v. delivery were significantly decreased in α6-integrin siRNA-transfected HemSC. In 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 antihemangioma therapy.

  10. Endogenous BMPR-IB signaling is required for early osteoblast differentiation of human bone cells.

    PubMed

    Singhatanadgit, Weerachai; Olsen, Irwin

    2011-03-01

    Osteoblast differentiation is tightly regulated by a number of cytokines and growth factors, including bone morphogenetic proteins (BMP) which stimulate osteoblast differentiation by signal transduction via three BMP receptors (BMPR-IA, -IB and -II). Although the mechanisms which regulate osteoblast differentiation are not fully understood, it is possible that endogenous BMPR signaling could play an important part in this process. To test this hypothesis, we have examined the expression and the functional significance of BMPR during osteoblast differentiation of primary human bone cells. The results showed that although the expression of BMPR-IA and -II transcripts were constantly expressed while the bone cells underwent osteoblast differentiation, the level of BMPR-IB mRNA was transiently, but significantly, up-regulated by threefold on day 3. This increase in BMPR-IB expression was found to be associated with the significant up-regulation of core binding factor alpha 1 (Cbfa1) and alkaline phosphatase (ALP) transcripts as well as the ALP activity, the well-established early markers of osteoblast differentiation. Transfection of bone cells with BMPR-IB small interfering RNA (siRNA) was found to significantly ablate the expression of BMPR-IB which subsequently resulted in reduction of Cbfa1 and ALP mRNA as well as the ALP activity. Moreover, exogenously added BMP-2 failed to rescue osteoblast differentiation of BMPR-IB siRNA-transfected bone cells. In conclusion, the present study has shown that endogenous BMPR-IB signaling is required for early phase of osteoblast differentiation of human bone cells in vitro, suggesting that BMPR-IB could be a therapeutic target for initiating bone healing in vivo.

  11. 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.

  12. Transendothelial migration of effector T cells across inflamed endothelial barriers does not require heparan sulfate proteoglycans.

    PubMed

    Stoler-Barak, Liat; Barzilai, Sagi; Zauberman, Ayelet; Alon, Ronen

    2014-06-01

    Leukocyte diapedesis is a chemotactic multistep process that requires optimal chemoattractant presentation by the endothelial barrier. Recent studies have described a critical role for heparan sulfate glycosaminoglycans (HSGAGs) in the presentation and functions of chemokines essential for lymphocyte interactions with the lymph node vasculature. We wished to test whether HS expression by a prototypic endothelial cell type, i.e. human umbilical vein endothelial cells (HUVECs), is critical for their ability to support neutrophil and lymphocyte adhesion and transendothelial migration (TEM) under shear flow. We found that HUVECs deposit HS GAGs mainly at their basolateral compartments in both their resting and inflamed states. We next inactivated the key enzyme involved in HS biosynthesis, exostosin-1 (Ext1). Silencing Ext1 resulted in a complete loss of HS biosynthesis; nonetheless, TNF-α and IL-1β stimulation of key adhesion molecules and inflammatory chemokines necessary for neutrophil or lymphocyte adhesion and TEM remained intact. Ext1 silencing reduced neutrophil arrest and markedly impaired TEM, consistent with a role of basolateral HS GAGs in directing neutrophil crossing of inflamed endothelial barriers. Strikingly, however, the TEM of effector T cells across identically Ext1-silenced HUVECs remained normal. Importantly, the biosynthesis of the main promigratory chemokines for effector T cells and neutrophils, respectively, CCL2 and CXCL1, and their vesicle distributions were also Ext1 independent. These results suggest that transmigrating neutrophils must respond to chemokines transiently presented by apical and basolateral endothelial HS GAGs. In contrast, effector T cells can integrate chemotactic TEM signals directly from intra-endothelial chemokine stores rather than from externally deposited chemokines.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. Chronic Myelogenous Leukemia- Initiating Cells Require Polycomb Group Protein EZH2.

    PubMed

    Xie, Huafeng; Peng, Cong; Huang, Jialiang; Li, Bin E; Kim, Woojin; Smith, Elenoe C; Fujiwara, Yuko; Qi, Jun; Cheloni, Giulia; Das, Partha P; Nguyen, Minh; Li, Shaoguang; Bradner, James E; Orkin, Stuart H

    2016-11-01

    Tyrosine kinase inhibitors (TKI) have revolutionized chronic myelogenous leukemia (CML) management. Disease eradication, however, is hampered by innate resistance of leukemia-initiating cells (LIC) to TKI-induced killing, which also provides the basis for subsequent emergence of TKI-resistant mutants. We report that EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is overexpressed in CML LICs and required for colony formation and survival and cell-cycle progression of CML cell lines. A critical role for EZH2 is supported by genetic studies in a mouse CML model. Inactivation of Ezh2 in conventional conditional mice and through CRISPR/Cas9-mediated gene editing prevents initiation and maintenance of disease and survival of LICs, irrespective of BCR-ABL1 mutational status, and extends survival. Expression of the EZH2 homolog EZH1 is reduced in EZH2-deficient CML LICs, creating a scenario resembling complete loss of PRC2. EZH2 dependence of CML LICs raises prospects for improved therapy of TKI-resistant CML and/or eradication of disease by addition of EZH2 inhibitors.

  19. 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.

  20. 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.

  1. 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

  2. Conformational Changes Required for Reovirus Cell Entry are Sensitive to pH

    PubMed Central

    Thete, Deepti; Danthi, Pranav

    2015-01-01

    During cell entry, reovirus particles disassemble to generate ISVPs. ISVPs undergo conformational changes to form ISVP* and this conversion is required for membrane penetration. In tissues where ISVP formation occurs within endosomes, ISVP-to-ISVP* conversion occurs at low pH. In contrast, in tissues where ISVP formation occurs extracellularly, ISVP-to-ISVP* transition occurs at neutral pH. Whether these two distinct pH environments influence the efficiency of cell entry is not known. In this study, we used Ouabain to lower the endosomal pH and determined its effect on reovirus infection. We found that Ouabain treatment blocks reovirus infection. In cells treated with Ouabain, virus attachment, internalization, and ISVP formation were unaffected but the efficiency of ISVP*s formation was diminished. Low pH also diminished the efficiency of ISVP-to-ISVP* conversion in vitro. Thus, the pH of the compartment where ISVP-to-ISVP* conversion takes place may dictate the efficiency of reovirus infection. PMID:26004253

  3. Molecular requirements for actin-based lamella formation in Drosophila S2 cells.

    PubMed

    Rogers, Stephen L; Wiedemann, Ursula; Stuurman, Nico; Vale, Ronald D

    2003-09-15

    Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of approximately 90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells.

  4. CARMA1 is required for Akt-mediated NF-kappaB activation in T cells.

    PubMed

    Narayan, Preeti; Holt, Brittany; Tosti, Richard; Kane, Lawrence P

    2006-03-01

    Many details of the generic pathway for induction of NF-kappaB have been delineated, but it is still not clear how multiple, diverse receptor systems are able to converge on this evolutionarily conserved family of transcription factors. Recent studies have shown that the CARMA1, Bcl10, and MALT1 proteins are critical for coupling the common elements of the NF-kappaB pathway to the T-cell receptor (TCR) and CD28. We previously demonstrated a role for the serine/threonine kinase Akt in CD28-mediated NF-kappaB induction. Using a CARMA1-deficient T-cell line, we have now found that the CARMA complex is required for induction of NF-kappaB by Akt, in cooperation with protein kinase C activation. Furthermore, using a novel selective inhibitor of Akt, we confirm that Akt plays a modulatory role in NF-kappaB induction by the TCR and CD28. Finally, we provide evidence for a physical and functional interaction between Akt and CARMA and for Akt-dependent phosphorylation of Bcl10. Therefore, in T cells, Akt impinges upon NF-kappaB signaling through at least two separate mechanisms.

  5. An interbacterial NAD(P)+ glycohydrolase toxin requires elongation factor Tu for delivery to target cells

    DOE PAGES

    Whitney, John C.; Quentin, Dennis; Sawai, Shin; ...

    2015-10-08

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD+ and NADP+. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). Thesemore » proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Lastly, visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.« less

  6. 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

  7. Rnd3-induced cell rounding requires interaction with Plexin-B2

    PubMed Central

    McColl, Brad; Garg, Ritu; Riou, Philippe; Riento, Kirsi

    2016-01-01

    ABSTRACT Rnd proteins are atypical members of the Rho GTPase family that induce actin cytoskeletal reorganization and cell rounding. Rnd proteins have been reported to bind to the intracellular domain of several plexin receptors, but whether plexins contribute to the Rnd-induced rounding response is not known. Here we show that Rnd3 interacts preferentially with plexin-B2 of the three plexin-B proteins, whereas Rnd2 interacts with all three B-type plexins, and Rnd1 shows only very weak interaction with plexin-B proteins in immunoprecipitations. Plexin-B1 has been reported to act as a GAP for R-Ras and/or Rap1 proteins. We show that all three plexin-B proteins interact with R-Ras and Rap1, but Rnd proteins do not alter this interaction or R-Ras or Rap1 activity. We demonstrate that plexin-B2 promotes Rnd3-induced cell rounding and loss of stress fibres, and enhances the inhibition of HeLa cell invasion by Rnd3. We identify the amino acids in Rnd3 that are required for plexin-B2 interaction, and show that mutation of these amino acids prevents Rnd3-induced morphological changes. These results indicate that plexin-B2 is a downstream target for Rnd3, which contributes to its cellular function. PMID:27656111

  8. Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells.

    PubMed

    Ly, Dalam; Mi, Qing-Sheng; Hussain, Shabbir; Delovitch, Terry L

    2006-09-15

    Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.

  9. 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

  10. Retrieval of HDEL proteins is required for growth of yeast cells

    PubMed Central

    1994-01-01

    The ERD2 gene of Saccharomyces cerevisiae encodes the receptor which retrieves HDEL-containing containing ER proteins from the Golgi apparatus. Viable erd2 mutants have been isolated that show no obvious HDEL-dependent retention of the luminal ER protein BiP, suggesting that retrieval of HDEL proteins is not essential for growth. However, cells that lack Erd2p completely have a defective Golgi apparatus and cannot grow. This observation led to the suggestion that the receptor had a second function, possibly related to its ability to recycle from Golgi to ER. In this paper we investigate the requirements for Erd2p to support growth. We show that mutations that block its recycling also prevent growth. In addition, we show that all mutant receptors that can support growth have a residual ability to retrieve BiP, which is detectable when they are overexpressed. Mere recycling of an inactive form of the receptor, mediated by a cytoplasmic KKXX sequence, is not sufficient for growth. Furthermore, saturation of the receptor by expression of an HDEL-tagged version of pro-alpha factor inhibits growth, even of strains that do not show obvious BiP retention. We conclude that growth requires the HDEL-dependent retrieval of one or more proteins, and that these proteins can be recognized even under conditions where BiP is secreted. Genetic screens have failed to identify any one protein whose loss could account for the Erd2p requirement. Therefore, a growth may require the retention of multiple HDEL proteins in the ER, or alternatively the removal of such proteins from the Golgi apparatus. PMID:7929564

  11. 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

  12. The posterior determinant gene nanos is required for the maintenance of the adult germline stem cells during Drosophila oogenesis.

    PubMed

    Bhat, K M

    1999-04-01

    In a variety of tissues in eukaryotes, multipotential stem cells are responsible for maintaining a germinal population and generating a differentiated progeny. The Drosophila germline is one such tissue where a continuous supply of eggs or sperm relies on the normal functioning of stem cells. Recent studies have implicated a possible role for the posterior determinant gene nanos (nos) in stem cells. Here, I report that nanos is required in the Drosophila female germline as well as in the male germline. In the female, nos is required for the functioning of stem cells. In nos mutants, while the stem cells are specified, these cells divide only a few times at the most and then degenerate. The loss of germline stem cells in nos mutant mothers appears to be due to a progressive degeneration of the plasma membrane. Furthermore, following germ cell loss, the germaria in the nos mutant mothers appear to carry on massive mitochondrial biogenesis activity. Thus, the syncytia of such germaria are filled with mitochondria. In the male germline, the male fertility assay indicates that nos appears to be also required for the maintenance of stem cells. In these mutant males, spermatogenesis is progressively affected and these males eventually become sterile. These results indicate novel requirements for nos in the Drosophila germline.

  13. 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

  14. 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

  15. 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.

  16. T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1.

    PubMed

    Staal, Jens; Driege, Yasmine; Bekaert, Tine; Demeyer, Annelies; Muyllaert, David; Van Damme, Petra; Gevaert, Kris; Beyaert, Rudi

    2011-05-04

    The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

  17. Sororin pre-mRNA splicing is required for proper sister chromatid cohesion in human cells.

    PubMed

    Watrin, Erwan; Demidova, Maria; Watrin, Tanguy; Hu, Zheng; Prigent, Claude

    2014-09-01

    Sister chromatid cohesion, which depends on cohesin, is essential for the faithful segregation of replicated chromosomes. Here, we report that splicing complex Prp19 is essential for cohesion in both G2 and mitosis, and consequently for the proper progression of the cell through mitosis. Inactivation of splicing factors SF3a120 and U2AF65 induces similar cohesion defects to Prp19 complex inactivation. Our data indicate that these splicing factors are all required for the accumulation of cohesion factor Sororin, by facilitating the proper splicing of its pre-mRNA. Finally, we show that ectopic expression of Sororin corrects defective cohesion caused by Prp19 complex inactivation. We propose that the Prp19 complex and the splicing machinery contribute to the establishment of cohesion by promoting Sororin accumulation during S phase, and are, therefore, essential to the maintenance of genome stability.

  18. A wheat embryo cell-free protein synthesis system not requiring an exogenous supply of GTP.

    PubMed

    Koga, Hirohisa; Misawa, Satoru; Shibui, Tatsuro

    2009-01-01

    Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous-flow wheat embryo cell-free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC-based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 microM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C-terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B(1-320)) gave almost the same result. The wheat embryo cell-free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48-h period at 26 degrees C.

  19. A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms.

    PubMed

    Néant, I; Charbonneau, M; Guerrier, P

    1989-04-01

    Populations of hormone-stimulated starfish oocytes and fertilized sea urchin eggs undergo synchronous meiotic and mitotic divisions. We have studied the requirement for protein phosphorylation during these events by testing the effects of 6-dimethylaminopurine (6-DMAP) upon the incorporation of [32P]orthophosphate. It was found that 6-DMAP blocked meiosis reinitiation and early cleavage and simultaneously inhibited protein phosphorylation, without changing the rate of [35S]methionine incorporation or pattern of protein synthesis. The protein, cyclin (54 kDa in starfish and 57 kDa in sea urchin), continues to be synthesized in the presence of 6-DMAP. This protein is destroyed at first and second cell cycles when 6-DMAP is added 30 min following fertilization but not when this drug is present before fertilization. Thus, cyclin breakdown does not depend on the completion of the nuclear events of M-phase, and its time of breakdown is set at an early step between fertilization and first cleavage. Using tubulin immunostaining, we found that 6-DMAP did not affect the cortical microtubules and resting female centrioles of prophase-arrested starfish oocytes, whereas it induced a precocious disappearance of spindle fibers when applied to hormone-stimulated oocytes. While an early addition of 6-DMAP precluded nuclear breakdown and spindle formation in both systems, a late treatment always allowed chromosome separation and centriole separation. Under these conditions pericentriolar tubulin persisted and could organize new spindles after the inhibitor was removed. It is suggested that (1) the assembly of cortical and centriolar-associated microtubules is not controlled by the same factors as spindle-associated tubulin; (2) specific proteins which are required for the cell to enter the following M-phase can become operative only via a process depending upon protein phosphorylation; (3) microtubule-associated kinases may play an important role in MPF function and spindle dynamics.

  20. Plac8 is required for white adipocyte differentiation in vitro and cell number control in vivo.

    PubMed

    Jimenez-Preitner, Maria; Berney, Xavier; Thorens, Bernard

    2012-01-01

    Plac8 belongs to an evolutionary conserved family of proteins, mostly abundant in plants where they control fruit weight through regulation of cell number. In mice, Plac8 is expressed both in white and brown adipose tissues and we previously showed that Plac8(-/-) mice develop late-onset obesity, with abnormal brown fat differentiation and reduced thermogenic capacity. We also showed that in brown adipocytes, Plac8 is an upstream regulator of C/EBPβ expression. Here, we first assessed the role of Plac8 in white adipogenesis in vitro. We show that Plac8 is induced early after induction of 3T3-L1 adipocytes differentiation, a process that is prevented by Plac8 knockdown; similarly, embryonic fibroblasts obtained from Plac8 knockout mice failed to form adipocytes upon stimulation of differentiation. Knockdown of Plac8 in 3T3-L1 was associated with reduced expression of C/EBPβ, Krox20, and Klf4, early regulators of the white adipogenic program, and we show that Plac8 could transactivate the C/EBPβ promoter. In vivo, we show that absence of Plac8 led to increased white fat mass with enlarged adipocytes but reduced total number of adipocytes. Finally, even though Plac8(-/-) mice showed impaired thermogenesis due to brown fat dysfunction, this was not associated with changes in glycemia or plasma free fatty acid and triglyceride levels. Collectively, these data indicate that Plac8 is an upstream regulator of C/EBPβ required for adipogenesis in vitro. However, in vivo, Plac8 is dispensable for the differentiation of white adipocytes with preserved fat storage capacity but is required for normal fat cell number regulation.

  1. Peripherally induced tolerance depends on pTreg cells that require Hopx to inhibit intrinsic IL-2 expression

    PubMed Central

    Jones, Andrew; Opejin, Adeleye; Henderson, Jacob G.; Gross, Cindy; Jain, Rajan; Epstein, Jonathan A.; Flavell, Richard A.; Hawiger, Daniel

    2015-01-01

    Dendritic Cells (DCs) can induce peripheral immune tolerance that prevents autoimmune responses. Antigen presentation by peripheral DCs under steady state conditions leads to a conversion of some peripheral CD4+ T cells into Treg cells that require Homeodomain Only Protein (Hopx) to mediate T cell unresponsiveness. However, the roles of these peripheral (p)Treg cells in averting autoimmune responses as well as immunological mechanisms of Hopx remain unknown. Here we report that Hopx+ pTreg cells converted by DCs from Hopxneg T cells are indispensible to sustain tolerance that prevents autoimmune responses directed at self-antigens during experimental acute encephalomyelitis (EAE). Our studies further reveal that Hopx inhibits intrinsic IL-2 expression in pTreg cells after antigenic re-challenge. In the absence of Hopx, increased levels of IL-2 lead to death and decreased numbers of pTreg cells. Therefore formation of Hopx+ pTreg cells represents a crucial pathway of sustained tolerance induced by peripheral DCs and the maintenance of such pTreg cells and tolerance requires functions of Hopx to block intrinsic IL-2 production in pTreg cells. PMID:26170384

  2. HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores.

    PubMed

    Monel, Blandine; Beaumont, Elodie; Vendrame, Daniela; Schwartz, Olivier; Brand, Denys; Mammano, Fabrizio

    2012-04-01

    Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.

  3. Minimal requirements for exocytosis. A study using PC 12 cells permeabilized with staphylococcal alpha-toxin

    SciTech Connect

    Ahnert-Hilger, G.; Bhakdi, S.; Gratzl, M.

    1985-10-15

    The membrane-permeabilizing effects of streptolysin O, staphylococcal alpha-toxin, and digitonin on cultured rat pheochromocytoma cells were studied. All three agents perturbed the plasma membrane, causing release of intracellular YWRb and uptake of trypan blue. In addition, streptolysin O and digitonin also damaged the membranes of secretory vesicles, including a parallel release of dopamine. In contrast, the effects of alpha-toxin appeared to be strictly confined to the plasma membrane, and no dopamine release was observed with this agent. The exocytotic machinery, however, remained intact and could be triggered by subsequent introduction of micromolar concentrations of Ca2+ into the medium. Dopamine release was entirely Ca2+ specific and occurred independent of the presence or absence of other cations or anions including K+ glutamate, K+ acetate, or Na+ chloride. Ca2+-induced exocytosis did not require the presence of Mg2+-ATP in the medium. The process was insensitive to pH alterations in the range pH 6.6-7.2, and appeared optimal at an osmolarity of 300 mosm/kg. Toxin permeabilization seems to be an excellent method for studying the minimal requirements for exocytosis.

  4. Live imaging and modeling of inner nuclear membrane targeting reveals its molecular requirements in mammalian cells

    PubMed Central

    Boni, Andrea; Politi, Antonio Z.; Strnad, Petr; Xiang, Wanqing; Hossain, M. Julius

    2015-01-01

    Targeting of inner nuclear membrane (INM) proteins is essential for nuclear architecture and function, yet its mechanism remains poorly understood. Here, we established a new reporter that allows real-time imaging of membrane protein transport from the ER to the INM using Lamin B receptor and Lap2β as model INM proteins. These reporters allowed us to characterize the kinetics of INM targeting and establish a mathematical model of this process and enabled us to probe its molecular requirements in an RNA interference screen of 96 candidate genes. Modeling of the phenotypes of genes involved in transport of these INM proteins predicted that it critically depended on the number and permeability of nuclear pores and the availability of nuclear binding sites, but was unaffected by depletion of most transport receptors. These predictions were confirmed with targeted validation experiments on the functional requirements of nucleoporins and nuclear lamins. Collectively, our data support a diffusion retention model of INM protein transport in mammalian cells. PMID:26056140

  5. Endocytosis of megalin by visceral endoderm cells requires the Dab2 adaptor protein.

    PubMed

    Maurer, Meghan E; Cooper, Jonathan A

    2005-11-15

    Rapid endocytosis of lipoprotein receptors involves NPxY signals contained in their cytoplasmic tails. Several proteins, including ARH and Dab2, can bind these sequences, but their importance for endocytosis may vary in different cell types. The lipoprotein receptor megalin is expressed in the visceral endoderm (VE), a polarized epithelium that supplies maternal nutrients to the early mammalian embryo. Dab2 is also expressed in the VE, and is required for embryo growth and gastrulation. Here, we show that ARH is absent from the VE, and Dab2 is required for uptake of megalin, its co-receptor cubilin, and a cubilin ligand, transferrin, from the brush border of the VE into intracellular vesicles. By making isoform-specific knock-in mice, we show that the p96 splice form of Dab2, which binds endocytic proteins, can fully rescue endocytosis. The more abundant p67 isoform, which lacks some endocytic protein binding sites, only partly rescues endocytosis. Endocytosis of cubilin is also impaired in VE and in mid-gestation visceral yolk sac when p96 is absent. These studies suggest that Dab2 p96 mediates endocytosis of megalin in the VE. In addition, rescue of embryonic viability correlates with endocytosis, suggesting that endocytosis mediated by Dab2 is important for normal development.

  6. 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

  7. whmD is an essential mycobacterial gene required for proper septation and cell division

    PubMed Central

    Gomez, James E.; Bishai, William R.

    2000-01-01

    A study of potential mycobacterial regulatory genes led to the isolation of the Mycobacterium smegmatis whmD gene, which encodes a homologue of WhiB, a Streptomyces coelicolor protein required for sporulation. Unlike its Streptomyces homologue, WhmD is essential in M. smegmatis. The whmD gene could be disrupted only in the presence of a plasmid supplying whmD in trans. A plasmid that allowed chemically regulated expression of the WhmD protein was used to generate a conditional whmD mutant. On withdrawal of the inducer, the conditional whmD mutant exhibited irreversible, filamentous, branched growth with diminished septum formation and aberrant septal placement, whereas WhmD overexpression resulted in growth retardation and hyperseptation. Nucleic acid synthesis and levels of the essential cell division protein FtsZ were unaltered by WhmD deficiency. Together, these phenotypes indicate a role for WhmD in mycobacterial septum formation and cell division. PMID:10880571

  8. The deubiquitinating enzyme complex BRISC is required for proper mitotic spindle assembly in mammalian cells

    PubMed Central

    Yan, Kaowen; Li, Li; Wang, Xiaojian; Hong, Ruisha; Zhang, Ying; Yang, Hua; Lin, Ming; Zhang, Sha; He, Qihua; Zheng, Duo; Tang, Jun; Yin, Yuxin

    2015-01-01

    Deubiquitinating enzymes (DUBs) negatively regulate protein ubiquitination and play an important role in diverse physiological processes, including mitotic division. The BRCC36 isopeptidase complex (BRISC) is a DUB that is specific for lysine 63–linked ubiquitin hydrolysis; however, its biological function remains largely undefined. Here, we identify a critical role for BRISC in the control of mitotic spindle assembly in cultured mammalian cells. BRISC is a microtubule (MT)-associated protein complex that predominantly localizes to the minus ends of K-fibers and spindle poles and directly binds to MTs; importantly, BRISC promotes the assembly of functional bipolar spindle by deubiquitinating the essential spindle assembly factor nuclear mitotic apparatus (NuMA). The deubiquitination of NuMA regulates its interaction with dynein and importin-β, which are required for its function in spindle assembly. Collectively, these results uncover BRISC as an important regulator of the mitotic spindle assembly and cell division, and have important implications for the development of anticancer drugs targeting BRISC. PMID:26195665

  9. Wnt secretion is required to maintain high levels of Wnt activity in colon cancer cells.

    PubMed

    Voloshanenko, Oksana; Erdmann, Gerrit; Dubash, Taronish D; Augustin, Iris; Metzig, Marie; Moffa, Giusi; Hundsrucker, Christian; Kerr, Grainne; Sandmann, Thomas; Anchang, Benedikt; Demir, Kubilay; Boehm, Christina; Leible, Svenja; Ball, Claudia R; Glimm, Hanno; Spang, Rainer; Boutros, Michael

    2013-01-01

    Aberrant regulation of the Wnt/β-catenin pathway has an important role during the onset and progression of colorectal cancer, with over 90% of cases of sporadic colon cancer featuring mutations in APC or β-catenin. However, it has remained a point of controversy whether these mutations are sufficient to activate the pathway or require additional upstream signals. Here we show that colorectal tumours express elevated levels of Wnt3 and Evi/Wls/GPR177. We found that in colon cancer cells, even in the presence of mutations in APC or β-catenin, downstream signalling remains responsive to Wnt ligands and receptor proximal signalling. Furthermore, we demonstrate that truncated APC proteins bind β-catenin and key components of the destruction complex. These results indicate that cells with mutations in APC or β-catenin depend on Wnt ligands and their secretion for a sufficient level of β-catenin signalling, which potentially opens new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls.

  10. Glucocorticoid Modulation of Mitochondrial Function in Hepatoma Cells Requires the Mitochondrial Fission Protein Drp1

    PubMed Central

    Hernández-Alvarez, María Isabel; Paz, José C.; Sebastián, David; Muñoz, Juan Pablo; Liesa, Marc; Segalés, Jessica; Palacín, Manuel

    2013-01-01

    Abstract Aims: Glucocorticoids, such as dexamethasone, enhance hepatic energy metabolism and gluconeogenesis partly through changes in mitochondrial function. Mitochondrial function is influenced by the balance between mitochondrial fusion and fission events. However, whether glucocorticoids modulate mitochondrial function through the regulation of mitochondrial dynamics is currently unknown. Results: Here, we report that the effects of dexamethasone on mitochondrial function and gluconeogenesis in hepatoma cells are dependent on the mitochondrial fission protein dynamin-related protein 1 (Drp1). Dexamethasone increased routine oxygen consumption, maximal respiratory capacity, superoxide anion, proton leak, and gluconeogenesis in hepatoma cells. Under these conditions, dexamethasone altered mitochondrial morphology, which was paralleled by a large increase in Drp1 expression, and reduced mitofusin 1 (Mfn1) and Mfn2. In vivo dexamethasone treatment also enhanced Drp1 expression in mouse liver. On the basis of these observations, we analyzed the dependence on the Drp1 function of dexamethasone effects on mitochondrial respiration and gluconeogenesis. We show that the increase in mitochondrial respiration and gluconeogenesis induced by dexamethasone are hampered by the inhibition of Drp1 function. Innovation: Our findings provide the first evidence that the effects of glucocorticoids on hepatic metabolism require the mitochondrial fission protein Drp1. Conclusion: In summary, we demonstrate that the mitochondrial effects of dexamethasone both on mitochondrial respiration and on the gluconeogenic pathway depend on Drp1. Antioxid. Redox Signal. 19, 366–378. PMID:22703557

  11. 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.

  12. 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.

  13. putzig is required for cell proliferation and regulates notch activity in Drosophila.

    PubMed

    Kugler, Sabrina J; Nagel, Anja C

    2007-10-01

    We have identified the gene putzig (pzg) as a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that was found earlier within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. Here, we provide the first in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. Our findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation.

  14. S1P3 confers differential S1P migration by autoreactive and non-autoreactive immature B cells and is required for normal B cell development

    PubMed Central

    Donovan, Erin E.; Pelanda, Roberta; Torres, Raul M.

    2010-01-01

    SUMMARY During B cell development, immature B cell fate is determined by whether the B cell antigen receptor is engaged in the bone marrow. Immature B cells that are non-autoreactive continue maturation and emigrate from the marrow whereas autoreactive immature B cells remain and are tolerized. However, the microenvironment where these events occur and the chemoattractants responsible for immature B cell trafficking within and out of the bone marrow remain largely undefined. Sphingosine 1-phosphate (S1P) is a chemoattractant that directs lymphocyte trafficking and thymocyte egress and in this study we investigated whether S1P contributed to B cell development, egress and positioning within the bone marrow. Our findings show that immature B cells are chemotactic towards S1P but that this response is dependent on antigen receptor specificity: non-autoreactive, but not autoreactive, immature B cells migrate towards S1P and are shown to require S1P3 receptor for this response. Despite this response, S1P3 is shown not to facilitate immature B cell egress but is required for normal B cell development including the positioning of transitional B cells within bone marrow sinusoids. These data indicate that S1P3 signaling directs immature B cells to a bone marrow microenvironment important for both tolerance induction and maturation. PMID:20039302

  15. 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.

  16. Mapping of ESE-1 subdomains required to initiate mammary epithelial cell transformation via a cytoplasmic mechanism

    PubMed Central

    2011-01-01

    Background The ETS family transcription factor ESE-1 is often overexpressed in human breast cancer. ESE-1 initiates transformation of MCF-12A cells via a non-transcriptional, cytoplasmic process that is mediated by a unique 40-amino acid serine and aspartic acid rich (SAR) subdomain, whereas, ESE-1's nuclear transcriptional property is required to maintain the transformed phenotype of MCF7, ZR-75-1 and T47D breast cancer cells. Results To map the minimal functional nuclear localization (NLS) and nuclear export (NES) signals, we fused in-frame putative NLS and NES motifs between GFP and the SAR domain. Using these GFP constructs as reporters of subcellular localization, we mapped a single NLS to six basic amino acids (242HGKRRR247) in the AT-hook and two CRM1-dependent NES motifs, one to the pointed domain (NES1: 102LCNCALEELRL112) and another to the DNA binding domain (DBD), (NES2: 275LWEFIRDILI284). Moreover, analysis of a putative NLS located in the DBD (316GQKKKNSN323) by a similar GFP-SAR reporter or by internal deletion of the DBD, revealed this sequence to lack NLS activity. To assess the role of NES2 in regulating ESE-1 subcellular localization and subsequent transformation potency, we site-specifically mutagenized NES2, within full-length GFP-ESE-1 and GFP-NES2-SAR reporter constructs. These studies show that site-specific mutation of NES2 completely abrogates ESE-1 transforming activity. Furthermore, we show that exclusive cytoplasmic targeting of the SAR domain is sufficient to initiate transformation, and we report that an intact SAR domain is required, since block mutagenesis reveals that an intact SAR domain is necessary to maintain its full transforming potency. Finally, using a monoclonal antibody targeting the SAR domain, we demonstrate that the SAR domain contains a region accessible for protein - protein interactions. Conclusions These data highlight that ESE-1 contains NLS and NES signals that play a critical role in regulating its subcellular

  17. Supporting cell division is not required for regeneration of auditory hair cells after ototoxic injury in vitro.

    PubMed

    Shang, Jialin; Cafaro, Jon; Nehmer, Rachel; Stone, Jennifer

    2010-06-01

    In chickens, nonsensory supporting cells divide and regenerate auditory hair cells after injury. Anatomical evidence suggests that supporting cells can also transdifferentiate into hair cells without dividing. In this study, we characterized an organ culture model to study auditory hair cell regeneration, and we used these cultures to test if direct transdifferentiation alone can lead to significant hair cell regeneration. Control cultures (organs from posthatch chickens maintained without streptomycin) showed complete hair cell loss in the proximal (high-frequency) region by 5 days. In contrast, a 2-day treatment with streptomycin induced loss of hair cells from all regions by 3 days. Hair cell regeneration proceeded in culture, with the time course of supporting cell division and hair cell differentiation generally resembling in vivo patterns. The degree of supporting cell division depended upon the presence of streptomycin, the epithelial region, the type of culture media, and serum concentration. On average, 87% of the regenerated hair cells lacked the cell division marker BrdU despite its continuous presence, suggesting that most hair cells were regenerated via direct transdifferentiation. Addition of the DNA polymerase inhibitor aphidicolin to culture media prevented supporting cell division, but numerous hair cells were regenerated nonetheless. These hair cells showed signs of functional maturation, including stereociliary bundles and rapid uptake of FM1-43. These observations demonstrate that direct transdifferentiation is a significant mechanism of hair cell regeneration in the chicken auditory after streptomycin damage in vitro.

  18. Proliferating cell nuclear antigen (PCNA) is required for cell cycle-regulated silent chromatin on replicated and nonreplicated genes.

    PubMed

    Miller, Andrew; Chen, Jiji; Takasuka, Taichi E; Jacobi, Jennifer L; Kaufman, Paul D; Irudayaraj, Joseph M K; Kirchmaier, Ann L

    2010-11-05

    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.

  19. WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

    PubMed

    Sakakibara, Keiko; Reisewitz, Pascal; Aoyama, Tsuyoshi; Friedrich, Thomas; Ando, Sayuri; Sato, Yoshikatsu; Tamada, Yosuke; Nishiyama, Tomoaki; Hiwatashi, Yuji; Kurata, Tetsuya; Ishikawa, Masaki; Deguchi, Hironori; Rensing, Stefan A; Werr, Wolfgang; Murata, Takashi; Hasebe, Mitsuyasu; Laux, Thomas

    2014-04-01

    Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

  20. 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

  1. The scaffold protein MEK Partner 1 is required for the survival of estrogen receptor positive breast cancer cells.

    PubMed

    Marina, Mihaela; Wang, Limin; Conrad, Susan E

    2012-07-09

    MEK Partner 1 (MP1 or MAPKSP1) is a scaffold protein that has been reported to function in multiple signaling pathways, including the ERK, PAK and mTORC pathways. Several of these pathways influence the biology of breast cancer, but MP1's functional significance in breast cancer cells has not been investigated. In this report, we demonstrate a requirement for MP1 expression in estrogen receptor (ER) positive breast cancer cells. MP1 is widely expressed in both ER-positive and negative breast cancer cell lines, and in non-tumorigenic mammary epithelial cell lines. However, inhibition of its expression using siRNA duplexes resulted in detachment and apoptosis of several ER-positive breast cancer cell lines, but not ER-negative breast cancer cells or non-tumorigenic mammary epithelial cells. Inhibition of MP1 expression in ER-positive MCF-7 cells did not affect ERK activity, but resulted in reduced Akt1 activity and reduced ER expression and activity. Inhibition of ER expression did not result in cell death, suggesting that decreased ER expression is not the cause of cell death. In contrast, pharmacological inhibition of PI3K signaling did induce cell death in MCF-7 cells, and expression of a constitutively active form of Akt1 partially rescued the cell death observed when the MP1 gene was silenced in these cells. Together, these results suggest that MP1 is required for pro-survival signaling from the PI3K/Akt pathway in ER-positive breast cancer cells.

  2. Activated Notch signaling is required for hepatitis B virus X protein to promote proliferation and survival of human hepatic cells.

    PubMed

    Wang, Fan; Zhou, Haiyan; Xia, Xiumei; Sun, Qian; Wang, Ying; Cheng, Bin

    2010-12-01

    Hepatitis B virus X protein (HBx) is a multifunctional oncoprotein which plays a crucial role in the pathogenesis of hepatocellular carcinoma (HCC). However, the exact mechanisms remain controversial. Here we show that HBx strongly stimulated cell growth, promoted cell cycle progression and inhibited apoptosis of human non-tumor hepatic cell line L02 cells. It also accelerated tumor formation of L02 cells in BALB/c nude mice. Furthermore, Notch signaling components were upregulated in HBx-expressing L02 cells compared to normal L02 cells. However, blocking Notch signaling with a γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) attenuated cell growth, shortened the S phase of cell cycle and promoted apoptosis of HBx-expressing L02 cell in a dose- and time-dependent manner, but normal L02 cells were not significantly affected by Notch signaling blocking. Therefore, our findings demonstrate that HBx could promote the growth of human non-tumor hepatic cell line L02 cells both in vitro and in vivo, which may require the activation of Notch signaling pathway.

  3. Class A scavenger receptor-mediated cell adhesion requires the sequential activation of Lyn and PI3-kinase.

    PubMed

    Nikolic, Dejan M; Cholewa, Jill; Gass, Cecelia; Gong, Ming C; Post, Steven R

    2007-04-01

    Class A scavenger receptors (SR-A) participate in multiple macrophage functions including macrophage adhesion to modified proteins. SR-A-mediated adhesion may therefore contribute to chronic inflammation by promoting macrophage accumulation at sites of protein modification. The mechanisms that couple SR-A binding to modified proteins with increased cell adhesion have not been defined. In this study, SR-A expressing HEK cells and SR-A+/+ or SR-A-/- macrophages were used to delineate the signaling pathways required for SR-A-mediated adhesion to modified protein. Inhibiting G(i/o) activation, which decreases initial SR-A-mediated cell attachment, did not prevent the subsequent spreading of attached cells. In contrast, inhibition of Src kinases or PI3-kinase abolished SR-A-dependent cell spreading without affecting SR-A-mediated cell attachment. Consistent with these results, the Src kinase Lyn and PI3-kinase were sequentially activated during SR-A-mediated cell spreading. Furthermore, activation of both Lyn and PI3-kinase was required for enhancing paxillin phosphorylation. Activation of a Src kinase-PI3-kinase-Akt pathway was also observed in cells expressing a truncated SR-A protein that does not internalize indicating that SR-A-mediated activation of intracellular signaling cascades following adhesion to MDA-BSA is independent of receptor internalization. Thus SR-A binding to modified protein activates signaling cascades that have distinct roles in regulating initial cell attachment and subsequent cell spreading.

  4. Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal.

    PubMed

    Solana, Jordi; Lasko, Paul; Romero, Rafael

    2009-04-15

    Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.

  5. Peyer's Patch M Cells Derived from Lgr5+ Stem Cells Require SpiB and Are Induced by RankL in Cultured “Miniguts”

    PubMed Central

    de Lau, Wim; Kujala, Pekka; Schneeberger, Kerstin; Middendorp, Sabine; Li, Vivian S. W.; Barker, Nick; Martens, Anton; Hofhuis, Frans; DeKoter, Rodney P.; Peters, Peter J.; Nieuwenhuis, Edward

    2012-01-01

    Peyer's patches consist of domains of specialized intestinal epithelium overlying gut-associated lymphoid tissue (GALT). Luminal antigens reach the GALT by translocation through epithelial gatekeeper cells, the so-called M cells. We recently demonstrated that all epithelial cells required for the digestive functions of the intestine are generated from Lgr5-expressing stem cells. Here, we show that M cells also derive from these crypt-based Lgr5 stem cells. The Ets family transcription factor SpiB, known to control effector functions of bone marrow-derived immune cells, is specifically expressed in M cells. In SpiB−/− mice, M cells are entirely absent, which occurs in a cell-autonomous fashion. It has been shown that Tnfsf11 (RankL) can induce M cell development in vivo. We show that in intestinal organoid (“minigut”) cultures, stimulation with RankL induces SpiB expression within 24 h and expression of other M cell markers subsequently. We conclude that RankL-induced expression of SpiB is essential for Lgr5 stem cell-derived epithelial precursors to develop into M cells. PMID:22778137

  6. Lipid rafts are required for GLUT4 internalization in adipose cells

    PubMed Central

    Ros-Baró, Anna; López-Iglesias, Carmen; Peiró, Sandra; Bellido, David; Palacín, Manuel; Zorzano, Antonio; Camps, Marta

    2001-01-01

    It has been recently reported that insulin recruits a novel signaling machinery to lipid rafts required for insulin-stimulated GLUT4 translocation [Baumann, A., Ribon, V., Kanzaki, M., Thurmond, D. C., Mora, S., Shigematsu, S., Bickel, P. E., Pessin, J. E. & Saltiel, A. R. (2001) Nature 407, 202–207, 2000; Chiang, S. H., Baumann, C. A., Kanzaki, M., Thurmond, D. C., Watson, R. T., Neudauer, C. L., Macara, I. G., Pessin, J. E. & Saltiel, A. R. (2001) Nature 410, 944–948]. We have assessed the role of lipid rafts on GLUT4 traffic in adipose cells. High GLUT4 levels were detected in caveolae from adipocytes by two approaches, the mechanical isolation of purified caveolae from plasma membrane lawns and the immunogold analysis of plasma membrane lawns followed by freeze-drying. The role of lipid rafts in GLUT4 trafficking was studied by adding nystatin or filipin at concentrations that specifically disrupt caveolae morphology and inhibit caveolae function without altering clathrin-mediated endocytosis. These caveolae inhibitors did not affect the insulin-stimulated glucose transport. However, they blocked both the GLUT4 internalization and the down-regulation of glucose transport triggered by insulin removal in 3T3-L1 adipocytes. Our data indicate that lipid rafts are crucial for GLUT4 internalization after insulin removal. Given that high levels of GLUT4 were detected in caveolae from insulin-treated adipose cells, this transporter may be internalized from caveolae or caveolae may operate as an obligatory transition station before internalization. PMID:11593015

  7. CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis

    PubMed Central

    Ruhlmann, Jeffrey M.; Kram, Brian W.; Carter, Clay J.

    2010-01-01

    To date, no genes have been reported to directly affect the de novo production of floral nectar. In an effort to identify genes involved in nectar production, the Affymetrix® ATH1 GeneChip was previously used to examine global gene expression profiles in Arabidopsis thaliana nectaries. One of the genes displaying highly enriched expression in nectaries was CELL WALL INVERTASE 4 (AtCWINV4, At2g36190), which encodes an enzyme that putatively catalyses the hydrolysis of sucrose into glucose and fructose. RT-PCR was used to confirm the nectary-enriched expression of AtCWINV4, as well as an orthologue from Brassica rapa. To probe biological function, two independent Arabidopsis cwinv4 T-DNA mutants were isolated. Unlike wild-type plants, cwinv4 lines did not produce nectar. While overall nectary morphology appeared to be normal, cwinv4 flowers accumulated higher than normal levels of starch in the receptacle, but not within the nectaries themselves. Conversely, wild-type, but not cwinv4, nectarial stomata stained intensely for starch. Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar. Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within nectaries, thus allowing them to accumulate the sugars necessary for nectar production. In addition, CWINV4 is probably responsible for the hexose-rich composition observed for many Brassicaceae nectars. PMID:19861655

  8. Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells.

    PubMed Central

    Paz, P E; Wang, S; Clarke, H; Lu, X; Stokoe, D; Abo, A

    2001-01-01

    T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110alpha and phospholipase Cgamma1 (PLCgamma1). Our results indicate that Tyr(226) and Tyr(191) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and PLCgamma1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr(191) and Tyr(171) are required for T-cell activation and Tyr(132) is required for the activation of PLCgamma1 and Ras signalling pathways. PMID:11368773

  9. Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

    PubMed Central

    Herman, P K; Rine, J

    1997-01-01

    Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression. PMID:9321396

  10. SAP expression in invariant NKT cells is required for cognate help to support B-cell responses.

    PubMed

    Detre, Cynthia; Keszei, Marton; Garrido-Mesa, Natividad; Kis-Toth, Katalin; Castro, Wilson; Agyemang, Amma F; Veerapen, Natacha; Besra, Gurdyal S; Carroll, Michael C; Tsokos, George C; Wang, Ninghai; Leadbetter, Elizabeth A; Terhorst, Cox

    2012-07-05

    One of the manifestations of X-linked lymphoproliferative disease (XLP) is progressive agammaglobulinemia, caused by the absence of a functional signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) in T, invariant natural killer T (NKT) cells and NK cells. Here we report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. Whereas NKT cell-dependent B cell responses were absent in SAP(-/-).B6 mice that completely lack NKT cells, the small number of SAP-deficient NKT cells in SAP(-/-).BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted after development in SAP(fl/fl).tgCreERT2.B6 mice. We find that NKT cell intrinsic expression of SAP is dispensable for noncognate helper functions, but is critical for providing cognate help to antigen-specific B cells. These results demonstrate that SLAM-family receptor-regulated cell-cell interactions are not limited to T-B cell conjugates. We conclude that in the absence of SAP, several routes of NKT cell-mediated antibody production are still accessible. The latter suggests that residual NKT cells in XLP patients might contribute to variations in dysgammaglobulinemia.

  11. T suppressor cells are required for the maintenance of the antigen-induced B-cell unresponsive state in humans

    SciTech Connect

    Benveniste, E.; Stevens, R.H.

    1983-04-01

    Tetanus toxoid immunization of humans generates circulating B cells which secrete IgG anti-tetanus toxoid antibodies (IgG-Tet) when stimulated in vitro with T cells and pokeweed mitogen (PWM). A unique property of these cells is the inhibition of maturation into antibody-secreting plasma cells following a 1-hr in vitro pulse with tetanus toxoid. Studies were undertaken to determine if different T-cell subsets could modulate the in vitro generated B-cell unresponsive state. The addition of OKT4+/OKT8- cells to antigen-treated B cells resulted in a partial reversal of the antigen-induced inhibition of IgG-Tet synthesis. The addition of OKT4-/OKT8+ cells to the treated B cells caused a suppression of IgG-Tet synthesis comparable to that seen in cultures containing unfractionated T cells. These results indicate that (1) the B-cell unresponsive state generated by antigen treatment is not absolute, (2) the degree of B-cell unresponsiveness results from a balance of suppressor and helper signals, and (3) T-suppressor cells need to be present to induce and maintain the B-cell unresponsive state.

  12. 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.

  13. Myc and Fgf Are Required for Zebrafish Neuromast Hair Cell Regeneration.

    PubMed

    Lee, Sang Goo; Huang, Mingqian; 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.

  14. 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

    2016-03-25

    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.

  15. Differential requirements for the Ets transcription factor Elf-1 in the development of NKT cells and NK cells.

    PubMed

    Choi, Hak-Jong; Geng, Yanbiao; Cho, Hoonsik; Li, Sha; Giri, Pramod Kumar; Felio, Kyrie; Wang, Chyung-Ru

    2011-02-10

    E26 Transformation specific (Ets) family transcription factors control the expression of a large number of genes regulating hematopoietic cell development and function. Two such transcription factors, Ets-1 and myeloid Elf-1-like factor (MEF), have been shown to play critical roles in both natural killer (NK)- and NKT-cell development, but not in the development of conventional T cells. In this study, we address the role of E74-like factor 1 (Elf-1), another Ets family transcription factor that is closely related to MEF but divergent from Ets-1, in NK- and NKT-cell development using Elf-1-deficient (Elf-1(-/-)) mice. Whereas the proportion of NK cells in Elf-1(-/-) mice was normal, the proportion of NKT cells was significantly reduced in the thymus and periphery of Elf-1(-/-) mice compared with wild-type (WT) mice. Although Ets-1-deficient mice lack NKT cells altogether, Elf-1(-/-) mice exhibited only a partial block in NKT-cell development caused by a cell-intrinsic defect in the selection, survival, and maturation of NKT cells. In addition, residual NKT cells found in Elf-1(-/-) mice produced less cytokine upon antigen stimulation compared with WT NKT cells. Our data demonstrate that Elf-1 plays an important and nonredundant role in the development and function of NKT cells, but is not involved in NK-cell development.

  16. 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.

  17. 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.

  18. PKCδ is required for Jagged-1 induction of human mesenchymal stem cell osteogenic differentiation.

    PubMed

    Zhu, Fengchang; Sweetwyne, Mariya T; Hankenson, Kurt D

    2013-06-01

    JAG1, the gene for the Jagged-1 ligand (Jag1) in the Notch signaling pathway, is variably mutated in Alagille Syndrome (ALGS). ALGS patients have skeletal defects, and additionally JAG1 has been shown to be associated with low bone mass through genome-wide association studies. Plating human osteoblast precursors (human mesenchymal stem cells-hMSCs) on Jag1 is sufficient to induce osteoblast differentiation; however, exposure of mouse MSC (mMSC) to Jag1 actually inhibits osteoblastogenesis. Overexpression of the notch-2 intracellular domain (NICD2) is sufficient to mimic the effect of Jag1 on hMSC osteoblastogenesis, while blocking Notch signaling with a γ-secretase inhibitor or with dominant-negative mastermind inhibits Jag1-induced hMSC osteoblastogenesis. In pursuit of interacting signaling pathways, we discovered that treatment with a protein kinase C δ (PKCδ) inhibitor abrogates Jag1-induced hMSC osteoblastogenesis. Jag1 results in rapid PKCδ nuclear translocation and kinase activation. Furthermore, Jag1 stimulates the physical interaction of PKCδ with NICD. Collectively, these results suggest that Jag1 induces hMSC osteoblast differentiation through canonical Notch signaling and requires concomitant PKCδ signaling. This research also demonstrates potential deficiencies in using mouse models to study ALGS bone abnormalities.

  19. AMP-activated protein kinase is required for cell survival and growth in HeLa-S3 cells in vivo.

    PubMed

    Song, Xuhong; Huang, Dongyang; Liu, Yanmin; Pan, Xiaokang; Zhang, Jing; Liang, Bin

    2014-06-01

    Activation of the AMP-dependent protein kinase (AMPK) is linked to cancer cell survival in a variety of cancer cell lines, particularly under conditions of stress. As a potent activator of AMPK, metformin has become a hot topic of discussion for its effect on cancer cell. Here, we report that AMPK activated by metformin promotes HeLa-S3 cell survival and growth in vivo. Our results show that metformin inhibited cell proliferation in MCF-7 cells, but not in LKB1-deficient HeLa-S3 cells. Re-expression of LKB-1 in HeLa-S3 cells restored the growth inhibitory effect of metformin, indicating a requirement for LKB-1 in metformin-induced growth inhibition. Moreover, AMPK activation exerted a protective effect in HeLa-S3 cells by relieving ER stress, modulating ER Ca(2+) storage, and finally contributing to cellular adaptation and resistance to apoptosis. Our findings identify a link between AMPK activation and cell survival in HeLa-S3 cells, which demonstrates a beneficial effect of AMPK activated by metformin in cancer cell, and suggests a discrete re-evaluation on the role of metformin/AMPK activation on tumor cell growth, proliferation, and on clinical application in cancer therapy.

  20. 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.

  1. Dynamic filopodia are required for chemokine-dependent intracellular polarization during guided cell migration in vivo.

    PubMed

    Meyen, Dana; Tarbashevich, Katsiaryna; Banisch, Torsten U; Wittwer, Carolina; Reichman-Fried, Michal; Maugis, Benoît; Grimaldi, Cecilia; Messerschmidt, Esther-Maria; Raz, Erez

    2015-04-15

    Cell migration and polarization is controlled by signals in the environment. Migrating cells typically form filopodia that extend from the cell surface, but the precise function of these structures in cell polarization and guided migration is poorly understood. Using the in vivo model of zebrafish primordial germ cells for studying chemokine-directed single cell migration, we show that filopodia distribution and their dynamics are dictated by the gradient of the chemokine Cxcl12a. By specifically interfering with filopodia formation, we demonstrate for the first time that these protrusions play an important role in cell polarization by Cxcl12a, as manifested by elevation of intracellular pH and Rac1 activity at the cell front. The establishment of this polarity is at the basis of effective cell migration towards the target. Together, we show that filopodia allow the interpretation of the chemotactic gradient in vivo by directing single-cell polarization in response to the guidance cue.

  2. CD31 is required on CD4+ T cells to promote T cell survival during Salmonella infection.

    PubMed

    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

    2011-08-15

    Hematopoietic cells constitutively express CD31/PECAM1, a signaling 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 on 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 postinfection compared with wild-type 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, because 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.

  3. 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

  4. GATA-4 is required for sex steroidogenic cell development in the fetal mouse

    PubMed Central

    Bielinska, Malgorzata; Seehra, Amrita; Toppari, Jorma; Heikinheimo, Markku; Wilson, David B.

    2007-01-01

    The transcription factor GATA-4 is expressed in Sertoli cells, steroidogenic Leydig cells, and other testicular somatic cells. Previous studies have established that interaction between GATA-4 and its cofactor FOG-2 is necessary for proper Sry expression and all subsequent steps in testicular organogenesis, including testis cord formation and differentiation of both Sertoli and fetal Leydig cells. Since fetal Leydig cell differentiation depends on Sertoli cell-derived factors, it has remained unclear whether GATA-4 has cell autonomous role in Leydig cell development. We used two experimental systems to explore the role of GATA-4 in the ontogeny of testicular steroidogenic cells. First, chimeric mice were generated by injection of Gata4−/− ES cells into Rosa26 blastocysts. Analysis of the resultant chimeras showed that in developing testis Gata4−/− cells can contribute to fetal germ cells and interstitial fibroblasts but not fetal Leydig cells. Second, wild-type or Gata4−/− ES cells were injected into the flanks of intact or gonadectomized nude mice and the resultant teratomas examined for expression of steroidogenic markers. Wild-type but not Gata4−/− ES cells were capable of differentiating into gonadal-type steroidogenic lineages in teratomas grown in gonadectomized mice. In chimeric teratomas derived from mixtures of GFP-tagged Gata4+/+ ES cells and unlabeled Gata4−/− ES cells, sex steroidogenic cell differentiation was restricted to GFP-expressing cells. Collectively these data suggest that GATA-4 plays an integral role in the development of testicular steroidogenic cells. PMID:17096405

  5. Yeast acetic acid-induced programmed cell death can occur without cytochrome c release which requires metacaspase YCA1.

    PubMed

    Guaragnella, Nicoletta; Bobba, Antonella; Passarella, Salvatore; Marra, Ersilia; Giannattasio, Sergio

    2010-01-04

    To investigate the role of cytochrome c (cyt c) release in yeast acetic acid-induced programmed cell death (AA-PCD), wild type (wt) and cells lacking metacaspase (Deltayca1), cytochrome c (Deltacyc1,7) and both (Deltacyc1,7Deltayca1) were compared for AA-PCD occurrence, hydrogen peroxide (H(2)O(2)) production and caspase activity. AA-PCD occurs in Deltacyc1,7 and Deltacyc1,7Deltayca1 cells slower than in wt, but similar to that in Deltayca1 cells, in which no cytochrome c release occurs. Both H(2)O(2) production and caspase activation occur in these cells with early and extra-activation in Deltacyc1,7 cells. We conclude that alternative death pathways can be activated in yeast AA-PCD, one dependent on cyt c release, which requires YCA1, and the other(s) independent on it.

  6. The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death.

    PubMed

    Sakagami, H; Kuribayashi, N; Iida, M; Hagiwara, T; Takahashi, H; Yoshida, H; Shiota, F; Ohata, H; Momose, K; Takeda, M

    1996-01-01

    The requirement for and mobilization of Ca2+ ions during induction of cell death by sodium ascorbate were compared with those during induction of cell death by hydrogen peroxide. When HL-60 cells were incubated with sodium ascorbate, a rapid increase in the intracellular concentration of Ca2+ ions and subsequent apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and cleavage of internucleosomal DNA to yield fragments that were multiples of 180-200 base pairs, were induced. However, these effects of sodium ascorbate were significantly reduced in Ca2+-depleted medium. By contrast, hydrogen peroxide induced similar apoptosis associated phenomena in the presence and in the absence of extracellular Ca2+ ions. The intracellular concentration of the reduced form of glutathione was not significantly affected and glutathione disulfide was undetectable during the early stages of apoptosis. These data suggest that sodium ascorbate and hydrogen peroxide initiate cell death by different mechanisms.

  7. ELT-1, a GATA-like transcription factor, is required for epidermal cell fates in Caenorhabditis elegans embryos.

    PubMed

    Page, B D; Zhang, W; Steward, K; Blumenthal, T; Priess, J R

    1997-07-01

    Epidermal cells are generated during Caenorhabditis elegans embryogenesis by several distinct lineage patterns. These patterns are controlled by maternal genes that determine the identities of early embryonic blastomeres. We show that the embryonically expressed gene elt-1, which was shown previously to encode a GATA-like transcription factor, is required for the production of epidermal cells by each of these lineages. Depending on their lineage history, cells that become epidermal in wild-type embryos become either neurons or muscle cells in elt-1 mutant embryos. The ELT-1 protein is expressed in epidermal cells and in their precursors. We propose that elt-1 functions at an early step in the specification of epidermal cell fates.

  8. Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow

    PubMed Central

    Yumoto, Kenji; Eber, Matthew R.; Wang, Jingcheng; Cackowski, Frank C.; Decker, Ann M.; Lee, Eunsohl; Nobre, Ana Rita; Aguirre-Ghiso, Julio A.; Jung, Younghun; Taichman, Russell S.

    2016-01-01

    Disseminated prostate cancer (PCa) cells in the marrow survive for years without evidence of proliferation, while maintaining the capacity to develop into metastatic lesions. These dormant disseminated tumor cells (DTCs) may reside in close proximity to osteoblasts, while expressing high levels of Axl, one of the tyrosine kinase receptors for growth arrest specific 6 (Gas6). Yet how Axl regulates DTC proliferation in marrow remains undefined. Here, we explored the impact of the loss of Axl in PCa cells (PC3 and DU145) on the induction of their dormancy when they are co-cultured with a pre-osteoblastic cell line, MC3T3-E1. MC3T3-E1 cells dramatically decrease the proliferation of PCa cells, however this suppressive effect of osteoblasts is significantly reduced by the reduction of Axl expression in PCa cells. Interestingly, expression of both TGF-β and its receptors were regulated by Axl expression in PCa cells, while specific blockade of TGF-β signaling limited the ability of the osteoblasts to induce dormancy of PCa cells. Finally, we found that both Gas6 and Axl are required for TGF-β2-mediated cell growth suppression. Taken together, these data suggest that a loop between the Gas6/Axl axis and TGF-β2 signaling plays a significant role in the induction of PCa cell dormancy. PMID:27819283

  9. 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

  10. Antioxidants Complement the Requirement for Protein Chaperone Function to Maintain β-Cell Function and Glucose Homeostasis.

    PubMed

    Han, Jaeseok; Song, Benbo; Kim, Jiun; Kodali, Vamsi K; Pottekat, Anita; Wang, Miao; Hassler, Justin; Wang, Shiyu; Pennathur, Subramaniam; Back, Sung Hoon; Katze, Michael G; Kaufman, Randal J

    2015-08-01

    Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58(IPK)/DnajC3, an ER luminal co-chaperone. P58(IPK-/-) mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58(IPK-/-) mice, suggesting P58(IPK) deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58(IPK-/-) mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes.

  11. Generation of a conditionally immortalized myeloid progenitor cell line requiring the presence of both interleukin-3 and stem cell factor to survive and proliferate.

    PubMed

    Lee, Candy; Evans, Caroline A; Spooncer, Elaine; Pierce, Andrew; Mottram, Rachel; Whetton, Anthony D

    2003-09-01

    The H-2Kappab temperature-sensitive (ts) A58 transgenic (Immorto) mouse has been used previously to generate conditionally immortalized cells from a number of tissues. The present study aimed to investigate characteristics of primitive myeloid precursor cells derived from H-2Kappab-tsA58 bone marrow. Cell populations were enriched for granulocyte/macrophage progenitors by centrifugal elutriation, and were cultured in the presence and absence of cytokines at the permissive and restrictive temperatures for the A58 oncogene. Cells derived from H-2Kappab-tsA58 mice required both A58 activation and the growth factors, stem cell factor (SCF) and interleukin-3 (IL-3), for long-term cell survival and growth; cells were maintained for > 300 d in culture under these conditions. IL-3- and SCF-dependent clonal cell lines were derived with a phenotype (lin-, Sca-1+, CD34+, ER-MP 58+, ER-MP 12+, ER-MP 20-) characteristic of primitive myeloid progenitors. These cells differentiated on addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) and acquired mature cell morphology with some upregulation of differentiation markers. In conclusion, the A58 oncogene can immortalize haemopoietic progenitor cells. These cells require two cytokines for growth, IL-3 and SCF; as such, they constitute a useful resource for the study of synergistic interactions between growth factors. The ability to develop monocytic cell characteristics also permits the investigation of cytokine-mediated early haemopoietic progenitor cell development.

  12. Characterization of asgC a Gene Required for Cell-Cell Signaling Early Development of Myxococcus Xanthus

    DTIC Science & Technology

    2007-11-02

    Plamann, Lynda and Heidi B. Kaplan. 1998. Cell-density sensing during early development in Myxococcus xanthus.. In G. Dunney and S. Winans (ed...and Heidi B. Kaplan. 1998. Cell-density sensing during early development in Myxococcus xanthus.. In G. Dunney and S. Winans (ed.), Cell-cell...conjugative transfer of plasmid RP4. J Bacteriol 176: 4285-4295. Bibb, M.J., Findlay , P.R., and Johnson, M.W. (1984) The relationship between base

  13. Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation.

    PubMed

    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; Dzierzak, Elaine

    2015-01-12

    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.

  14. 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

  15. Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium

    PubMed Central

    Gerbe, François; van Es, Johan H.; Makrini, Leila; Brulin, Bénédicte; Mellitzer, Georg; Robine, Sylvie; Romagnolo, Béatrice; Shroyer, Noah F.; Bourgaux, Jean-François; Pignodel, Christine; Clevers, Hans

    2011-01-01

    The unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cell types of the intestinal epithelium and describe the first marker signature allowing their unambiguous identification. We demonstrate that although mature tuft cells express DCLK1, a putative marker of quiescent stem cells, they are post-mitotic, short lived, derive from Lgr5-expressing epithelial stem cells, and are found in mouse and human tumors. We show that whereas the ATOH1/MATH1 transcription factor is essential for their differentiation, Neurog3, SOX9, GFI1, and SPDEF are dispensable, which distinguishes these cells from enteroendocrine, Paneth, and goblet cells, and raises from three to four the number of secretory cell types in the intestinal epithelium. Moreover, we show that tuft cells are the main source of endogenous intestinal opioids and are the only epithelial cells that express cyclooxygenase enzymes, suggesting important roles for these cells in the intestinal epithelium physiopathology. PMID:21383077

  16. Renin expression in developing zebrafish is associated with angiogenesis and requires the Notch pathway and endothelium

    PubMed Central

    Mullins, Linda J.; Verdon, Rachel F.; MacRae, Calum A.; Mullins, John J.

    2015-01-01

    Although renin is a critical regulatory enzyme of the cardiovascular system, its roles in organogenesis and the establishment of cardiovascular homeostasis remain unclear. Mammalian renin-expressing cells are widespread in embryonic kidneys but are highly restricted, specialized endocrine cells in adults. With a functional pronephros, embryonic zebrafish are ideal for delineating the developmental functions of renin-expressing cells and the mechanisms governing renin transcription. Larval zebrafish renin expression originates in the mural cells of the juxtaglomerular anterior mesenteric artery and subsequently at extrarenal sites. The role of renin was determined by assessing responses to renin-angiotensin system blockade, salinity variation, and renal perfusion ablation. Renin expression did not respond to renal flow ablation but was modulated by inhibition of angiotensin-converting enzyme and altered salinity. Our data in larval fish are consistent with conservation of renin's physiological functions. Using transgenic renin reporter fish, with mindbomb and cloche mutants, we show that Notch signaling and the endothelium are essential for developmental renin expression. After inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts. Arising from separate lineages, but relying on mutual interplay with endothelial cells, renin-expressing cells are among the earliest mural cells observed in larval fish, performing both endocrine and paracrine functions. PMID:26202224

  17. Blood vessel endothelium-directed tumor cell streaming in breast tumors requires the HGF/C-Met signaling pathway.

    PubMed

    Leung, E; Xue, A; Wang, Y; Rougerie, P; Sharma, V P; Eddy, R; Cox, D; Condeelis, J

    2016-11-28

    During metastasis to distant sites, tumor cells migrate to blood vessels. In vivo, breast tumor cells utilize a specialized mode of migration known as streaming, where a linear assembly of tumor cells migrate directionally towards blood vessels on fibronectin-collagen I-containing extracellular matrix (ECM) fibers in response to chemotactic signals. We have successfully reconstructed tumor cell streaming in vitro by co-plating tumors cells, macrophages and endothelial cells on 2.5 μm thick ECM-coated micro-patterned substrates. We found that tumor cells and macrophages, when plated together on the micro-patterned substrates, do not demonstrate sustained directional migration in only one direction (sustained directionality) but show random bi-directional walking. Sustained directionality of tumor cells as seen in vivo was established in vitro when beads coated with human umbilical vein endothelial cells were placed at one end of the micro-patterned 'ECM fibers' within the assay. We demonstrated that these endothelial cells supply the hepatocyte growth factor (HGF) required for the chemotactic gradient responsible for sustained directionality. Using this in vitro reconstituted streaming system, we found that directional streaming is dependent on, and most effectively blocked, by inhibiting the HGF/C-Met signaling pathway between endothelial cells and tumor cells. Key observations made with the in vitro reconstituted system implicating C-Met signaling were confirmed in vivo in mammary tumors using the in vivo invasion assay and intravital multiphoton imaging of tumor cell streaming. These results establish HGF/C-Met as a central organizing signal in blood vessel-directed tumor cell migration in vivo and highlight a promising role for C-Met inhibitors in blocking tumor cell streaming and metastasis in vivo, and for use in human trials.Oncogene advance online publication, 28 November 2016; doi:10.1038/onc.2016.421.

  18. CD4+ T cells are not required for the induction of dengue virus-specific CD8+ T cell or antibody responses but contribute to protection after vaccination.

    PubMed

    Yauch, Lauren E; Prestwood, Tyler R; May, Monica M; Morar, Malika M; Zellweger, Raphaël M; Peters, Bjoern; Sette, Alessandro; Shresta, Sujan

    2010-11-01

    The contribution of T cells to the host response to dengue virus (DENV) infection is not well understood. We previously demonstrated a protective role for CD8(+) T cells during primary DENV infection using a mouse-passaged DENV strain and IFN-α/βR(-/-) C57BL/6 mice, which are susceptible to DENV infection. In this study, we examine the role of CD4(+) T cells during primary DENV infection. Four I-A(b)-restricted epitopes derived from three of the nonstructural DENV proteins were identified. CD4(+) T cells expanded and were activated after DENV infection, with peak activation occurring on day 7. The DENV-specific CD4(+) T cells expressed intracellular IFN-γ, TNF, IL-2, and CD40L, and killed peptide-pulsed target cells in vivo. Surprisingly, depletion of CD4(+) T cells before DENV infection had no effect on viral loads. Consistent with this observation, CD4(+) T cell depletion did not affect the DENV-specific IgG or IgM Ab titers or their neutralizing activity, or the DENV-specific CD8(+) T cell response. However, immunization with the CD4(+) T cell epitopes before infection resulted in significantly lower viral loads. Thus, we conclude that whereas CD4(+) T cells are not required for controlling primary DENV infection, their induction by immunization can contribute to viral clearance. These findings suggest inducing anti-DENV CD4(+) T cell responses by vaccination may be beneficial.

  19. 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.

  20. A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

    PubMed Central

    Toret, Christopher P.; D’Ambrosio, Michael V.; Vale, Ronald D.; Simon, Michael A.

    2014-01-01

    Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling. PMID:24446484

  1. 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.

  2. Role of antigen-presenting cells in activation of human T cells by the streptococcal M protein superantigen: requirement for secreted and membrane-associated costimulatory factors.

    PubMed Central

    Majumdar, G; Ohnishi, H; Tomai, M A; Geller, A M; Wang, B; Dockter, M E; Kotb, M

    1993-01-01

    The requirements for T-cell activation by the streptococcal superantigen (SAg), pepsin-extracted M protein from type 5 streptococci (pep M5), were studied by monitoring Ca2+ influx and cell proliferation. Cells from a pep M5-specific T-cell line showed no change in intracellular Ca2+ levels in response to pep M5 when added alone or with freshly isolated autologous antigen-presenting cells (APC). However, after being incubated with pep M5 overnight, the APC secreted soluble factors that together with pep M5 induced a marked increase in intracellular Ca2+ levels in pep M5-specific T cells or freshly isolated, purified T cells. Removal of the SAg from the overnight APC-derived supernatants resulted in loss of the Ca(2+)-mobilizing activity, which was restored within seconds of addition of SAg, suggesting that both the SAg and the soluble factors synergize to induce the Ca2+ influx. Induction of cell proliferation required additional signals inasmuch as the activated APC-derived supernatant failed to synergize with pep M5 to induce the proliferation of purified T cells and required the presence of phorbol myristate acetate for this activity. Metabolically inactive, fixed APC were impaired in their ability to present pep M5 to T cells. Presentation of pep M5 by fixed APC was, however, restored when the APC-derived soluble costimulatory factors were added to the culture. Our data suggest that pep M5-induced activation of T cells is dependent on APC-derived soluble factors and an APC membrane-associated costimulatory molecule(s). These interactions may be important in regulating the in vivo responses to M proteins, could contribute to the severity or progression of infections with Streptococcus pyogenes, and may influence the susceptibility of individuals to its associated nonsuppurative autoimmune sequelae. PMID:8423107

  3. Primary mesenchyme cell migration requires a chondroitin sulfate/dermatan sulfate proteoglycan.

    PubMed

    Lane, M C; Solursh, M

    1991-02-01

    Primary mesenchyme cell migration in the sea urchin embryo is inhibited by sulfate deprivation and exposure to exogenous beta-D-xylosides, two treatments known to disrupt proteoglycan synthesis. We show that in the developing sea urchin, exogenous xyloside affects the synthesis by the primary mesenchyme cells of a very large, cell surface chondroitin sulfate/dermatan sulfate proteoglycan. This proteoglycan is present in a partially purified fraction that restores migratory ability to defective cells in vitro. The integrity of this chondroitin sulfate/dermatan sulfate proteoglycan appears essential for primary mesenchyme cell migration since treatment of actively migrating cells with chondroitinase ABC reversibly inhibited their migration in vitro.

  4. 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

  5. Immune stimulatory receptor CD40 is required for T-cell suppression and T regulatory cell activation mediated by myeloid-derived suppressor cells in cancer.

    PubMed

    Pan, Ping-Ying; Ma, Ge; Weber, Kaare J; Ozao-Choy, Junko; Wang, George; Yin, Bingjiao; Divino, Celia M; Chen, Shu-Hsia

    2010-01-01

    Immune tolerance to tumors is often associated with accumulation of myeloid-derived suppressor cells (MDSC) and an increase in the number of T-regulatory cells (Treg). In tumor-bearing mice, MDSCs can themselves facilitate the generation of tumor-specific Tregs. In this study, we demonstrate that expression of the immune stimulatory receptor CD40 on MDSCs is required to induce T-cell tolerance and Treg accumulation. In an immune reconstitution model, adoptive transfer of Gr-1+CD115+ monocytic MDSCs derived from CD40-deficient mice failed to recapitulate the ability of wild-type MDSCs to induce tolerance and Treg development in vivo. Agonistic anti-CD40 antibodies phenocopied the effect of CD40 deficiency and also improved the therapeutic efficacy of IL-12 and 4-1BB immunotherapy in the treatment of advanced tumors. Our findings suggest that CD40 is essential not only for MDSC-mediated immune suppression but also for tumor-specific Treg expansion. Blockade of CD40-CD40L interaction between MDSC and Treg may provide a new strategy to ablate tumoral immune suppression and thereby heighten responses to immunotherapy.

  6. Roles of VRK1 as a new player in the control of biological processes required for cell division.

    PubMed

    Valbuena, Alberto; Sanz-García, Marta; López-Sánchez, Inmaculada; Vega, Francisco M; Lazo, Pedro A

    2011-08-01

    Cell division, in addition to an accurate transmission of genetic information to daughter cells, also requires the temporal and spatial coordination of several biological processes without which cell division would not be feasible. These processes include the temporal coordination of DNA replication and chromosome segregation, regulation of nuclear envelope disassembly and assembly, chromatin condensation and Golgi fragmentation for its redistribution into daughter cells, among others. However, little is known regarding regulatory proteins and signalling pathways that might participate in the coordination of all these different biological functions. Such regulatory players should directly have a role in the processes leading to cell division. VRK1 (Vaccinia-related kinase 1) is an early response gene required for cyclin D1 expression, regulates p53 by a specific Thr18 phosphorylation, controls chromatin condensation by histone phosphorylation, nuclear envelope assembly by phosphorylation of BANF1, and participates in signalling required for Golgi fragmentation late in the G2 phase. We propose that VRK1, a Ser-Thr kinase, might be a candidate to play an important coordinator role in these cell division processes as part of a novel signalling pathway.

  7. 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.

  8. Somatic cell lineage is required for differentiation and not maintenance of germline stem cells in Drosophila testes.

    PubMed

    Lim, Jaclyn G Y; Fuller, Margaret T

    2012-11-06

    Adult stem cells are believed to be maintained by a specialized microenvironment, the niche, which provides short-range signals that either instruct stem cells to self-renew or inhibit execution of preprogrammed differentiation pathways. In Drosophila testes, somatic cyst stem cells (CySCs) and the apical hub form the niche for neighboring germline stem cells (GSCs), with CySCs as the proposed source of instructive self-renewal signals [Leatherman JL, Dinardo S (2010) Nat Cell Biol 12(8):806-811]. In contrast to this model, we show that early germ cells with GSC characteristics can be maintained over time after ablation of CySCs and their cyst cell progeny. Without CySCs and cyst cells, early germ cells away from the hub failed to initiate differentiation. Our results suggest that CySCs do not have a necessary instructive role in specifying GSC self-renewal and that the differentiated progeny of CySCs provide an environment necessary to trigger GSC differentiation. This work highlights the complex interaction between different stem cell populations in the same niche and how the state of one stem cell population can influence the fate of the other.

  9. Hormone signaling requirements for the conversion of non-mammary mouse cells to mammary cell fate(s) in vivo.

    PubMed

    Boulanger, Corinne A; Rosenfield, Sonia M; George, Andrea L; Smith, Gilbert H

    2015-06-01

    Mammotropic hormones and growth factors play a very important role in mammary growth and differentiation. Here, hormones including Estrogen, Progesterone, Prolactin, their cognate receptors, and the growth factor Amphiregulin, are tested with respect to their roles in signaling non-mammary cells from the mouse to redirect to mammary epithelial cell fate(s). This was done in the context of glandular regeneration in pubertal athymic female mice. Our previous studies demonstrated that mammary stem cell niches are recapitulated during gland regeneration in vivo. During this process, cells of exogenous origin cooperate with mammary epithelial cells to form mammary stem cell niches and thus respond to normal developmental signals. In all cases tested with the possible exception of estrogen receptor alpha (ER-α), hormone signaling is dispensable for non-mammary cells to undertake mammary epithelial cell fate(s), proliferate, and contribute progeny to chimeric mammary outgrowths. Importantly, redirected non-mammary cell progeny, regardless of their source, have the ability to self-renew and contribute offspring to secondary mammary outgrowths derived from transplanted chimeric mammary fragments; thus suggesting that some of these cells are capable of mammary stem cell/progenitor functions.

  10. 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.

  11. Male germline stem cell division and spermatocyte growth require insulin signaling in Drosophila.

    PubMed

    Ueishi, Satoru; Shimizu, Hanako; H Inoue, Yoshihiro

    2009-01-01

    Spermatogenesis in Drosophila commences with cell division of germline stem cells (GSCs) to produce male germline cells at the tip of the testis. However, molecular mechanisms inducing division of male GSCs have not been reported. Insulin-like peptides are known to play an essential role in stimulation of proliferation and growth of somatic cells, and it has recently been reported that such peptides promote cell division in female Drosophila GSCs. However, their effects on male germline cells have not been characterized. We found that inhibition of insulin production and insulin signaling mutations resulted in decreased numbers of germline cells in Drosophila testes. GSC numbers were maintained in young mutant males, with a gradual decrease in abundance of GSCs with age. Furthermore, in mutants, fewer germline cysts originated from GSCs and a lower frequency of GSC division was seen. Insulin signaling was found to promote cell cycle progression of the male GSCs at the G(2)/M phase. The cell volume of spermatocytes increases up to 25 times before initiation of meiosis in Drosophila. We examined whether insulin signaling extrinsically induces the greatest cell growth in Drosophila diploid cells and found that spermatocyte growth was affected in mutants. The results indicate that in addition to its function in somatic cells, insulin signaling plays an essential role in cell proliferation and growth during male Drosophila gametogenesis and that sperm production is regulated by hormonal control via insulin-like peptides.

  12. 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

  13. 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

  14. Runx2 Expression in Smooth Muscle Cells Is Required for Arterial Medial Calcification in Mice

    PubMed Central

    Lin, Mu-En; Chen, Theodore; Leaf, Elizabeth M.; Speer, Mei Y.; Giachelli, Cecilia M.

    2016-01-01

    Arterial medial calcification (AMC) is a hallmark of aging, diabetes, and chronic kidney disease. Smooth muscle cell (SMC) transition to an osteogenic phenotype is a common feature of AMC, and is preceded by expression of runt-related transcription factor 2 (Runx2), a master regulator of bone development. Whether SMC-specific Runx2 expression is required for osteogenic phenotype change and AMC remains unknown. We therefore created an improved targeting construct to generate mice with floxed Runx2 alleles (Runx2f/f) that do not produce truncated Runx2 proteins after Cre recombination, thereby preventing potential off-target effects. SMC-specific deletion using SM22–recombinase transgenic allele mice (Runx2ΔSM) led to viable mice with normal bone and arterial morphology. After vitamin D overload, arterial SMCs in Runx2f/f mice expressed Runx2, underwent osteogenic phenotype change, and developed severe AMC. In contrast, vitamin D–treated Runx2ΔSM mice had no Runx2 in blood vessels, maintained SMC phenotype, and did not develop AMC. Runx2 deletion did not affect serum calcium, phosphate, fibroblast growth factor-23, or alkaline phosphatase levels. In vitro, Runx2f/f SMCs calcified to a much greater extent than those derived from Runx2ΔSM mice. These data indicate a critical role of Runx2 in SMC osteogenic phenotype change and mineral deposition in a mouse model of AMC, suggesting that Runx2 and downstream osteogenic pathways in SMCs may be useful therapeutic targets for treating or preventing AMC in high-risk patients. PMID:25987250

  15. BMP2 signals loss of epithelial character in epicardial cells but requires the Type III TGFβ receptor to promote invasion.

    PubMed

    Hill, Cynthia R; Sanchez, Nora S; Love, Joseph D; Arrieta, Julian A; Hong, Charles C; Brown, Christopher B; Austin, Anita F; Barnett, Joey V

    2012-05-01

    Coronary vessel development depends on a subpopulation of epicardial cells that undergo epithelial to mesenchymal transformation (EMT) and invade the subepicardial space and myocardium. These cells form the smooth muscle of the vessels and fibroblasts, but the mechanisms that regulate these processes are poorly understood. Mice lacking the Type III Transforming Growth Factor β Receptor (TGFβR3) die by E14.5 due to failed coronary vessel development accompanied by reduced epicardial cell invasion. BMP2 signals via TGFβR3 emphasizing the importance of determining the relative contributions of the canonical BMP signaling pathway and TGFβR3-dependent signaling to BMP2 responsiveness. Here we examined the role of TGFβR3 in BMP2 signaling in epicardial cells. Whereas TGFβ induced loss of epithelial character and smooth muscle differentiation, BMP2 induced an ALK3-dependent loss of epithelial character and modestly inhibited TGFβ-stimulated differentiation. Tgfbr3(-/-) cells respond to BMP2 indicating that TGFβR3 is not required. However, Tgfbr3(-/-) cells show decreased invasion in response to BMP2 and overexpression of TGFβR3 in Tgfbr3(-/-) cells rescued invasion. Invasion was dependent on ALK5, ALK2, ALK3, and Smad4. Expression of TGFβR3 lacking the 3 C-terminal amino acids required to interact with the scaffolding protein GIPC (GAIP-interacting protein, C terminus) did not rescue. Knockdown of GIPC in Tgfbr3(+/+) or Tgfbr3(-/-) cells rescued with TGFβR3 decreased BMP2-stimulated invasion confirming a requirement for TGFβR3/GIPC interaction. Our results reveal the relative roles of TGFβR3-dependent and TGFβR3-independent signaling in the actions of BMP2 on epicardial cell behavior and demonstrate the critical role of TGFβR3 in mediating BMP2-stimulated invasion.

  16. Different cryopreservation requirements in foetal versus adult skin cells from an endangered mammal, the Iberian lynx (Lynx pardinus).

    PubMed

    León-Quinto, Trinidad; Simón, Miguel A; Cadenas, Rafael; Martínez, Africa; Serna, Arturo

    2014-04-01

    Cryobanking somatic foetal cells acquire much relevance in endangered species for biodiversity conservation purposes. Such cells could be later used to reintroduce the lost genes into the breeding pool, by inducing pluripotency and/or nuclear transfer if necessary. Since requirements for preserving foetal cells are not always the same as for adult ones, we evaluated the cryosensitivity of foetal skin cells in comparison with adult ones from the critically endangered Iberian lynx. Responses to cryoinjury were analyzed in both thawed cell types by means of cell viability and functionality (by analyzing their membrane integrity, metabolic activity, glycosaminoglycan content and proliferative activity). Freezing media included the permeating cryoprotectant Me2SO, either alone or along with the non-permeating cryoprotectant sucrose at 0.1 or 0.2M. When Me2SO was the only cryoprotectant, survival rate fell in thawed foetal cells to 54±4% (against 89±6% for thawed adult ones) and both proliferative and metabolic activities remained significantly lower than values for thawed adult cells. However, the combination of sucrose (both 0.1 as 0.2) and Me2SO in foetal cells significantly increased their survival rates (to 71±4% and 73±5%, respectively), proliferative activities (partially at day 7 and completely at day 14 after thawing) and metabolic activities. Our findings clearly show a difference between foetal and adult cells concerning their cryopreservation sensitivity and requirements, as well as their recovery time after thawing. These results are of relevance for the cryopreservation of foetal and adult cells from the Iberian lynx and could be also useful for other mammals.

  17. Murine complement receptor 1 is required for germinal center B cell maintenance but not initiation.

    PubMed

    Donius, Luke R; Weis, Janis J; Weis, John H

    2014-06-01

    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.

  18. CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell maintenance.

    PubMed

    Greenbaum, Adam; Hsu, Yen-Michael S; Day, Ryan B; Schuettpelz, Laura G; Christopher, Matthew J; Borgerding, Joshua N; Nagasawa, Takashi; Link, Daniel C

    2013-03-14

    Haematopoietic stem cells (HSCs) primarily reside in the bone marrow where signals generated by stromal cells regulate their self-renewal, proliferation and trafficking. Endosteal osteoblasts and perivascular stromal cells including endothelial cells, CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin-green fluorescent protein (GFP)-positive mesenchymal progenitors have all been implicated in HSC maintenance. However, it is unclear whether specific haematopoietic progenitor cell (HPC) subsets reside in distinct niches defined by the surrounding stromal cells and the regulatory molecules they produce. CXCL12 (chemokine (C-X-C motif) ligand 12) regulates both HSCs and lymphoid progenitors and is expressed by all of these stromal cell populations. Here we selectively deleted Cxcl12 from candidate niche stromal cell populations and characterized the effect on HPCs. Deletion of Cxcl12 from mineralizing osteoblasts has no effect on HSCs or lymphoid progenitors. Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, results in constitutive HPC mobilization and a loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from endothelial cells results in a modest loss of long-term repopulating activity. Strikingly, deletion of Cxcl12 from nestin-negative mesenchymal progenitors using Prx1-cre (Prx1 also known as Prrx1) is associated with a marked loss of HSCs, long-term repopulating activity, HSC quiescence and common lymphoid progenitors. These data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow, and that expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

  19. 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.

  20. Protein kinase C-associated kinase is required for NF-kappaB signaling and survival in diffuse large B-cell lymphoma cells.

    PubMed

    Kim, Sang-Woo; Oleksyn, David W; Rossi, Randall M; Jordan, Craig T; Sanz, Ignacio; Chen, Luojing; Zhao, Jiyong

    2008-02-01

    Diffuse large B-cell lymphoma (DLBCL) is an aggressive and the most common type of non-Hodgkin lymphoma. Despite recent advances in treatment, less than 50% of the patients are cured with current multiagent chemotherapy. Abnormal NF-kappaB activity not only contributes to tumor development but also renders cancer cells resistant to chemotherapeutic agents. Identifying and targeting signaling molecules that control NF-kappaB activation in cancer cells may thus yield more effective therapy for DLBCL. Here, we show that while overexpression of protein kinase C-associated kinase (PKK) activates NF-kappaB signaling in DLBCL cells, suppression of PKK expression inhibits NF-kappaB activity in these cells. In addition, we show that NF-kappaB activation induced by B cell-activating factor of tumor necrosis factor family (BAFF) in DLBCL cells requires PKK. Importantly, we show that knockdown of PKK impairs the survival of DLBCL cells in vitro and inhibits tumor growth of xenografted DLBCL cells in mice. Suppression of PKK expression also sensitizes DLBCL cells to treatment with chemotherapeutic agents. Together, these results indicate that PKK plays a pivotal role in the survival of human DLBCL cells and represents a potential target for DLBCL therapy.

  1. Phoenix is required for mechanosensory hair cell regeneration in the zebrafish lateral line.

    PubMed

    Behra, Martine; Bradsher, John; Sougrat, Rachid; Gallardo, Viviana; Allende, Miguel L; Burgess, Shawn M

    2009-04-01

    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