scholarly journals Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

2020 ◽  
Vol 21 (20) ◽  
pp. 7747
Author(s):  
Ananth Kumar Kammala ◽  
Samantha Sheller-Miller ◽  
Enkhtuya Radnaa ◽  
Talar Kechichian ◽  
Hariharan Subramanian ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Cell Types ◽  
Fetal Membrane ◽  
Regulatory Factor ◽  
Rna Targeting ◽  
Sodium Hydrogen Exchanger ◽  
Functional Relevance ◽  
Interfering Rna ◽  
Different Cell Types ◽  
Phosphate Buffered Saline

The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.

scholarly journals Efficiency of RNA Interference in the Mouse Hematopoietic System Varies between Cell Types and Developmental Stages

2005 ◽  
Vol 25 (10) ◽  
pp. 3896-3905 ◽  
Author(s):  
Philipp Oberdoerffer ◽  
Chryssa Kanellopoulou ◽  
Vigo Heissmeyer ◽  
Corinna Paeper ◽  
Christine Borowski ◽  
...  
Keyword(s):  
Rna Interference ◽  
Gene Targeting ◽  
Developmental Stages ◽  
Cell Types ◽  
Gene Inactivation ◽  
Loss Of Function ◽  
Homologous Genes ◽  
Family Gene ◽  
Interfering Rna ◽  
Different Cell Types

ABSTRACT RNA interference (RNAi) is a naturally occurring posttranscriptional gene-silencing mechanism that has been adapted as a genetic tool for loss-of-function studies of a variety of organisms. It is more widely applicable than classical gene targeting and allows for the simultaneous inactivation of several homologous genes with a single transgene. Recently, RNAi has been used for conditional and conventional gene inactivation in mice. Unlike gene targeting, RNAi is a dynamic process, and its efficiency may vary both between cell types and throughout development. Here we demonstrate that RNAi can be used to target three separately encoded isoforms of the bcl-2 family gene bfl-1/A1 in a conditional manner in mice. The extent of gene inactivation varies between different cell types and is least efficient in mature lymphocytes. Our data suggest that RNAi is affected by factors beyond small interfering RNA-mRNA stoichiometry.

Turnover of the carboxy-terminal tyrosine of alpha-tubulin and means of reaching elevated levels of detyrosination in living cells

1987 ◽  
Vol 88 (2) ◽  
pp. 185-203
Author(s):  
J. Wehland ◽  
K. Weber
Keyword(s):  
Primary Cultures ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Morphological Transformation ◽  
Alpha Tubulin ◽  
Tubulin Antibodies ◽  
Cell Processes ◽  
Carboxy Terminal ◽  
Almost All ◽  
Different Cell Types

Monoclonal antibodies specific for either the tyrosinated (Tyr) or the detyrosinated (Glu) form of alpha-tubulin were elicited with synthetic peptides spanning the carboxy-terminal sequences of the two forms. While almost all microtubules (MTs) are usually of the Tyr-tubulin type (Tyr-rich MTs) some MTs containing noticeable amounts of Glu-tubulin (Glu-rich MTs) were found in many but not all cell lines studied. Glu-rich MTs seemed absent from proliferating CHO and N115 neuroblastoma cells. When differentiation of these cells was initiated by the addition of forskolin for CHO, or by serum deprivation for N115, elevated levels of microtubular Glu-tubulin were observed. In differentiated N115 cells Glu-tubulin was restricted to MT of elongated cell processes and was not found in growth cones and many MT of the cell body. Elevated levels of Glu-tubulin were also characteristic of other differentiated cell types, including neurones and myotubes but were not found in glial cells, astrocytes and fibroblasts in the same primary cultures. Additional experiments suggested that the restricted distribution of Glu-tubulin is the result of MT subsets with different stabilities. Results with mitotic drugs indicated that detyrosination occurs on MTs rather than on soluble tubulin and that stabilization of MTs usually favours the detyrosination process. Evidence for a functional alpha-tubulin cycle involving an inherent carboxypeptidase and a recharging ligase was apparent in 3T3 cells from the preponderance of Glu-rich MTs induced by taxol treatment or the micro-injection of certain antibodies either protecting the detyrosinated form (Glu-tubulin antibodies) or inhibiting retyrosination (ligase antibodies). As the same treatment of CHO cells resulted in comparable arrays of Glu-rich MTs only when forskolin was also present, different cell types may differ in the level of active carboxypeptidase. The results are discussed in terms of possible functions of the tyrosination/detyrosination cycle of alpha-tubulin. While most results can be explained on the basis of ‘older’ and, consequently, more detyrosinated MTs, others raise the possibility that cyclic-AMP-dependent events and certain environmental influences known to induce either a morphological transformation or a differentiation event may influence the carboxypeptidase inherent in the alpha-tubulin cycle.

scholarly journals Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 743-751 ◽  
Author(s):  
S C Mizrak ◽  
F Renault-Mihara ◽  
M Párraga ◽  
J Bogerd ◽  
H J G van de Kant ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Meiotic Prophase ◽  
Primary Cultures ◽  
Cell Types ◽  
Mouse Testis ◽  
C Terminus ◽  
Specific Step ◽  
Different Cell Types

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA byin situhybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) andPEA-15−/−mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.

scholarly journals Lineage-Specific Modulation of Interleukin 4 Signaling by Interferon Regulatory Factor 4

1999 ◽  
Vol 190 (12) ◽  
pp. 1837-1848 ◽  
Author(s):  
Sanjay Gupta ◽  
Man Jiang ◽  
Alissa Anthony ◽  
Alessandra B. Pernis
Keyword(s):  
Biological Activities ◽  
Cell Types ◽  
Interferon Regulatory Factor ◽  
Lymphoid Cells ◽  
Interleukin 4 ◽  
Regulatory Factor ◽  
Immunoregulatory Cytokine ◽  
Interferon Regulatory Factor 4 ◽  
Inducible Genes ◽  
Different Cell Types

Interleukin (IL)-4 is an immunoregulatory cytokine that exerts distinct biological activities on different cell types. Our studies indicate that interferon regulatory factor (IRF)-4 is both a target and a modulator of the IL-4 signaling cascade. IRF-4 expression is strongly upregulated upon costimulation of B cells with CD40 and IL-4. Furthermore, we find that IRF-4 can interact with signal transducer and activator of transcription (Stat)6 and drive the expression of IL-4–inducible genes. The transactivating ability of IRF-4 is blocked by the repressor factor BCL-6. Since expression of IRF-4 is mostly confined to lymphoid cells, these data provide a potential mechanism by which IL-4–inducible genes can be regulated in a lineage-specific manner.

scholarly journals Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation

2017 ◽  
Vol 313 (1) ◽  
pp. C11-C26 ◽  
Author(s):  
Aline M. S. Yamashita ◽  
Maryana T. C. Ancillotti ◽  
Luciana P. Rangel ◽  
Marcio Fontenele ◽  
Cicero Figueiredo-Freitas ◽  
...  
Keyword(s):  
Guanylyl Cyclase ◽  
Primary Cultures ◽  
Soluble Guanylyl Cyclase ◽  
Cell Types ◽  
Myoblast Fusion ◽  
No Synthase ◽  
Myoblast Proliferation ◽  
Cgmp Signaling ◽  
Different Cell Types

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor – GSNORi – or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

scholarly journals Joint reconstruction of cis-regulatory interaction networks across multiple tissues using single-cell chromatin accessibility data

2019 ◽  
Author(s):  
Kangning Dong ◽  
Shihua Zhang
Keyword(s):  
Single Cell ◽  
Regulatory Network ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Computational Method ◽  
Regulatory Mechanisms ◽  
Regulatory Interactions ◽  
Mouse Tissues ◽  
Functional Relevance ◽  
Different Cell Types

ABSTRACTThe rapid accumulation of single-cell chromatin accessibility data offers a unique opportunity to investigate common and specific regulatory mechanisms across different cell types. However, existing methods for cis-regulatory network reconstruction using single-cell chromatin accessibility data were only designed for cells belonging to one cell type, and resulting networks may be incomparable directly due to diverse cell numbers of different cell types. Here, we adopt a computational method to jointly reconstruct cis-regulatory interaction maps (JRIM) of multiple cell populations based on patterns of co-accessibility in single-cell data. We applied JRIM to explore common and specific regulatory interactions across multiple tissues from single-cell ATAC-seq dataset containing ~80,000 cells across 13 mouse tissues. Reconstructed common interactions among 13 tissues indeed relate to basic biological functions, and individual cis-regulatory network shows strong tissue specificity and functional relevance. More importantly, tissue-specific regulatory interactions are mediated by coordination of histone modifications and tissue related TFs, and many of them reveal novel regulatory mechanisms (e.g., a kidney-specific promoter-enhancer loop of clock-controlled gene Gys2).

scholarly journals Mutagenesis of human cytomegalovirus glycoprotein L disproportionately disrupts gH/gL/gO over gH/gL/pUL128-131.

2021 ◽  
Author(s):  
Eric P. Schultz ◽  
Qin Yu ◽  
Cora Stegmann ◽  
Le Zhang Day ◽  
Jean-Marc Lanchy ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Transient Expression ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Cell Types ◽  
Regulatory Factor ◽  
Bac Clones ◽  
Dependent Cell ◽  
Different Cell Types ◽  
Cell Spread

Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but the how the gH/gL portion of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB dependent cell-cell fusion, but were still able to form gH/gL/pUL128-131 and induce receptor-interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. Effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRa, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a “hyperactive” gH/gL/gO. Recently published crystallography and cryo-EM studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB.

scholarly journals Transcriptomic profile of cystic fibrosis airway epithelial cells undergoing repair

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Alice Zoso ◽  
Aderonke Sofoluwe ◽  
Marc Bacchetta ◽  
Marc Chanson
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Airway Epithelium ◽  
Primary Cultures ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Molecular Signature ◽  
Airway Epithelial ◽  
Transcriptomic Profile ◽  
Different Cell Types

Abstract Pathological remodeling of the airway epithelium is commonly observed in Cystic Fibrosis (CF). The different cell types that constitute the airway epithelium are regenerated upon injury to restore integrity and maintenance of the epithelium barrier function. The molecular signature of tissue repair in CF airway epithelial cells has, however, not well been investigated in primary cultures. We therefore collected RNA-seq data from well-differentiated primary cultures of bronchial human airway epithelial cells (HAECs) of CF (F508del/F508del) and non-CF (NCF) origins before and after mechanical wounding, exposed or not to flagellin. We identified the expression changes with time of repair of genes, the products of which are markers of the different cell types that constitute the airway epithelium (basal, suprabasal, intermediate, secretory, goblet and ciliated cells as well as ionocytes). Researchers in the CF field may benefit from this transcriptomic profile, which covers the initial steps of wound repair and revealed differences in this process between CF and NCF cultures.

Morphology and locomotion of individual epithelial cells in culture

1985 ◽  
Vol 78 (1) ◽  
pp. 105-115 ◽  
Author(s):  
R.M. Brown ◽  
C.A. Middleton
Keyword(s):  
Epithelial Cells ◽  
Primary Cultures ◽  
Time Lapse ◽  
Cell Types ◽  
Epidermal Cells ◽  
Cell Type ◽  
Isolated Cells ◽  
Cell Behaviour ◽  
Significant Movement ◽  
Different Cell Types

The behaviour in culture of epithelial cells derived from chick embryo pigmented retina epithelium (PRE), corneal epithelium (CE) and epidermis has been studied using time-lapse cinemicrography. The analysis concentrated on the morphology and movement of individual isolated cells, lacking contacts with other cells, during a 24h period starting 1–3 h after the cells were plated out in primary cultures. Isolated cells from all three sources could change morphology and reversibly exhibited either a poorly spread or a well-spread morphology. While poorly spread, the different cell types all appeared similar and all blebbed vigorously. In contrast, while well spread, the cells did not bleb significantly but there were other differences between them. Well-spread CE cells were always polarized by the presence of a dominant leading lamella but well-spread PRE cells were always unpolarized. Well-spread epidermal cells exhibited both a polarized and an unpolarized morphology. The tendency of individual isolated cells to change morphology varied with cell type. PRE cells were the most stable. Nearly 80% of them retained the same morphology throughout the period of analysis and only 1% of them showed three or more changes in morphology during this period. In contrast, 22% of CE cells and 37% of epidermal cells showed three or more changes in morphology during the period of observation. Isolated cells of all three types spent a greater proportion of the time exhibiting a poorly spread morphology than they spent exhibiting any alternative well-spread morphology. The analysis revealed a relationship between the morphology of isolated cells and the speed of their locomotion. Only cells with a well-spread polarized morphology showed significant movement. CE and epidermal cells with this morphology moved three to four times faster than their counter-parts with a poorly spread morphology or, in the case of epidermal cells, with a well-spread but unpolarized morphology. Actively moving PRE cells were not seen and this correlates with the absence of cells with a well-spread polarized morphology from cultures of this type. These findings are discussed in the light of similar investigations of cell behaviour in other epithelial cell types and fibroblasts.

scholarly journals Neuron-glia cross talk revealed in reverberating networks by simultaneous extracellular recording of spikes and astrocytes' glutamate transporter and K+ currents

2016 ◽  
Vol 116 (6) ◽  
pp. 2706-2719 ◽  
Author(s):  
Enzo Wanke ◽  
Francesca Gullo ◽  
Elena Dossi ◽  
Gaetano Valenza ◽  
Andrea Becchetti
Keyword(s):  
Cross Talk ◽  
Primary Cultures ◽  
Glutamate Transporter ◽  
Extracellular Recording ◽  
Cell Types ◽  
Spike Rate ◽  
Multielectrode Arrays ◽  
Different Cell Types ◽  
Inwardly Rectifying ◽  
K Currents

Astrocytes uptake synaptically released glutamate with electrogenic transporters (GluT) and buffer the spike-dependent extracellular K+ excess with background K+ channels. We studied neuronal spikes and the slower astrocytic signals on reverberating neocortical cultures and organotypic slices from mouse brains. Spike trains and glial responses were simultaneously captured from individual sites of multielectrode arrays (MEA) by splitting the recorded traces into appropriate filters and reconstructing the original signal by deconvolution. GluT currents were identified by using dl-threo-β-benzyloxyaspartate (TBOA). K+ currents were blocked by 30 μM Ba2+, suggesting a major contribution of inwardly rectifying K+ currents. Both types of current were tightly correlated with the spike rate, and their astrocytic origin was tested in primary cultures by blocking glial proliferation with cytosine β-d-arabinofuranoside (AraC). The spike-related, time-locked inward and outward K+ currents in different regions of the astrocyte syncytium were consistent with the assumptions of the spatial K+ buffering model. In organotypic slices from ventral tegmental area and prefrontal cortex, the GluT current amplitudes exceeded those observed in primary cultures by several orders of magnitude, which allowed to directly measure transporter currents with a single electrode. Simultaneously measuring cell signals displaying widely different amplitudes and kinetics will help clarify the neuron-glia interplay and make it possible to follow the cross talk between different cell types in excitable as well as nonexcitable tissue.

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