Selective reduction of dormant maternal mRNAs in mouse oocytes by RNA interference

Development ◽  
2000 ◽  
Vol 127 (19) ◽  
pp. 4147-4156 ◽  
Author(s):  
P. Svoboda ◽  
P. Stein ◽  
H. Hayashi ◽  
R.M. Schultz
Keyword(s):  
Rna Interference ◽  
Map Kinase ◽  
Selective Reduction ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Double Stranded Rna ◽  
Mouse Oocytes ◽  
Tissue Plasminogen ◽  
Maternal Mrnas ◽  
Specific Mrna

Specific mRNA degradation mediated by double-stranded RNA (dsRNA), which is termed RNA interference (RNAi), is a useful tool with which to study gene function in several systems. We report here that in mouse oocytes, RNAi provides a suitable and robust approach to study the function of dormant maternal mRNAs. Mos (originally known as c-mos) and tissue plasminogen activator (tPA, Plat) mRNAs are dormant maternal mRNAs that are recruited during oocyte maturation; translation of Mos mRNA results in the activation of MAP kinase. dsRNA directed towards Mos or Plat mRNAs in mouse oocytes effectively results in the specific reduction of the targeted mRNA in both a time- and concentration-dependent manner. Moreover, dsRNA is more potent than either sense or antisense RNAs. Targeting the Mos mRNA results in inhibiting the appearance of MAP kinase activity and can result in parthenogenetic activation. Mos dsRNA, therefore, faithfully phenocopies the Mos null mutant. Targeting the Plat mRNA with Plat dsRNA results in inhibiting production of tPA activity. Finally, effective reduction of the Mos and Plat mRNA is observed with stoichiometric amounts of Mos and Plat dsRNA, respectively.

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 309-316 ◽  
Author(s):  
Carsten Krischek ◽  
Burkhard Meinecke
Keyword(s):  
Map Kinase ◽  
Protein Kinases ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Histone H1 ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation

2004 ◽  
Vol 286 (4) ◽  
pp. G645-G652 ◽  
Author(s):  
Keishi Kojima ◽  
Mark W. Musch ◽  
Mark J. Ropeleski ◽  
David L. Boone ◽  
Averil Ma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Map Kinase ◽  
Enteric Bacteria ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Filamentous Actin ◽  
Concentration Dependent Manner ◽  
Epithelial Integrity

Protection of colonic epithelial integrity and function is critical, because compromises in mucosal functions can lead to adverse and potentially life-threatening effects. The gut flora may contribute to this protection, in part, through the sustained induction of cytoprotective heat shock proteins (HSPs) in surface colonocytes. In this study, we investigated whether Escherichia coli LPS mediates bacteria-induced HSP by using cultured young adult mouse colon (YAMC) cells, an in vitro model of the colonic epithelium. E. coli LPS led to an epithelial cell-type specific induction of HSP25 in a time- and concentration-dependent manner, an effect that did not involve changes in HSP72. YAMC cells expressed the toll-like receptors (TLR)2 and TLR4 but not the costimulatory CD14 molecule. Whereas LPS stimulated both the p38 and ERK1/2 but not the stress-activated protein kinase/c-Jun NH2-terminal kinase, signaling pathways in the YAMC cells, all three were stimulated in RAW macrophage cells (in which no LPS-induced HSP25 expression was observed). The p38 inhibitor SB-203580 and the MAP kinase kinase-1 inhibitor PD-98059 inhibited HSP25 induction by LPS. LPS treatment also conferred protection against actin depolymerization induced by the oxidant monochloramine. The HSP25 dependence of the LPS protective effect was outlined in inhibitor studies and through adenovirus-mediated overexpression of HSP25. In conclusion, LPS may be an important mediator of enteric bacteria-induced expression of intestinal epithelial HSP25, an effect that may contribute to filamentous actin stabilization under physiological as well as pathophysiological conditions and thus protection of colonic epithelial integrity.

scholarly journals Signal regulatory protein α1 is involved in the inhibitory effect of glucocorticoid receptor on the proliferation of murine macrophage RAW264.7 cell and mouse peritoneal macrophage

2008 ◽  
Vol 41 (5) ◽  
pp. 393-403 ◽  
Author(s):  
Xiaohui Wang ◽  
Yidong Li ◽  
Xiaoyan Zhu ◽  
Yan Wang ◽  
Fei Diao ◽  
...  
Keyword(s):  
Rna Interference ◽  
Peritoneal Macrophage ◽  
Inflammatory Responses ◽  
Regulatory Protein ◽  
Mouse Peritoneal Macrophage ◽  
Raw264.7 Cells ◽  
Dependent Manner ◽  
Proliferation Inhibition ◽  
Murine Macrophage ◽  
Concentration Dependent Manner

Glucocorticoid (GC) effectively suppresses immune and inflammatory responses and inhibits the growth of several types of cells, but the role of GC and its receptor on macrophage proliferation is unclear. In our previous work, we found RAW-GR(−) cells (murine macrophage RAW264.7 cells stably transfected with GR-siRNA expression vector by RNA interference) grew faster by about twofold. In this study, we further explored the role and mechanisms of GC/GR on the proliferation of macrophage. We found that the growth of RAW264.7 cells was inhibited by dexamethasone (Dex) in a concentration-dependent manner. The mRNA and protein levels of signal regulatory protein α1 (SIRPA) were induced by GC/GR in RAW264.7 cells and SIRPA expression was decreased remarkably in RAW-GR(−) cells. Overexpression of SIRPA negatively regulated the proliferation of RAW-GR(−) cells, and inhibition of SIRPA expression by a small from RNA interference attenuated Dex-induced proliferation inhibition in RAW264.7 cells. The proliferation inhibition of GC/GR was also found in mouse peritoneal macrophage, which was associated with the increase in SIRPA induced by GC/GR as well. In addition, elevation of the expression of CDK2, cyclinD1, and cyclinB1, but not phosphorylated ERK1/2 and p38, was found in RAW-GR(−) cells. In conclusion, we provided the novel evidences that GC/GR inhibited the growth of RAW264.7 cells and mouse peritoneal macrophage, and the antiproliferative effect of GC/GR on these cells was at least in part a result from GC/GR-induced SIRPA expression. Up-regulation of CDK2, cyclinD1, and cyclinB1 was also related to the increased proliferation of RAW-GR(−) cells.

scholarly journals Binding of dsRNA by D. melanogaster Dicer-2 is substrate-dependent and regulated by Loquacious-PD

2020 ◽  
Author(s):  
M. Jonely ◽  
R. K. Singh ◽  
B. L. Bass ◽  
R. Noriega
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Interference ◽  
Molecular Recognition ◽  
Rna Binding ◽  
Regulatory Proteins ◽  
Dependent Manner ◽  
Biochemical Activity ◽  
Double Stranded Rna ◽  
Molecular Scale ◽  
Local Binding

ABSTRACTDrosophila melanogaster Dicer-2 is a large, multidomain protein that cleaves double-stranded RNA (dsRNA) into small interfering RNAs in a terminus-dependent manner as part of the RNA interference pathway. We characterize the local binding environment involved in this substrate-selective molecular recognition event by monitoring the time-resolved photophysics of a cyanine dye linked to the dsRNA terminus. We observe substantial changes in the molecular rigidity and local freedom of motion of the probe as a function of distinct conformations of the biomolecular complex between Dicer-2 and dsRNA as a function of dsRNA termini, the presence of regulatory proteins, and the addition of a biochemical energy source (ATP) or a non-hydrolysable equivalent (ATP-γS). With a clustering analysis based solely on these molecular-scale measures of the local binding environment at the dsRNA terminus, we identify sub-populations of similar conformations that define distinct modes of molecular recognition which are correlated with biochemical activity. These observations reveal the important role of substrate-selective molecular recognition properties for proteins with multiple domains that can bind RNA, regulatory proteins, and cofactors.STATEMENT OF SIGNIFICANCEThe molecular-scale determinants of protein-RNA binding remain elusive, particularly when different subunits of a single protein confer specificity toward small structural differences of their RNA partners. An important case is that of Drosophila melanogaster Dicer-2, a critical component of the antiviral RNA interference response. Dicer-2 discriminates between double stranded RNA with blunt or 3’ overhang termini, a feature suggested to mediate recognition of “self” vs. “non-self” substrates. We study these interactions at the binding site with a fluorescent label at the RNA terminus, monitoring intramolecular and collective measures of flexibility to report on the local environment. Dicer-2 has distinct modes of molecular recognition which are regulated by accessory proteins and ATP, leading to different conformations and tuning biochemical activity.

scholarly journals Absence of non-specific effects of RNA interference triggered by long double-stranded RNA in mouse oocytes

2005 ◽  
Vol 286 (2) ◽  
pp. 464-471 ◽  
Author(s):  
Paula Stein ◽  
Fanyi Zeng ◽  
Hua Pan ◽  
Richard M. Schultz
Keyword(s):  
Rna Interference ◽  
Double Stranded Rna ◽  
Mouse Oocytes ◽  
Specific Effects

A 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes

2007 ◽  
Vol 293 (3) ◽  
pp. C897-C905 ◽  
Author(s):  
Yongchang Qian ◽  
Ying Zheng ◽  
Deanna Weber ◽  
Evelyn Tiffany-Castiglioni
Keyword(s):  
Fluorescent Protein ◽  
Affinity Column ◽  
Chimeric Proteins ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Double Stranded Rna ◽  
Imaging Probes ◽  
The Central Nervous System ◽  
Green Fluorescent ◽  
Glucose Regulated Protein

Interleukin (IL)-6 is a cytokine produced mainly by microglia and astrocytes and plays a pleiotropic role in the central nervous system. In this study, we cloned rat IL-6 cDNA into an enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRed2) vector and rat 78-kDa glucose-regulated protein (GRP78) cDNA into an EGFP vector to construct IL-6-EGFP, IL-6-DsRed2, and GRP78-EGFP chimeras for the investigation of the mechanism of IL-6 secretion from astrocytes. The data showed that constructed IL-6-EGFP and IL-6-DsRed2 chimeras retained the secretory property, and the secretion of IL-6-EGFP from astrocytes could be attenuated by GRP78 depletion with double-stranded RNA interference. Coexpression of IL-6-DsRed2 and dysfunctional GRP78-EGFP abolished IL-6-DsRed2 secretion, and two chimeric proteins colocalized inside living astrocytes. Coimmunoprecipitation analysis indicated that IL-6 and GRP78 resided in the same complex. The data further revealed that IL-6-EGFP secretion from astrocytes was blocked by the heavy metal lead (Pb) in a concentration-dependent manner. Analysis of the Pb interaction with protein on a Pb-affinity column demonstrated that Pb bound to GRP78 but failed to bind to IL-6. Therefore, these data suggest that IL-6-EGFP or IL-6-DsRed2 chimeras can be used as imaging probes to study IL-6 secretion from living cells, that GRP78 is involved in IL-6 secretion from astrocytes, and that Pb can block IL-6 secretion from astrocytes via targeting GRP78.

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom

1993 ◽  
Vol 69 (03) ◽  
pp. 286-292 ◽  
Author(s):  
Che-Ming Teng ◽  
Feng-Nien Ko ◽  
Inn-Ho Tsai ◽  
Man-Ling Hung ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Inositol Phosphate ◽  
Shape Change ◽  
Platelet Activating Factor ◽  
Atp Release ◽  
Platelet Membrane ◽  
Thromboxane B2 ◽  
Dependent Manner ◽  
Concentration Dependent Manner

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.

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