The H3K27me3 Demethylase dUTX Is a Suppressor of Notch- and Rb-Dependent Tumors in Drosophila

ABSTRACT Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and can be demethylated by the JmjC domain of UTX. Excessive H3K27me3 levels can cause tumorigenesis, but little is known about the mechanisms leading to those cancers. Mutants of the Drosophila H3K27me3 demethylase dUTX display some characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. We show that a disruption of the JmjC domain of dUTX results in a growth advantage for mutant cells over adjacent wild-type tissue due to increased proliferation. The growth advantage of dUTX mutant tissue is caused, at least in part, by increased Notch activity, demonstrating that dUTX is a Notch antagonist. Furthermore, the inactivation of Retinoblastoma (Rbf in Drosophila) contributes to the growth advantage of dUTX mutant tissue. The excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila melanogaster and may provide a model for UTX-dependent tumorigenesis in humans.

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The STK Receptor Tyrosine Kinase Regulates the Response of Primary Macrophages to LPS in a Ligand-Independent Manner.

Blood ◽

10.1182/blood.v104.11.3436.3436 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3436-3436

Author(s):

Pamela Correll ◽

Qingping Liu Liu

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Macrophage Activation ◽

Endotoxic Shock ◽

Peritoneal Macrophages ◽

Dependent Manner ◽

Wild Type ◽

Independent Manner ◽

Retroviral Transduction

Abstract We have shown previously that activation of the STK/RON receptor tyrosine kinase expressed on tissue resident macrophages, by it’s ligand macrophage stimulating protein (MSP), results in the inhibition of NFkB activation, inducible nitric oxide synthase (iNOS) expression and TNFa production, as well as the induction of arginase expression, suggesting a role for this receptor in the regulation of classical vs. alternative macrophage activation. Furthermore, mice with a targeted deletion in this receptor exhibit increased sensitivity to endotoxic shock and DTH responses. More recently, we have demonstrated that MSP stimulation of primary peritoneal macrophages inhibits the production of IL-12. In order to map the domains of STK responsible for the inhibition of classical macrophage activation by MSP, we generated mutant forms of the receptor and expressed wild-type and mutant receptors in primary bone marrow derived macrophages by retroviral transduction. Expression of wild-type STK in these primary cells resulted in the ligand-independent reduction in IL-12p40 production in response to LPS stimulation, which was further inhibited by MSP treatment. This is consistent with the lack of a requirement for MSP in regulating responses to endotoxin in vivo. Surprisingly, a kinase dead receptor, which fails to signal in 293T cells, was fully functional in this assay, suggesting that the kinase activity of the receptor is not required for the inhibition of IL-12p40 under these conditions. However, the docking site tyrosines in the c-terminal tail of the receptor are essential for the inhibition of IL-12p40 by STK, suggesting that STK may be phosphorylated by an another kinase in this system. STK/RON has been shown to associate both physically and functionally with a number of other cell-surface receptors including EpoR, IL-3R bc, EGFR, MET as well as a number of integrins and cadherins. We have shown previously that STK regulates the activity of the aMb2 integrin (CR3) in peritoneal macrophages in a PI3K, PKCz-dependent manner. Here we show that STK also physically associates with CR3, as well as CD14, in RAW264.7 cells in the absence of ligand. Both CR3 and CD14 are capable of directly binding to LPS. Thus, we speculate that STK may exist as part of a receptor complex in macrophages and that signalling through STK might be induced directly by LPS. This would provide a means by which STK could temper the response of tissue-resident macrophages to LPS thereby preventing damage to host tissues.

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Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Endocrinology ◽

10.1210/en.2004-0061 ◽

2004 ◽

Vol 145 (12) ◽

pp. 5525-5531 ◽

Cited By ~ 50

Author(s):

Gary M. Leong ◽

Sofia Moverare ◽

Jesena Brce ◽

Nathan Doyle ◽

Klara Sjögren ◽

...

Keyword(s):

Promoter Activity ◽

Hepatoma Cells ◽

Cytokine Signaling ◽

Mrna Levels ◽

Dependent Manner ◽

Wild Type ◽

Hepatic Expression ◽

Suppressors Of Cytokine Signaling

Abstract Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-α, ERβ, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERβ knockout mice but not in those lacking ERα or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides −1862 and −855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ERα, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.

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A Possible Role of Allatostatin C in Inhibiting Ecdysone Biosynthesis Revealed in the Mud Crab Scylla paramamosain

Frontiers in Marine Science ◽

10.3389/fmars.2021.740251 ◽

2021 ◽

Vol 8 ◽

Author(s):

An Liu ◽

Wenyuan Shi ◽

Dongdong Lin ◽

Haihui Ye

Keyword(s):

Scylla Paramamosain ◽

Dependent Manner ◽

Mud Crab ◽

Methyl Farnesoate ◽

Independent Manner ◽

Wide Range ◽

Negative Effect

C-type allatostatins (C-type ASTs) are a family of structurally related neuropeptides found in a wide range of insects and crustaceans. To date, the C-type allatostatin receptor in crustaceans has not been deorphaned, and little is known about its physiological functions. In this study, we aimed to functionally define a C-type ASTs receptor in the mud crab, Scylla paramamosian. We showed that C-type ASTs receptor can be activated by ScypaAST-C peptide in a dose-independent manner and by ScypaAST-CCC peptide in a dose-dependent manner with an IC50 value of 6.683 nM. Subsequently, in vivo and in vitro experiments were performed to investigate the potential roles of ScypaAST-C and ScypaAST-CCC peptides in the regulation of ecdysone (20E) and methyl farnesoate (MF) biosynthesis. The results indicated that ScypaAST-C inhibited biosynthesis of 20E in the Y-organ, whereas ScypaAST-CCC had no effect on the production of 20E. In addition, qRT-PCR showed that both ScypaAST-C and ScypaAST-CCC significantly decreased the level of expression of the MF biosynthetic enzyme gene in the mandibular organ, suggesting that the two neuropeptides have a negative effect on the MF biosynthesis in mandibular organs. In conclusion, this study provided new insight into the physiological roles of AST-C in inhibiting ecdysone biosynthesis. Furthermore, it was revealed that AST-C family peptides might inhibit MF biosynthesis in crustaceans.

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Murine monoclonal antibodies against RBD of SARS-CoV-2 neutralize authentic wild type SARS-CoV-2 as well as B.1.1.7 and B.1.351 viruses and protect in vivo in a mouse model in a neutralization dependent manner

10.1101/2021.04.05.438547 ◽

2021 ◽

Author(s):

Fatima Amanat ◽

Shirin Strohmeier ◽

Wen-Hsin Lee ◽

Sandhya Bangaru ◽

Andrew B Ward ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Mouse Model ◽

Neutralizing Antibodies ◽

Dependent Manner ◽

Receptor Binding Domain ◽

Wild Type ◽

Murine Monoclonal Antibodies

After first emerging in December 2019 in China, severe acute respiratory syndrome 2 (SARS-CoV-2) has since caused a pandemic leading to millions of infections and deaths worldwide. Vaccines have been developed and authorized but supply of these vaccines is currently limited. With new variants of the virus now emerging and spreading globally, it is essential to develop therapeutics that are broadly protective and bind conserved epitopes in the receptor binding domain (RBD) or the whole spike of SARS-CoV-2. In this study, we have generated mouse monoclonal antibodies (mAbs) against different epitopes on the RBD and assessed binding and neutralization against authentic SARS-CoV-2. We have demonstrated that antibodies with neutralizing activity, but not non-neutralizing antibodies, lower viral titers in the lungs when administered in a prophylactic setting in vivo in a mouse challenge model. In addition, most of the mAbs cross-neutralize the B.1.351 as well as the B.1.1.7 variants in vitro.

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An alkaline phosphatase mutant of Pseudomonas aeruginosa. 1. Effects of regulatory, structural, and environmental shifts on enzyme function

Canadian Journal of Microbiology ◽

10.1139/m78-070 ◽

1978 ◽

Vol 24 (4) ◽

pp. 427-432 ◽

Cited By ~ 1

Author(s):

M. L. Marceau-Day ◽

D. F Day ◽

J. M. Ingram

Keyword(s):

Pseudomonas Aeruginosa ◽

Alkaline Phosphatase ◽

Core Region ◽

Significant Activity ◽

Wild Type ◽

Glycerol Phosphate ◽

The Core ◽

Nitrophenyl Phosphate ◽

Mutant Cells

An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both β-glycerol phosphate (βGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of βGP versus pNPP, whereas this ratio was reversed to 1:9 βGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide(LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.

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Abstract 5566: Recovery of Erk Signaling Restores Defective Angiogenesis and Arteriogenesis in Synectin-Deficient Animals

Circulation ◽

10.1161/circ.118.suppl_18.s_576-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Bin Ren ◽

Arpita Mukhopadhyay* ◽

Anthony A Lanahan ◽

Zhen W Zhuang ◽

Karen L Moodie ◽

...

Keyword(s):

Endothelial Cells ◽

Branching Morphogenesis ◽

Erk Signaling ◽

Dependent Manner ◽

Wild Type ◽

Erk Activation ◽

Arterial Development ◽

Constitutively Active

Background : Arterial morphogenesis is an important and poorly understood process. We have previously demonstrated that disruption of synectin gene expression in mice and zebrafish results in impaired arterial development and branching morphogenesis. Synectin null endothelial cells demonstrate reduced VEGF responsiveness in terms of migration, proliferation and differentiation and ERK-1/2 activation (Chittenden et al, Dev Cell 2006). Since ERK has been established as major participants in the regulation of cell growth and differentiation and Erk activation has been previously linked to arterial morphogenesis, we evaluated whether activation of Erk signaling in synectin disrupted mice and zebrafish as well as synectin KO arterial endothelial cells (ECs) would restore defective migration, arterial differentiation, angiogenesis and arteriogenesis. To stimulate ERK signaling we used partial inhibition of PI3-K activity to reduce Akt-dependent suppression of Raf1 activation or introduction of constitutively active ERK construct. Methods : In vitro studies were conducted with primary arterial ECs isolated from synectin wild type (WT) and knock out (KO) mice. In vivo studies were carried out in WT and synectin deficient mice and synectin knockdown zebrafish embryos. Results: Exposure of synectin −/− arterial EC to two selective PI3K inhibitors GS4898 or LY294002 in vitro restored ERK activation in a dose-dependent manner and returned cell migration and in vitro branching morphogenesis to wild type levels. Transduction of a constitutively active ERK construct in vitro or in a Matrigel model in vivo had similar effect. Systemic treatment of synectin −/− mice with GS4898 fully restored impaired angiogenesis and arterial morphogenesis in adult animals in the setting of hindlimb ischemia. Similar treatment nearly completely restored arterial development defects in zebrafish treated with a synectin morpholino. Conclusions: ERK activation plays a key role in arteriogenesis both in adult tissues and during embryonic development. Activation of compromised ERK-1/2 signaling may be a novel therapeutic intervention to stimulate arteriogenesis.

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Abstract 123: Age-Related Endothelial Senescence is Driven by Thrombospondin-1-Activated NADPH Oxidase Nox1

Hypertension ◽

10.1161/hyp.66.suppl_1.123 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Daniel N Meijles ◽

Imad Al Ghouleh ◽

Sanghamitra Sahoo ◽

Jefferson H Amaral ◽

Heather Knupp ◽

...

Keyword(s):

Oxidative Stress ◽

Nadph Oxidase ◽

Vascular Diseases ◽

Dependent Manner ◽

Wild Type ◽

Molecular Control ◽

Redox Biology ◽

Age Related ◽

P53 Activation

Organismal aging represents an independent risk factor underlying many vascular diseases, including systemic and pulmonary hypertension, and atherosclerosis. While the mechanisms driving aging are largely elusive, a steady persistent increase in tissue oxidative stress has been associated with senescence. Previously we showed TSP1 elicits NADPH oxidase (Nox)-dependent vascular smooth muscle cell oxidative stress. However mechanisms by which TSP1 affects endothelial redox biology are unknown. Here, we tested the hypothesis that TSP1 induces endothelial oxidative stress-linked senescence in aging. Using rapid autopsy disease-free human pulmonary (PA) artery, we identified a significant positive correlation between age, protein levels of TSP1, Nox1 and the cell-cycle repressor p21cip (p<0.05). Age also positively associated with increased Amplex Red-detected PA hydrogen peroxide levels (p<0.05). Moreover, treatment of human PA endothelial cells (HPAEC) with TSP1 (2.2nM; 24h) increased expression (~1.9 fold; p<0.05) and activation of Nox1 (~1.7 fold; p<0.05) compared to control, as assessed by Western blot and SOD-inhibitable cytochrome c reduction. Western blotting and immunofluorescence showed a TSP1-mediated increase in p53 activation, indicative of the DNA damage response. Moreover, TSP1 significantly increased HPAEC senescence in a p53/p21cip/Rb-dependent manner, as assessed by immunofluorescent detection of subcellular localization and senescence-associated β-galactosidase staining. To explore this pathway in vivo, middle-aged (8-10 month) wild-type and TSP1-null mice were utilized. In the TSP1-null, reduced lung senescence, oxidative stress, Nox1 levels and p21cip expression were observed compared to wild-type supporting findings in human samples and cell experiments. Finally, prophylactic treatment with specific Nox1 inhibitor NoxA1ds (10μM) attenuated TSP1-induced HPAEC ROS, p53 activation, p21cip expression and senescence. Taken together, our results provide molecular insight into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence, with implications for molecular control of the aging process.

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The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/3264858 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 6

Author(s):

V. Helfinger ◽

K. Palfi ◽

A. Weigert ◽

K. Schröder

Keyword(s):

Ex Vivo ◽

Macrophage Polarization ◽

Dependent Manner ◽

Oxygen Species ◽

Superoxide Anions ◽

Wild Type ◽

Macrophage Population ◽

The Family ◽

High Level

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.

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Piquiá Shells (Caryocar villosum): A Fruit by-Product with Antioxidant and Antiaging Properties in Caenorhabditis elegans

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/7590707 ◽

2020 ◽

Vol 2020 ◽

pp. 1-19 ◽

Cited By ~ 1

Author(s):

Mariana Roxo ◽

Herbenya Peixoto ◽

Pille Wetterauer ◽

Emerson Lima ◽

Michael Wink

Keyword(s):

Caenorhabditis Elegans ◽

Lethal Dose ◽

Pcr Analysis ◽

Transcriptional Level ◽

Dependent Manner ◽

Wild Type ◽

Fluorescent Reporter ◽

Triterpenoid Saponins ◽

Promising Source

In a context of rising demand for sustainable antiaging interventions, fruit processing by-products are a promising source of bioactive compounds for the production of antiaging dietary supplements. Piquiá (Caryocar villosum) is a native Amazonian fruit consisting of 65% nonedible shells. In the present study, the phytochemical profile of a hydroalcoholic extract of piquiá shells (CV) was characterized by LC-MS/MS analysis. Its antioxidant and antiaging activities were investigated using the nematode Caenorhabditis elegans as an in vivo model. CV is mainly composed by hydrolysable tannins and triterpenoid saponins. The extract enhanced stress resistance of wild-type and mutant worms by reducing the intracellular levels of reactive oxygen species (ROS) and by increasing their survival against a lethal dose of the prooxidant juglone. These effects involved the upregulation of sod-3 and downregulation of gst-4 and hsp-16.2, studied through the GFP fluorescent reporter intensity and at the transcriptional level by qRT-PCR analysis. CV extended the lifespan of wild-type worms in a DAF-16/FoxO- and SKN-1/Nrf-dependent manner. Taken together, our findings indicate piquiá shells as potential candidates for nutraceutical applications. Further studies are needed to validate the relevance of our findings to antiaging interventions in humans.

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Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster

Anesthesiology ◽

10.1097/00000542-200310000-00019 ◽

2003 ◽

Vol 99 (4) ◽

pp. 867-875 ◽

Cited By ~ 16

Author(s):

Sumiko Gamo ◽

Junya Tomida ◽

Katsuyuki Dodo ◽

Dai Keyakidani ◽

Hitoshi Matakatsu ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Double Mutant ◽

Developmental Stages ◽

Northern Blotting ◽

General Anesthetics ◽

Wild Type ◽

Mutant Strains ◽

Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

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