Homocysteine-Potentiated Keap1 Promotes Neuronal Senescence via Inhibiting Ubiquitination of β-Catenin

Abstract Background Elevated serum homocysteine (Hcy) is an independent risk factor of Alzheimer’s disease (AD). It has been reported that Hcy dramatically accelerates the aging of endothelial progenitor cells or endothelial cells. However, whether and how Hcy produces neuronal senescence is largely unknown. Methods Mouse neuroblastma 2a (N2a) cells were treated with Hcy, and senescence-associated β-galactosidase (SA-β-gal) staining was applied to assay senescence. Senescent markers and related proteins were examined by western blot, quantitative Polymerase Chain Reaction (qPCR), immunofluorescence staining. Methylation of promoter was assay by bisulfite sequencing PCR (BSP). Immunoprecipitation (IP) was applied to examine association between proteins. Rats were injected with homocysteine and examined neuronal senescence. Results In this study, we observed that Hcy significantly promoted the senescence of N2a cells with elevated β-catenin and Kelch like ECH-associated protein 1 (Keap1). Intriguingly, Hcy increased the interaction between Keap1 and Wilms tumor gene on X chromosome (WTX), but decreased β-catenin-WTX interaction simultaneously. Mechanistically, Hcy attenuated the methylation level of Keap1 promoter’s CqG island and activated the transcription of Keap1. While, slow degradation rate rather than transcriptional activation contributed to the high level of β-catenin. Hcy-increased Keap1 competed with β-catenin to bind to WTX. Knockdown of β-catenin and Keap1 both attenuated Hcy-induced senescence of N2a cells. Hcy-induced rats model also showed neuronal senescence in cortex along with elevated senescent markers. Conclusions Our data highlight a crucial role of Keap1-β-catenin pathway in Hcy-induced neuronal-like senescence and provide a promising target for AD treatment.

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Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation

Acta Neurochirurgica ◽

10.1007/s00701-021-04895-z ◽

2021 ◽

Author(s):

Sara Keränen ◽

Santeri Suutarinen ◽

Rahul Mallick ◽

Johanna P. Laakkonen ◽

Diana Guo ◽

...

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Rank Correlation ◽

Inflammatory Cells ◽

Vessel Remodeling ◽

Brain Avm ◽

Inflammatory Drugs ◽

Polymerase Chain ◽

The Brain ◽

Cox2 Expression

Abstract Background Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. Methods Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. Results COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels’ lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. Conclusion COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

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Type III Collagen is Required for Adipogenesis and Actin Stress Fibre Formation in 3T3-L1 Preadipocytes

Biomolecules ◽

10.3390/biom11020156 ◽

2021 ◽

Vol 11 (2) ◽

pp. 156

Author(s):

Mohammad Al Hasan ◽

Patricia E. Martin ◽

Xinhua Shu ◽

Steven Patterson ◽

Chris Bartholomew

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Type Iii Collagen ◽

Type Iii ◽

Actin Stress Fibre ◽

Matrix Gene ◽

Gene Transcripts ◽

Polymerase Chain ◽

Oil Red O Staining ◽

Oil Red O

GPR56 is required for the adipogenesis of preadipocytes, and the role of one of its ligands, type III collagen (ColIII), was investigated here. ColIII expression was examined by reverse transcription quantitative polymerase chain reaction, immunoblotting and immunostaining, and its function investigated by knockdown and genome editing in 3T3-L1 cells. Adipogenesis was assessed by oil red O staining of neutral cell lipids and production of established marker and regulator proteins. siRNA-mediated knockdown significantly reduced Col3a1 transcripts, ColIII protein and lipid accumulation in 3T3-L1 differentiating cells. Col3a1−/− 3T3-L1 genome-edited cell lines abolished adipogenesis, demonstrated by a dramatic reduction in adipogenic moderators: Pparγ2 (88%) and C/ebpα (96%) as well as markers aP2 (93%) and oil red O staining (80%). Col3a1−/− 3T3-L1 cells displayed reduced cell adhesion, sustained active β-catenin and deregulation of fibronectin (Fn) and collagen (Col4a1, Col6a1) extracellular matrix gene transcripts. Col3a1−/− 3T3-L1 cells also had dramatically reduced actin stress fibres. We conclude that ColIII is required for 3T3-L1 preadipocyte adipogenesis as well as the formation of actin stress fibres. The phenotype of Col3a1−/− 3T3-L1 cells is very similar to that of Gpr56−/− 3T3-L1 cells, suggesting a functional relationship between ColIII and Gpr56 in preadipocytes.

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The Only Function of Grauzone Required for Drosophila Oocyte Meiosis Is Transcriptional Activation of the cortex Gene

Genetics ◽

10.1093/genetics/155.4.1831 ◽

2000 ◽

Vol 155 (4) ◽

pp. 1831-1839

Author(s):

Emily Harms ◽

Tehyen Chu ◽

Gwénola Henrion ◽

Sidney Strickland

Keyword(s):

Zinc Finger ◽

Transcriptional Activation ◽

Single Amino Acid ◽

Primary Role ◽

Extra Copy ◽

Oocyte Meiosis ◽

Zinc Finger Motifs ◽

High Level ◽

Transcriptional Pathway

Abstract The grauzone and cortex genes are required for the completion of meiosis in Drosophila oocytes. The grauzone gene encodes a C2H2-type zinc-finger transcription factor that binds to the cortex promoter and is necessary for high-level activation of cortex transcription. Here we define the region of the cortex promoter to which Grauzone binds and show that the binding occurs through the C-terminal, zinc-finger-rich region of the protein. Mutations in two out of the five grauzone alleles result in single amino acid changes within different zinc-finger motifs. Both of these mutations result in the inability of Grauzone to bind DNA effectively. To determine the mechanism by which Grauzone regulates meiosis, transgenic flies were produced with an extra copy of the cortex gene in homozygous grauzone females. This transgene rescued the meiosis arrest of embryos from these mutants and allowed their complete development, indicating that activation of cortex transcription is the primary role of Grauzone during Drosophila oogenesis. These experiments further define a new transcriptional pathway that controls the meiotic cell cycle in Drosophila oocytes.

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Identification and Functional Characterization of a Cold-Related Protein, BcHHP5, in Pak-Choi (Brassica rapa ssp. chinensis)

International Journal of Molecular Sciences ◽

10.3390/ijms20010093 ◽

2018 ◽

Vol 20 (1) ◽

pp. 93

Author(s):

Jin Wang ◽

Feiyi Huang ◽

Xiong You ◽

Xilin Hou

Keyword(s):

Brassica Rapa ◽

Abiotic Stresses ◽

Quantitative Polymerase Chain Reaction ◽

Functional Characterization ◽

Regulation Mechanism ◽

Pak Choi ◽

Negative Effect ◽

Polymerase Chain ◽

Related Proteins

In plants, heptahelical proteins (HHPs) have been shown to respond to a variety of abiotic stresses, including cold stress. Up to the present, the regulation mechanism of HHP5 under low temperature stress remains unclear. In this study, BcHHP5 was isolated from Pak-choi (Brassica rapa ssp. chinensis cv. Suzhouqing). Sequence analysis and phylogenetic analysis indicated that BcHHP5 in Pak-choi is similar to AtHHP5 in Arabidopsis thaliana. Structure analysis showed that the structure of the BcHHP5 protein is relatively stable and highly conservative. Subcellular localization indicated that BcHHP5 was localized on the cell membrane and nuclear membrane. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) analysis showed that BcHHP5 was induced to express by cold and other abiotic stresses. In Pak-choi, BcHHP5-silenced assay, inhibiting the action of endogenous BcHHP5, indicated that BcHHP5-silenced might have a negative effect on cold tolerance, which was further confirmed. All of these results indicate that BcHHP5 might play a role in abiotic response. This work can serve as a reference for the functional analysis of other cold-related proteins from Pak-choi in the future.

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Decreased Expression of GPER1 Gene and Protein in Goiter

International Journal of Endocrinology ◽

10.1155/2015/869431 ◽

2015 ◽

Vol 2015 ◽

pp. 1-5 ◽

Cited By ~ 2

Author(s):

Raquel Weber ◽

Ana Paula Santin Bertoni ◽

Laura Walter Bessestil ◽

Ilma Simoni Brum ◽

Tania Weber Furlanetto

Keyword(s):

Estrogen Receptor ◽

Quantitative Polymerase Chain Reaction ◽

Thyroid Tissue ◽

Chain Reaction ◽

Normal Thyroid ◽

Protein Levels ◽

Protein Expressions ◽

Polymerase Chain ◽

G Protein Coupled

Goiter is more common in women, suggesting that estrogen could be involved in its physiopathology. The presence of classical estrogen receptors (ERαand ERβ) has been described in thyroid tissue, suggesting a direct effect of estrogen on the gland. A nonclassic estrogen receptor, the G-protein-coupled estrogen receptor (GPER1), has been described recently in several tissues. However, in goiter, the presence of this receptor has not been studied yet. We investigated GPER1 gene and protein expressions in normal thyroid and goiter using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. In normal thyroid (n=16) and goiter (n=19), GPER1 gene was expressed in all samples, while GPER1 protein was expressed in all samples of normal thyroid (n=15) but in only 72% of goiter samples (n=13). When comparing GPER1 gene and protein levels in both conditions, gene expression and protein levels were higher in normal thyroid than in goiter, suggesting a role of this receptor in this condition. Further studies are needed to elucidate the role of GPER1 in normal thyroid and goiter.

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C/EBPα directs monocytic commitment of primary myeloid progenitors

Blood ◽

10.1182/blood-2005-12-008763 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1223-1229 ◽

Cited By ~ 65

Author(s):

Dehua Wang ◽

Jenice D'Costa ◽

Curt I. Civin ◽

Alan D. Friedman

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Fold Increase ◽

Pcr Assay ◽

Gm Csf ◽

Specific Effects ◽

Proliferation And Apoptosis ◽

Polymerase Chain ◽

Lineage Markers ◽

Transcriptional Induction

Abstract C/EBPα is required for generation of granulocyte-monocyte progenitors, but the subsequent role of C/EBPα in myeloid lineage commitment remains uncertain. We transduced murine marrow cells with C/EBPα-estradiol receptor (ER) or empty vector and subjected these to lineage depletion just prior to culture in estradiol with myeloid cytokines. This protocol limits biases due to lineage-specific effects on developmental kinetics, proliferation, and apoptosis. Also, lowering the dose of estradiol reduced activated C/EBPα-ER to near the physiologic range. C/EBPα-ER increased Mac1+/Gr1–/MPO–/low monocytes 1.9-fold while reducing Mac1+/Gr1+/MPOhi granulocytes 2.5-fold at 48 hours, even in 0.01 μM estradiol. This pattern was confirmed morphologically and by quantitative polymerase chain reaction (PCR) assay of lineage markers. To directly assess effects on immature progenitors, transduced cells were cultured for 1 day with and then in methylcellulose without estradiol. A 2-fold increase in monocytic compared with granulocytic colonies was observed in IL-3/IL-6/SCF or GM-CSF, but not G-CSF, even in 0.01 μM estradiol. C/EBPα-ER induced PU.1 mRNA, and PU.1-ER stimulated monocytic development, suggesting that transcriptional induction of PU.1 by C/EBPα contributes to monopoiesis. A C/EBPα variant incapable of zippering with c-Jun did not induce monopoiesis, and a variant unable to bind NF-κB p50 stimulated granulopoiesis, suggesting their cooperation with C/EBPα during monocytic commitment.

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Promoter Methylation ofSFRP3Is Frequent in Hepatocellular Carcinoma

Disease Markers ◽

10.1155/2014/351863 ◽

2014 ◽

Vol 2014 ◽

pp. 1-10 ◽

Cited By ~ 9

Author(s):

Ya-Wen Lin ◽

Yu-Lueng Shih ◽

Gi-Shih Lien ◽

Fat-Moon Suk ◽

Chung-Bao Hsieh ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Chronic Hepatitis ◽

Mrna Expression ◽

Cell Lines ◽

Promoter Methylation ◽

Promoter Hypermethylation ◽

Polymerase Chain ◽

Related Proteins ◽

And Control

Oncogenic activation of the Wnt/β-catenin signaling pathway is common in human cancers. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications in carcinogenesis. Because there have been no reports about the role ofSFRP3in hepatocellular carcinoma (HCC), we investigated the level of methylation and transcription ofSFRP3. Four HCC cell lines, 60 HCCs, 23 cirrhosis livers, 37 chronic hepatitis livers, and 30 control livers were prescreened forSFRP3promoter methylation by methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing.SFRP3promoter methylation was observed in 100%, 60%, 39.1%, 16.2%, and 0% in HCC cell lines, primary HCCs, cirrhosis livers, chronic hepatitis livers, and control livers, respectively. Demethylation treatment with 5-aza-2′-deoxycytidine in HCC cells restored or increased theSFRP3mRNA expression. We next used quantitative MS-PCR (QMSP) to analyze the methylation level ofSFRP3in 60 HCCs and their corresponding nontumor tissues. Methylation ofSFRP3promoter region in HCCs increased significantly compared with control tissues. There is a positive correlation between promoter hypermethylation andSFRP3mRNA downregulation. Our data suggest that promoter hypermethylation ofSFRP3is a common event in HCCs and plays an important role in regulation ofSFRP3mRNA expression.

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Sinonasal Surfactant Protein A1, A2, and D Gene Expression in Cystic Fibrosis: A Preliminary Report

Otolaryngology ◽

10.1016/j.otohns.2007.01.025 ◽

2007 ◽

Vol 137 (1) ◽

pp. 34-38 ◽

Cited By ~ 18

Author(s):

Bradford A. Woodworth ◽

Rachel Wood ◽

John E. Baatz ◽

Rodney J. Schlosser

Keyword(s):

Gene Expression ◽

Cystic Fibrosis ◽

Nasal Polyposis ◽

Quantitative Polymerase Chain Reaction ◽

Surfactant Protein ◽

Fungal Rhinosinusitis ◽

Allergic Fungal Rhinosinusitis ◽

Novel Treatment ◽

Polymerase Chain

OBJECTIVE: To measure alterations in SPA1, A2, and D gene expression in various forms of inflammatory chronic rhinosinusitis (CRS). STUDY DESIGN AND SETTING: Sinus mucosal biopsies were performed in patients with allergic fungal rhinosinusitis (AFS), CRS with nasal polyposis, cystic fibrosis (CF), and controls. SP mRNA was measured with quantitative polymerase chain reaction. RESULTS: Patients with CF (n = 4) showed significantly increased SPA1 (82-fold), SPA2 (100-fold), and SPD (47-fold) mRNA ( P < 0.05) when compared with controls (n = 5). Patients with CRS with nasal polyposis (n = 5) also demonstrated elevated SPA1 (27-fold), SPA2 (13-fold), and SPD (13-fold). Patients with AFS (n = 7) had increased SPA1 (5-fold), SPA2 (9-fold), and SPD (17-fold), but were not statistically significant. CONCLUSION: SPA1, A2, and D are upregulated in various forms of CRS, but are significantly elevated in cystic fibrosis CRS. SIGNIFICANCE: Understanding the role of SPs in CRS will help develop novel treatment approaches for sinonasal pathoses.

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Activation of C-Type Lectin Receptor and (RIG)-I-Like Receptors Contributes to Proinflammatory Response in Middle East Respiratory Syndrome Coronavirus-Infected Macrophages

The Journal of Infectious Diseases ◽

10.1093/infdis/jiz483 ◽

2019 ◽

Cited By ~ 2

Author(s):

Xiaoyu Zhao ◽

Hin Chu ◽

Bosco Ho-Yin Wong ◽

Man Chun Chiu ◽

Dong Wang ◽

...

Keyword(s):

Middle East ◽

Quantitative Polymerase Chain Reaction ◽

Severe Disease ◽

Human Infection ◽

Middle East Respiratory Syndrome ◽

Proinflammatory Response ◽

Pharmacological Inhibition ◽

Lectin Receptor ◽

Polymerase Chain

Abstract Background Human infection with Middle East respiratory syndrome coronavirus (MERS-CoV) poses an ongoing threat to public health worldwide. The studies of MERS patients with severe disease and experimentally infected animals showed that robust viral replication and intensive proinflammatory response in lung tissues contribute to high pathogenicity of MERS-CoV. We sought to identify pattern recognition receptor (PRR) signaling pathway(s) that mediates the inflammatory cascade in human macrophages upon MERS-CoV infection. Methods The potential signaling pathways were manipulated individually by pharmacological inhibition, small interfering ribonucleic acid (siRNA) depletion, and antibody blocking. The MERS-CoV-induced proinflammatory response was evaluated by measuring the expression levels of key cytokines and/or chemokines. Reverse transcription-quantitative polymerase chain reaction assay, flow cytometry analysis, and Western blotting were applied to evaluate the activation of related PRRs and engagement of adaptors. Results MERS-CoV replication significantly upregulated C-type lectin receptor (CLR) macrophage-inducible Ca2+-dependent lectin receptor (Mincle). The role of Mincle for MERS-CoV-triggered cytokine/chemokine induction was established based on the results of antibody blockage, siRNA depletion of Mincle and its adaptor spleen tyrosine kinase (Syk), and Syk pharmacological inhibition. The cytokine and/or chemokine induction was significantly attenuated by siRNA depletion of retinoic acid-inducible-I-like receptors (RLR) or adaptor, indicating that RLR signaling also contributed to MERS-CoV-induced proinflammatory response. Conclusions The CLR and RLR pathways are activated and contribute to the proinflammatory response in MERS-CoV-infected macrophages.

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Possible Role of Trichothecene Mycotoxins in Virulence of Fusarium graminearum on Maize

Plant Disease ◽

10.1094/pdis.1999.83.10.954 ◽

1999 ◽

Vol 83 (10) ◽

pp. 954-960 ◽

Cited By ~ 130

Author(s):

L. J. Harris ◽

A. E. Desjardins ◽

R. D. Plattner ◽

P. Nicholson ◽

G. Butler ◽

...

Keyword(s):

Fusarium Graminearum ◽

Fungal Biomass ◽

Biosynthetic Pathway ◽

Quantitative Polymerase Chain Reaction ◽

Field Trials ◽

Ear Rot ◽

Inoculation Method ◽

Trichodiene Synthase ◽

Polymerase Chain

Trichothecene-producing and -nonproducing Fusarium graminearum strains were tested for their ability to cause Gibberella ear rot in field trials at two locations—Ottawa, Ontario, and Peoria, Illinois—in 1996. Maize ears were inoculated with wild-type or transgenic F. graminearum strains in which the trichothecene biosynthetic pathway had been disabled by the specific disruption of the trichodiene synthase gene and with a derivative revertant strain in which trichothecene production had been restored through recombination. A silk channel inoculation method was employed at both locations. In addition, a kernel puncture inoculation method was used at the Ontario location. Harvested maize ears were analyzed for visual disease severity, grain yield, deoxynivalenol (DON) concentration, and fungal biomass by quantitative polymerase chain reaction (PCR) and/or ergosterol quantitation. There was a significant correlation (r= 0.86) between data obtained from the two different methods of quantifying fungal biomass. The trichothecene-nonproducing strains were still pathogenic but appeared less virulent on maize than the trichothecene-producing progenitor and revertant strains, as assayed by most parameters. This suggests that the trichothecenes may act as virulence factors to enhance the spread of F. graminearum on maize.

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