scholarly journals Phosphorylation at Serines 157 and 161 Is Necessary for Preserving Cardiac Expression Level and Functions of Sarcomeric Z-Disc Protein Telethonin

2021 ◽  
Vol 12 ◽  
Author(s):  
Hannah R. Lewis ◽  
Seda Eminaga ◽  
Mathias Gautel ◽  
Metin Avkiran
Keyword(s):  
Protein Expression ◽  
Gene Dosage ◽  
Systolic Dysfunction ◽  
Ubiquitin Proteasome System ◽  
Expression Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Adrenergic Stimulation ◽  
Protein Expression Level

Aims: In cardiac myocytes, the sarcomeric Z-disc protein telethonin is constitutively bis-phosphorylated at C-terminal residues S157 and S161; however, the functional significance of this phosphorylation is not known. We sought to assess the significance of telethonin phosphorylation in vivo, using a novel knock-in (KI) mouse model generated to express non-phosphorylatable telethonin (TcapS157/161A).Methods and Results:TcapS157/161A and wild-type (WT) littermates were characterized by echocardiography at baseline and after sustained β-adrenergic stimulation via isoprenaline infusion. Heart tissues were collected for gravimetric, biochemical, and histological analyses. At baseline, TcapS157/161A mice did not show any variances in cardiac structure or function compared with WT littermates and mutant telethonin remained localized to the Z-disc. Ablation of telethonin phosphorylation sites resulted in a gene-dosage dependent decrease in the cardiac telethonin protein expression level in mice carrying the S157/161A alleles, without any alteration in telethonin mRNA levels. The proteasome inhibitor MG132 significantly increased the expression level of S157/161A telethonin protein in myocytes from TcapS157/161A mice, but not telethonin protein in myocytes from WT mice, indicating a role for the ubiquitin–proteasome system in the regulation of telethonin protein expression level. TcapS157/161A mice challenged with sustained β-adrenergic stimulation via isoprenaline infusion developed cardiac hypertrophy accompanied by mild systolic dysfunction. Furthermore, the telethonin protein expression level was significantly increased in WT mice following isoprenaline stimulation but this response was blunted in TcapS157/161A mice.Conclusion: Overall, these data reveal that telethonin protein turnover in vivo is regulated in a novel phosphorylation-dependent manner and suggest that C-terminal phosphorylation may protect telethonin against proteasomal degradation and preserve cardiac function during hemodynamic stress. Given that human telethonin C-terminal mutations have been associated with cardiac and skeletal myopathies, further research on their potential impact on phosphorylation-dependent regulation of telethonin protein expression could provide valuable mechanistic insight into those myopathies.

The Protein Expression Level of a Heterogeneous Gene Inserted in LIPI-1 of the Listeria ivanovii Genome Relies on Its Insertion Orientation

2017 ◽  
Vol 27 (5) ◽  
pp. 269-276
Author(s):  
Sijing Liu ◽  
Mingjuan Jiang ◽  
Lin Su ◽  
Tian Tang ◽  
Xiang Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Bacterial Genome ◽  
Expression Level ◽  
Foreign Gene ◽  
Protein Expression Level ◽  
B Genome ◽  
Listeria Ivanovii ◽  
Cellular Immune

Due to its capability to multiply in either phagocytic or nonphagocytic cells, and to subsequently elicit a robust cellular immune response, <i>Listeria ivanovii</i> (LI) is thought to be feasible for developing bacteria-based live attenuated vaccines. We previously generated several recombinant LI strains expressing <i>Mycobacterium tuberculosis</i> antigens. Since the expression level of heterogeneous protein was sometimes very low, we attempted to elucidate the principle of heterogeneous protein expression in such recombinant LI strains. In this study, we inserted the <i>M. tuberculosis </i>antigen gene <i>Rv0129c </i>into LI strains at the same site as the genome but with a different insertion orientation. RT-qPCR and Western blot showed that when the insertion orientation of the heterogeneous gene was opposite to the <i>LIorfXYZ </i>gene in the<i> Listeria </i>pathogenicity island 1 in the bacterial genome, the heterogeneous gene could be transcribed well but the protein expression level seemed limited, both in vitro and in vivo. When inserted at an orientation consistent with <i>LIorfXYZ</i> at the same site in the genome, the expected 43-kD protein was observed in vitro as well as in a mouse model. Bacterial virulence was found to have decreased after recombination. This work confirms that the protein expression level of the heterogenous gene in such genome-recombinant LI-based vaccines is related to its inserted orientation in the bacterial genome, and a foreign gene inserted at this position of LIPI-1 will abolish <i>Listeria</i> virulence without affecting its growth.

scholarly journals Glucolipotoxicity and GLP-1 secretion

2021 ◽  
Vol 9 (1) ◽  
pp. e001905
Author(s):  
Jung-Hee Hong ◽  
Dae-Hee Kim ◽  
Moon-Kyu Lee
Keyword(s):  
Glucose Transporter ◽  
Cell Function ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Gene Expressions ◽  
Simultaneous Treatment ◽  
L Cells ◽  
Triglyceride Accumulation

IntroductionThe concept of glucolipotoxicity refers to the combined, deleterious effects of elevated glucose and/or fatty acid levels.Research design and methodsTo investigate the effects of chronic glucolipotoxicity on glucagon-like peptide-1-(7-36) amide (GLP-1) secretion, we generated glucolipotoxic conditions in human NCI-H716 enteroendocrine cells using either 5 or 25 mM glucose with or without 500 µM palmitate for 72 hours. For in vivo study, we have established a chronic nutrient infusion model in the rat. Serial blood samples were collected for 2 hours after the consumption of a mixed meal to evaluate insulin sensitivity and β-cell function.ResultsChronic glucolipotoxic conditions decreased GLP-1 secretion and the expressions of pCREB, pGSK3β, β-catenin, and TCF7L2 in NCI-H716 cells. Glucolipotoxicity conditions reduced glucose transporter expression, glucose uptake, and nicotinamide-adenine dinucleotide phosphate (NADPH) levels in L-cells, and increased triglyceride accumulation. In contrast, PPARα and ATP levels were reduced, which correlated well with decreased levels of SUR1 and Kir6.2, cAMP contents and expressions of pCAMK2, EPAC and PKA. We also observed an increase in reactive oxygen species production, UCP2 expression and Complex I activity. Simultaneous treatment with insulin restored the GLP-1 secretion. Glucolipotoxic conditions decreased insulin secretion in a time-dependent manner in INS-1 cells, which was recovered with exendin-4 cotreatment. Glucose and SMOFlipid infusion for 6 hours decreased GLP-1 secretion and proglucagon mRNA levels as well as impaired the glucose tolerance, insulin and C-peptide secretion in rats.ConclusionThese results provide evidence for the first time that glucolipotoxicity could affect GLP-1 secretion through changes in glucose and lipid metabolism, gene expressions, and proglucagon biosynthesis and suggest the interrelationship between glucolipotoxicities of L-cells and β-cells which develops earlier than that of L-cells.

scholarly journals Comprehensive Analysis of ESRRA in Endometrial Cancer

2021 ◽  
Vol 20 ◽  
pp. 153303382199208
Author(s):  
Shufang Wang ◽  
Xinlong Huo
Keyword(s):  
Endometrial Cancer ◽  
Protein Expression ◽  
Immune Cell ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
The Cancer Genome Atlas ◽  
Expression Level ◽  
Operating Characteristics ◽  
Protein Expression Level ◽  
Normal Tissues

Background: Estrogen-related receptor alpha (ESRRA) was reported to play an important role in multiple biological processes of neoplastic diseases. The roles of ESRRA in endometrial cancer have not been fully investigated yet. Methods: Expression data and clinicopathological data of patients with uteri corpus endometrial carcinoma (UCEC) were obtained from The Cancer Genome Atlas (TCGA). Comprehensive bioinformatics analysis was performed, including receiver operating characteristics (ROC) curve analysis, Kaplan-Meier survival analysis, gene ontology (GO) enrichment analysis, and Gene Set Enrichment Analysis (GSEA). Immunohistochemistry was used to detect the protein expression level of ESRRA and CCK-8 assay was performed to evaluate the effect of ESRRA on the proliferation ability. Results: A total of 552 UCEC tissues and 35 normal tissues were obtained from the TCGA database. The mRNA and protein expression level of ESRRA was highly elevated in UCEC compared with normal tissues, and was closely associated with poor prognosis. ROC analysis indicated a very high diagnostic value of ESRRA for patients with UCEC. GO and GSEA functional analysis showed that ESRRA might be mainly involved in cellular metabolism processes, in turn, tumorigenesis and progression of UCEC. Knockdown of ESRRA inhibited the proliferation of UCEC cells in vitro. Further immune cell infiltration demonstrated that ESRRA enhanced the infiltration level of neutrophil cell and reduced that of T cell (CD4+ naïve), NK cell, and cancer associated fibroblast (CAF). The alteration of immune microenvironment will greatly help in developing immune checkpoint therapy for UCEC. Conclusions: Our study comprehensively analyzed the expression level, clinical value, and possible mechanisms of action of ESRRA in UCEC. These findings showed that ESRRA might be a potential diagnostic and therapeutic target.

scholarly journals Eap1p, an Adhesin That Mediates Candida albicans Biofilm Formation In Vitro and In Vivo

2007 ◽  
Vol 6 (6) ◽  
pp. 931-939 ◽  
Author(s):  
Fang Li ◽  
Michael J. Svarovsky ◽  
Amy J. Karlsson ◽  
Joel P. Wagner ◽  
Karen Marchillo ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Adhesion ◽  
Biofilm Formation ◽  
Fungal Infections ◽  
Gene Dosage ◽  
Venous Catheter ◽  
Flow Chamber ◽  
Dependent Manner

ABSTRACT Candida albicans is the leading cause of systemic fungal infections in immunocompromised humans. The ability to form biofilms on surfaces in the host or on implanted medical devices enhances C. albicans virulence, leading to antimicrobial resistance and providing a reservoir for infection. Biofilm formation is a complex multicellular process consisting of cell adhesion, cell growth, morphogenic switching between yeast form and filamentous states, and quorum sensing. Here we describe the role of the C. albicans EAP1 gene, which encodes a glycosylphosphatidylinositol-anchored, glucan-cross-linked cell wall protein, in adhesion and biofilm formation in vitro and in vivo. Deleting EAP1 reduced cell adhesion to polystyrene and epithelial cells in a gene dosage-dependent manner. Furthermore, EAP1 expression was required for C. albicans biofilm formation in an in vitro parallel plate flow chamber model and in an in vivo rat central venous catheter model. EAP1 expression was upregulated in biofilm-associated cells in vitro and in vivo. Our results illustrate an association between Eap1p-mediated adhesion and biofilm formation in vitro and in vivo.

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

Endocrinology ◽  
2004 ◽  
Vol 145 (12) ◽  
pp. 5525-5531 ◽  
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 &lt; 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P &lt; 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.

Expression of the Saccharomyces cerevisiae GAL7 gene on autonomous plasmids

1984 ◽  
Vol 4 (10) ◽  
pp. 2062-2071
Author(s):  
S M Baker ◽  
P G Okkema ◽  
J A Jaehning
Keyword(s):  
Gene Dosage ◽  
Constitutive Expression ◽  
Single Copy ◽  
Initial Time ◽  
Sufficient Information ◽  
Mrna Levels ◽  
Dna Encoding ◽  
Yeast Plasmid ◽  
Regulatory Factors

We used a combination of cloned DNA fragments encoding the GAL7 gene, yeast plasmid vectors, and chromosomal gal7 deletions to characterize the in vivo transcription of the GAL7 gene on autonomously replicating plasmids. Our results demonstrated that a plasmid-borne 3.1-kilobase DNA fragment containing the GAL7 gene provides sufficient information to mimic the regulated expression of the chromosomal location. Normal expression of GAL7 could occur in the absence of DNA encoding the functional genes of the GAL cluster region and was not altered when the gene was adjacent to other plasmid elements such as autonomously replicating sequences or centromeres. The chromosomal and single-copy centromeric plasmid locations of GAL7 were indistinguishable in their response to growth conditions (induction by galactose, repression by glucose) and positive and negative regulatory factors (GAL4 and GAL80). Increasing the gene dosage to more than 200 copies per cell resulted in constitutive expression of the GAL7 mRNA; fully induced mRNA levels were increased more than 10-fold at these high gene dosages. When cells were shifted from noninducing to inducing conditions, the initial time of appearance and the rate of accumulation of GAL7 mRNA were altered in cell populations containing multiple GAL7 genes. The induction kinetics and final accumulation of the chromosomal GAL10 mRNA were also affected by the presence of multiple copies of the GAL7 gene; these results are consistent with a model involving limiting amounts of regulatory factors.

scholarly journals Tumor-Stroma Crosstalk Enhances REG3A Expressions that Drive the Progression of Hepatocellular Carcinoma

2020 ◽  
Vol 21 (2) ◽  
pp. 472 ◽  
Author(s):  
Yuri Cho ◽  
Min Ji Park ◽  
Koeun Kim ◽  
Jae-Young Park ◽  
Jihye Kim ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Microarray Analysis ◽  
Cdna Microarray ◽  
Tumor Stroma ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Cdna Microarray Analysis ◽  
Hcc Cells

Abstract: Background: Crosstalk between tumors and their microenvironment plays a crucial role in the progression of hepatocellular carcinoma (HCC). However, there is little existing information about the key signaling molecule that modulates tumor-stroma crosstalk. Methods: Complementary DNA (cDNA) microarray analysis was performed to identify the key molecule in tumor-stroma crosstalk. Subcutaneous xenograft in vivo murine model, immunoblotting, immunofluorescence, and real-time polymerase chain reaction using HCC cells and tissues were performed. Results: The key molecule, regenerating gene protein-3A (REG3A), was most significantly enhanced when coculturing HCC cells and activated human hepatic stellate cells (HSCs) (+8.2 log) compared with monoculturing HCC cells using cDNA microarray analysis. Downregulation of REG3A using small interfering RNA significantly decreased the proliferation of HSC-cocultured HCC cells in vitro and in vivo, and enhanced deoxycholic acid-induced HCC cell apoptosis. Crosstalk-induced REG3A upregulation was modulated by platelet-derived growth factor ββ (PDGF-ββ) in p42/44-dependent manner. REG3A mRNA levels in human HCC tissues were upregulated 1.8-fold compared with non-tumor tissues and positively correlated with PDGF-ββ levels. Conclusions: REG3A/p42/44 pathway/PDGF-ββ signaling plays a significant role in hepatocarcinogenesis via tumor-stroma crosstalk. Targeting REG3A is a potential novel therapeutic target for the management of HCCs by inhibiting crosstalk between HCC cells and HSCs.

scholarly journals Evaluation of Patched-1 Protein Expression Level in Low Risk and High Risk Basal Cell Carcinoma Subtypes

2019 ◽  
Vol 20 (9) ◽  
pp. 2851-2857 ◽  
Author(s):  
Rowida Almomani ◽  
Mariam Khanfar ◽  
Khaldon Bodoor ◽  
Firas Al-Qarqaz ◽  
Mohammad Alqudah ◽  
...  
Keyword(s):  
High Risk ◽  
Basal Cell Carcinoma ◽  
Protein Expression ◽  
Cell Carcinoma ◽  
Basal Cell ◽  
Low Risk ◽  
Expression Level ◽  
Protein Expression Level ◽  
Patched 1
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