scholarly journals Ubiquitination-activating enzymes UBE1 and UBA6 regulate ubiquitination and expression of cardiac sodium channel Nav1.5

2020 ◽  
Vol 477 (9) ◽  
pp. 1683-1700
Author(s):  
Yushuang Hu ◽  
Xuemei Bai ◽  
Chi Zhang ◽  
Susmita Chakrabarti ◽  
Bo Tang ◽  
...  
Keyword(s):  
Heart Failure ◽  
Western Blot ◽  
Sodium Channel ◽  
Current Density ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Sodium Current ◽  
Pcr Analysis ◽  
Protein Ubiquitination ◽  
Cardiac Sodium Channel

Cardiac sodium channel Nav1.5 is associated with cardiac arrhythmias and heart failure. Protein ubiquitination is catalyzed by an E1–E2–E3 cascade of enzymes. However, the E1 enzyme catalyzing Nav1.5 ubiquitination is unknown. Here, we show that UBE1 and UBA6 are two E1 enzymes regulating Nav1.5 ubiquitination and expression. Western blot analysis and patch-clamping recordings showed that overexpression of UBE1 or UBA6 increased the ubiquitination of Nav1.5 and significantly reduced Nav1.5 expression and sodium current density, and knockdown of UBE1 or UBA6 expression significantly increased Nav1.5 expression and sodium current density in HEK293/Nav1.5 cells. Similar results were obtained in neonatal cardiomyocytes. Bioinformatic analysis predicted two ubiquitination sites at K590 and K591. Mutations of K590 and K591 to K590A and K591A abolished the effects of overexpression or knockdown of UBE1 or UBA6 on Nav1.5 expression and sodium current density. Western blot analysis showed that the effects of UBE1 or UBA6 overexpression on the ubiquitination and expression of Nav1.5 were abolished by knockdown of UBC9, a putative E2 enzyme reported for Nav1.5 ubiquitination by us. Interestingly, real-time RT-PCR analysis showed that the expression level of UBE1, but not UBA6, was significantly up-regulated in ventricular tissues from heart failure patients. These data establish UBE1 and UBA6 as the E1 enzymes involved in Nav1.5 ubiquitination, and suggest that UBE1 and UBA6 regulate ubiquitination of Nav1.5 through UBC9. Our study is the first to reveal the regulatory role of the UBE1 or UBA6 E1 enzyme in the ubiquitination of an ion channel and links UBE1 up-regulation to heart failure.

Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

2010 ◽  
Vol 299 (5) ◽  
pp. R1290-R1297 ◽  
Author(s):  
E. Zhao ◽  
Caleb L. Grey ◽  
Dapeng Zhang ◽  
Jan A. Mennigen ◽  
Ajoy Basak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Pcr Analysis ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Lh Release

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins ( R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression ( R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca2+ concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

Abstract 65: Desmin Preamyloid Oligomers in Experimental Heart Failure and Cultured Cardiac Myocytes

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Peter Rainer ◽  
Dong I Lee ◽  
Matteo Sorge ◽  
Carlo Guarnieri ◽  
Charles G Glabe ◽  
...  
Keyword(s):  
Heart Failure ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Molecular Mechanisms ◽  
Ventricular Myocytes ◽  
Preliminary Evidence ◽  
Cardiac Cells ◽  
Neonatal Rat ◽  
Genetic Mutations

Background: Heart Failure (HF) is one of the main causes of morbidity and mortality in westernized countries but the molecular mechanisms underlying its development are still unclear. A paradigm-shifting view focuses on the accumulation of preamyloid oligomers (PAOs), similar to those observed in Alzheimer disease, as a potential mechanism of cardiac toxicity. We reported that differential desmin phosphorylation at serines (S) 27 and 31 could drive the formation of PAOs in the heart, in the absence of genetic mutations. We sought to establish the identity of the molecular seed triggering the nucleation of cardiac PAOs in an experimental model of HF and in cultured cardiac cells. Methods: Mice were subjected to transverse aortic constriction (TAC) for 4 weeks (FS% = 29.3±2.6, P=0.0001). Alternatively, neonatal rat ventricular myocytes were transduced with lentiviral vectors carrying alanine (A) or phospho-mimetic aspartate (D) desmin double mutants at S27 and S31, fused with GFP. Protein homogenates were subjected to western blot analysis with fluorescent co-staining using the A11 anti-PAOs and anti-desmin antibodies. Transduced cells were also subjected to live imaging to assess phenotype. Results: Co-western blot analysis of both TAC mice and phospho-mimetic mutant cells revealed the colocalization of PAOs with desmin modified (potentially cleaved) forms. Preamyloid oligomers and a desmin fragment were both increased in TAC mice vs. controls (2.8-fold, P=0.023 and 1.8-fold, P=0.038, respectively). The DD mutant, mimicking the doubly phosphorylated desmin that we hypothesized is the physiological form, showed a healthier phenotype in terms of number of spontaneously contracting cells (P=0.041) and incorporation of GFP-desmin at the Z-discs (P=0.0027), whereas the mono-phosphomimetic mutant (AD) resulted in the increase of desmin positive aggregates (P=0.0014). Conclusions: This preliminary evidence suggests that desmin modified forms represent the seed triggering the formation of cardiac PAOs, in the absence of genetic mutations. The accumulation of desmin PAOs could therefore represent an overarching mechanism underlying the deterioration of cardiac function in HF.

scholarly journals MiR-221/222 Promote Dexamethasone Resistance of Multiple Myeloma through Inhibition of Autophagy By Targeting ATG12

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4469-4469
Author(s):  
Jian Xu ◽  
Yan Su ◽  
Aoshuang Xu ◽  
Fengjuan Fan ◽  
Haifan Huang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Conflicts Of Interest ◽  
Combined Treatment ◽  
Luciferase Reporter ◽  
Pcr Analysis ◽  
Expression Levels

Abstract Dexamethasone (Dex) is the most widely used chemotherapeutic drug in the treatment of multiple myeloma (MM). Inherent or acquired resistance to Dex is broadly associated with poor prognosis in MM. Many microRNAs are aberrantly expressed in MM, including miR-221/222, which have been reported to act as oncogenes in many cancer types. Recently, accumulating evidence has shown that miR-221/222 are involved in the development of chemoresistance in a variety of cancers. However, there is still a lack of valuable data regarding the role of miR-221/222 in the chemoresistance of MM. Here, we first evaluated the expression levels of miR-221/222 in plasma cells (PCs) from MM patients by qRT-PCR analysis. The results showed that miR-221/222 were markedly upregulated in PCs from newly diagnosed MM patients compared to healthy donors, and they were further upregulated in PCs from patients with relapsed MM. In addition, we found that the expression levels of miR-221/222 were inversely correlated with Dex-sensitivity of human MM cell lines (HMCLs). Importantly, enforced expression of miR-221/222 dramatically reduced the sensitivity of Dex-sensitive HMCLs to Dex, while inhibition of miR-221/222 re-sensitized Dex-resistant HMCLs to Dex. Previous studies have shown that Dex-induced cell death in lymphoid leukemia is mediated through initiation of autophagy. To study whether autophagy was involved in Dex-induced cell death in MM cells, HMCLs were exposed to Dex, and then autophagy in these cells was evaluated by the transmission electron microscopy and western blot analysis. The results showed that Dex induced the occurrence of autophagy in Dex-sensitive HMCLs, but not in Dex-resistant HMCLs. Moreover, pharmacological inhibitors of autophagy could significantly reduce Dex-induced cell death in Dex-sensitive HMCLs. These results reveal that autophagy is critical for the induction of cell death following Dex treatment in MM. MicroRNAs have been reported to play an important role in regulating autophagy. We therefore examined whether miR-221/222 can regulate autophagy in MM cells. Low miR-221/222 expressing MM.1S (Dex-sensitive) or high miR-221/222 expressing MM.1R (Dex-resistant) cells were transfected with agomir-221/222 or antagomir-221/222, respectively, and then the level of autophagy was evaluated. The results showed that overexpression of miR-221/222 reduced the level of autophagy in MM.1S cells, while inhibition of miR-221/222 elevated the level of autophagy in MM.1R cells. Using microRNA target prediction bioinformatics tools and dual-luciferase reporter assay, we confirmed that autophagy-related gene 12 (ATG12) was a novel target gene of miR-221/222. Indeed, miR-221/222 could negatively regulate the expression of ATG12 at both the mRNA and protein levels in MM cells. In addition, knockdown of ATG12 by siRNA markedly reduced the autophagy-inducing and Dex-sensitizing activity of miR-221/222 antagomirs in MM.1R cells. Of note, in MM.1S cells, Dex treatment could further decreased the expression of miR-221/222, accompanied by upregulated expression of ATG12, whereas silencing the expression of ATG12 could significantly inhibited Dex-induced autophagy and cell death. Thus, these results suggest that ATG12 is a key player in miR-221/222-mediated autophagy inhibition and Dex-resistance. Next, we evaluated whether miR-221/222 could regulate autophagy and Dex-sensitivity of MM cells invivo. NOD/SCID mice were subcutaneously injected with MM.1R cells to establish Dex-resistant MM xenografts. Combined treatment with antagomir-221/222 plus Dex showed a remarkable reduction of tumor size compared to antagomir-221/222 or Dex alone (397.6±55.08 mm3 VS 895.8±72.44 mm3 VS 987.3±68.49 mm3). Immunohistochemistry and western blot analysis of the retrieved xenografted tumors showed that combination treatment with antagomir-221/222 plus Dex induced upregulation of ATG12, as well as extended autophagy with increased p62 degradation and Beclin-1 expression. In conclusion, our data reveal that upregulation of miR-221/222 promotes Dex resistance of MM cells through inhibition of autophagy by targeting ATG12. Therefore, miR-221/222-ATG12 autophagy-regulatory axis may potentially be applied in glucocorticoid resistance prediction and treatment. Disclosures No relevant conflicts of interest to declare.

Ranolazine may be the Best and Safest Pharmacologic Therapy For Congestive Heart Failure, And Safe, Effective For Ventricular And Atrial Arrhythmias

2020 ◽  
Vol 1 (1) ◽  
Author(s):  
Gary L Murray
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Sodium Channel ◽  
Sodium Current ◽  
Left Ventricular ◽  
Left Ventricular Ejection ◽  
Pharmacologic Therapy ◽  
Ventricular Ejection Fraction ◽  
Late Sodium Current ◽  
Cardiac Sodium Channel

Background:Ranolazine (RAN) reduces cardiac sodium channel 1.5’s late sodium current(INaL ) in congestive heart failure (CHF), reducing myocardial calcium overload, potentially improving left ventricular ejection fraction(LVEF) and reducing arrhyth-mogenic after potentials. RAN blocks neuronal sodium channel 1.7(Nav 1.7), potentially altering parasympathetic and sympathetic (P&S) activity. RAN also selectively blocks inactivated atrial Nav 1.8, as well as ventricular IKr and ICaL ,affecting atrial and ventric-ular arrhythmias.

scholarly journals LYRM1, a novel gene promotes proliferation and inhibits apoptosis of preadipocytes

2009 ◽  
Vol 160 (2) ◽  
pp. 177-184 ◽  
Author(s):  
Jie Qiu ◽  
Chun-Lin Gao ◽  
Min Zhang ◽  
Rong-Hua Chen ◽  
Xia Chi ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Fluorescent Protein ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Omental Adipose Tissue ◽  
Novel Gene ◽  
Obese Subjects

ObjectiveTo characterize a novel gene, Homo sapiens LYR motif containing 1 (LYRM1), that is highly expressed in omental adipose tissue of obese subjects.Methods and resultsRT-PCR and western blot analysis confirmed that both mRNA and protein levels of LYRM1 were higher in omental adipose tissue of obese subjects than in normal weight subjects. RT-PCR analysis demonstrated that LYRM1 expression is widely distributed, with the highest levels of expression occurring in adipose tissue. A fusion protein of LYRM1 and green fluorescent protein as well as western blot analysis were used to identify the subcellular localization of LYRM1 in the nucleus. Based on Oil red O staining and the expression profile of specific differentiation markers, ectopic LYRM1 expression was not found to significantly affect adipogenesis. MTT assays and cell cycle analysis showed that LYRM1 promotes preadipocyte proliferation, and data from annexin V-FITC and caspase-3 activity assays further determined that LYRM1 can inhibit apoptosis of preadipocytes.ConclusionsBy increasing cell proliferation and lowering the rate of apoptosis, LYRM1 has the potential to modulate the size of the preadipocyte pool and influence adipose tissue homeostasis.

scholarly journals Antiviral activity of mitoxantrone dihydrochloride against human herpes simplex virus mediated by suppression of the viral immediate early genes

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Qiang Huang ◽  
Jue Hou ◽  
Peng Yang ◽  
Jun Yan ◽  
Xiaoliang Yu ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immediate Early Genes ◽  
Immediate Early ◽  
Pcr Analysis ◽  
Viral Gene ◽  
Chemical Library ◽  
Early Genes ◽  
Hsv 1

Abstract Background HSV-1 is a common pathogen that infects 50–90% of the human population worldwide. HSV-1 causes numerous infection-related diseases, some of which are severely life-threatening. There are antiviral medications with activity against HSV-1. However, with the emergence of drug-resistant mutant strains of HSV-1, there is an urgent need to develop new effective anti-HSV-1 agents. Methods Therefore, we screened a chemical library of approximately 1500 compounds to identify inhibitors of HSV-1-induced toxicity for further drug development. Moreover, we performed several experiments, including western blot analysis, Q-PCR analysis and luciferase activity assay, to explore the antiviral mechanism of the candidates. Results Here, we identified a small molecule, mitoxantrone dihydrochloride, with potency against HSV-1-induced toxicity. Furthermore, the viral titers and expression levels of HSV-1 viral proteins were potently reduced by the presence of MD in many cell lines. Using Q-PCR analysis, we found that MD efficiently reduced the transcription of viral genes that are essential for DNA synthesis, namely, UL5, UL9, UL29, UL30, UL42 and UL52. Notably, MD also significantly inhibited the transcription of the immediate early genes ICP0, ICP22, ICP27 and ICP47, all of which are required for the expression of early and late viral gene products. Using immunofluorescence and western blot analysis, we found that the antiviral effect of MD was independent of the activation of the NF-κB and MAPK pathways. Furthermore, we found that the reduction in the transcription of viral immediate early genes was not related to the promoter activities of ICP0. Conclusions Therefore, the identification of compound MD as an inhibitor of toxicity induced by HSV-1 highlights its potential use in the development of novel anti-HSV-1 drugs.

scholarly journals UBC9 regulates cardiac sodium channel Nav1.5 ubiquitination, degradation and sodium current density

2019 ◽  
Vol 129 ◽  
pp. 79-91 ◽  
Author(s):  
Bo Tang ◽  
Yushuang Hu ◽  
Zhijie Wang ◽  
Chen Cheng ◽  
Pengyun Wang ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Current Density ◽  
Sodium Current ◽  
Cardiac Sodium Channel

Voltage-gated sodium channel Nav1.5 promotes proliferation, migration and invasion of oral squamous cell carcinoma

2019 ◽  
Vol 51 (6) ◽  
pp. 562-570 ◽  
Author(s):  
Jie Zhang ◽  
Weijia Mao ◽  
Yongzheng Dai ◽  
Chengwei Qian ◽  
Yang Dong ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Oral Squamous Cell Carcinoma ◽  
Western Blot ◽  
Sodium Channel ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Migration And Invasion ◽  
Voltage Gated

Abstract The protein voltage-gated sodium channel Nav1.5 is highly upregulated in various types of cancer and, in general, promotes cancer cell invasiveness and metastatic progression. A previous study found that Nav1.5 was highly expressed in poorly differentiated oral squamous cell carcinoma (OSCC). However, whether Nav1.5 enhances invasiveness and metastasis of OSCC are still unknown. In this study, we found that Nav1.5 was highly expressed in OSCC cell lines compared with normal oral keratinocyte HOK cell line by using western blot analysis. CCK-8 assay results revealed that downregulation of Nav1.5 expression by its specific siRNA reduced proliferation of OSCC HSC-3 cells. Moreover, transwell assay results showed Nav1.5 knockdown significantly inhibited migration and invasion of HSC-3 cells. Meanwhile, qRT-PCR and western blot analysis results showed that epidermal growth factor (EGF) induced Nav1.5 expression in a time- and dose-dependent manner. In addition, EGF promoted proliferation, migration and invasion of HSC-3 cells. Importantly, the Nav1.5 inhibitor tetrodotoxin significantly inhibited the proliferation of HSC-3 cells and impeded the migration and invasion of HSC-3 cells. Furthermore, it was found that siRNA-mediated knockdown of Nav1.5 also lessened the proliferation of HSC-3 cells and blocked the migration and invasion of HSC-3 cells. Taken together, these results indicate that Nav1.5 is involved in the progression of OSCC and Nav1.5 promotes the proliferation, migration and invasion of OSCC cells.

scholarly journals Mixed Virus Infections of Garlic Determined by a Multivalent Polyclonal Antiserum and Virus Effects on Disease Symptoms

Plant Disease ◽  
2001 ◽  
Vol 85 (1) ◽  
pp. 71-75 ◽  
Author(s):  
Miyuki Takaichi ◽  
Takayuki Nagakubo ◽  
Kenji Oeda
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Severe Disease ◽  
Polyclonal Antiserum ◽  
Onion Yellow Dwarf Virus ◽  
Pcr Analysis ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Disease Symptoms

A garlic virus-specific polyclonal antiserum was developed against a mixture of flexuous rodshaped virus particles isolated from mosaic-diseased garlic plants (15). This antiserum was used in Western blot analysis against tissues from mosaic-diseased garlic plants, at least seven viral coat protein (CP) bands (from 38 to 32 kDa) were identified. Using Western blot analysis with Potyvirus-specific antibodies and reverse transcription-polymerase chain reaction (RT-PCR) analysis, we concluded that three of the seven bands corresponded to CPs of Leek yellow stripe virus (LYSV) (38 kDa) and two different Onion yellow dwarf virus (OYDV) strains (35.5 or 34 kDa). The 35 kDa band corresponded to the CP of GV1-Carlavirus, and the other four bands, 36, 35 (not GV1), 33, and 32 kDa, were identified as the CPs of four mite-borne viruses, based on RT-PCR analysis. Based on the molecular weights of CP, mixed infections of Potyvirus, Carlavirus, and mite-borne viruses were characterized. LYSV causes apparent disease symptoms in garlic plants, however, little reduction in bulb weights. Conversely, garlic plants infected with three different mite-borne viruses expressed weak symptoms and yield losses. Mixed infections of OYDV, the mite-borne viruses, and LYSV caused severe disease symptoms and considerable reduction of bulb weights.

scholarly journals Myocardial distribution and regulation of GRK and β-arrestin isoforms in congestive heart failure in rats

2001 ◽  
Vol 281 (6) ◽  
pp. H2490-H2499 ◽  
Author(s):  
Leif Erik Vinge ◽  
Erik Øie ◽  
Yvonne Andersson ◽  
Haakon K. Grøgaard ◽  
Geir Ø. Andersen ◽  
...  
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunohistochemical Analysis ◽  
Myocardial Tissue ◽  
Sham Operation ◽  
Cellular Distribution ◽  
Mrna Levels

Myocardial G protein-coupled receptor kinase 2 (GRK2) has been shown to be involved in the pathophysiology of congestive heart failure (CHF). However, the cellular distribution of this isoform, as well as the other isoforms of the GRK-arrestin system, has not been studied in myocardial tissue. Thus myocardial expression and cellular distribution of the different GRK and arrestin isoforms were investigated in a rat model of CHF. Rats subjected to ligation of the left coronary artery or sham operation were euthanized 2, 7, or 42 days after the surgical procedure. Myocardial GRK2, GRK5, β-arrestin-1, and β-arrestin-2 mRNA levels, but not that of GRK3, were induced in the failing hearts. Consistently, Western blot analysis of tissue extracts from the nonischemic region of the left ventricle revealed 3.0-, 2.6-, and 1.5-fold elevations of GRK2, GRK5, and β-arrestin-1, respectively, 7 days after induction of myocardial infarction compared with the sham-operated rats ( P < 0.05). Immunohistochemical analysis of myocardial tissue sections and Western blot analysis of isolated cells revealed localization of GRK2 and β-arrestin-1 predominantly in endothelial cells. Conversely, GRK3 was confined to cardiac myocytes. GRK5 immunostaining appeared to be homogeneously distributed in the cellular elements of the myocardium. In conclusion, myocardial mRNA and protein levels of GRK2, GRK5, and β-arrestin-1 are induced in postinfarction failure in rats. The immunohistochemical analysis suggests that GRK2 and β-arrestin-1 may act as primary regulators of endothelial function. Conversely, the cellular distribution of GRK3 and GRK5 implicates these isoforms as putative regulators of cardiac myocyte function.

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