scholarly journals Stimulation of iodide uptake by human chorionic gonadotropin in FRTL-5 cells: effects on sodium/iodide symporter gene and protein expression

2002 ◽  
pp. 655-661 ◽  
Author(s):  
F Arturi ◽  
I Presta ◽  
D Scarpelli ◽  
JM Bidart ◽  
M Schlumberger ◽  
...  
Keyword(s):  
Western Blot ◽  
Chorionic Gonadotropin ◽  
Human Chorionic Gonadotropin ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Iodide Uptake ◽  
Protein Levels

BACKGROUND: Various clinical and experimental findings support the concept that human chorionic gonadotropin (hCG) can stimulate iodide uptake in thyroid cells. DESIGN: We investigated the molecular mechanisms underlying the effects of hCG on iodide uptake, and particularly its action on the expression of Na+/I- symporter (NIS) mRNA and protein. METHODS: Iodide uptake was analyzed in FTRL-5 cells by measuring (125)I concentrations in cells after a 30-min exposure to 0.1 microCi carrier-free Na (125)I in the presence or absence of hCG or, for control purposes, TSH. Expression of NIS mRNA and NIS protein synthesis were evaluated, respectively, with a semiquantitative 'multiplex' RT-PCR method and Western blot analysis. RESULTS: Iodide uptake was increased by hCG in a dose- and time-dependent manner: maximal effects were observed after 72 h of stimulation. The effect was cAMP dependent and paralleled that of TSH, although it lacked the early cycloheximide-independent component seen with TSH, and its peak effect was lower. Semiquantitative multiplex RT-PCR revealed that hCG produced a significant increase in NIS mRNA levels that was detectable after 4 h and peaked after 48 h. In contrast, in TSH-stimulated FRTL-5 cells, maximum NIS mRNA expression was observed after 24 h of stimulation. Western blot analysis demonstrated that hCG also caused a 2.5-fold increase over basal values in NIS protein levels, which was similar to that observed after TSH stimulation although the peak effects of the latter hormone were less marked and occurred earlier. CONCLUSION: Our data demonstrated that hCG stimulates iodide uptake in FRTL-5 cells by increasing NIS mRNA and protein levels. Thus, the functional status of the thyroid may be influenced by hCG-dependent changes in NIS expression occurring during pregnancy.

scholarly journals Investigation into imbalance of Th1/Th2 cells in cirrhotic, hypersplenic rats

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051988944 ◽  
Author(s):  
Yunfu Lv ◽  
Yejuan Li ◽  
Ning Liu ◽  
Yonghong Dong ◽  
Jie Deng
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemokine Receptors ◽  
Immunohistochemical Staining ◽  
Th2 Cells ◽  
Intragastric Administration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chemokine Receptor Ccr3

Objectives To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3. Methods Experimental liver cirrhosis and hypersplenism were induced in rats by the intragastric administration of carbon tetrachloride (CCl4; 40% solution [0.3 ml/100g, twice/week for 8 weeks]) and confirmed by pathology and hemogram. Presence of the three chemokine receptors was investigated by real-time polymerase chain reaction (RT-PCR), immunohistochemical staining, and western blot analysis. Results By comparison with control animals (n=10), RT-PCR demonstrated that CXCR3 and CCR5-mRNA levels were significantly elevated in the hypersplenic rats (n=26) and CCR3-mRNA levels were lower. Immunohistochemical staining showed that by comparison with controls, the mean density of the Th1-associated CXCR3 and CCR5 receptors was significantly increased but there was no difference between groups in Th2-associated CCR3 receptors. Western blot analysis showed that by comparison with controls, hypersplenic rats had higher levels of CXCR3 and CCR5 protein but lower levels of CCR3 protein. Conclusions The abnormal expression of Th1-associated chemokine receptors in spleens of rats with cirrhosis and hypersplenism induced by CCL4 suggests that a functional imbalance between Th1/Th2 cells may play a role in the pathogenesis of hypersplenism.

scholarly journals Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4318-4318 ◽  
Author(s):  
Julian Baumeister ◽  
Nicolas Chatain ◽  
Annika Hubrich ◽  
Caroline Küstermann ◽  
Stephanie Sontag ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Protein Levels

Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

scholarly journals FGF21 Ameliorates Hepatic Fibrosis by Multiple Mechanisms

2021 ◽  
Author(s):  
Fanrui Meng ◽  
Mir Hassan Khoso ◽  
Kai Kang ◽  
Qi He ◽  
Yukai Cao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Hepatic Fibrosis ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Model ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Mrna And Protein Expression ◽  
Dose Dependent

Abstract Previous study reports that FGF21 could ameliorate hepatic fibrosis, but its mechanisms have not been fully investigated. In this study, three models were used to investigate the mechanism by which FGF21 alleviates liver fibrosis. CCL4 and DMN were respectively used to induce hepatic fibrosis animal models. Our results demonstrated that liver index and liver function were deteriorated in both models. HE and Masson’s staining showed that the damaged tissue architectonics were observed in the mice of both models. Treatment with FGF21 significantly ameliorated these changes. ELISA analysis showed that the serum levels of IL-1β, IL-6 and TNF-α were significantly elevated in both models. However, administration of FGF21 significantly reduced these inflammatory cytokines. RT-PCR and Western blot analysis showed that mRNA and protein expression of collagenI, α-SMA and TGF-β were significantly decreased by treatment with FGF21. PDGF-BB stimulant was used to establish the experimental cell model in HSCs. RT-PCR and Western blot analysis demonstrated that the expression of collagenI and α-SMA were significantly upregulated by this stimulant in model group. Interestingly, our results showed that mRNA and protein expression of leptin were also significantly induced in PDGF-BB treated HSCs. Administration of FGF21 could significantly reduce leptin expression in a dose dependent manner and these effects were reversed in siRNA (against β-klotho) transfected HSCs. Furthermore, the leptin signaling pathways related protein p-ERK/t-ERK, p-STAT3/STAT3 and TGF-β were significantly downregulated by FGF21 treatment in a dose dependent manner. The expression of SOCS3 and Nrf-2 were enhanced by treatment with FGF21. The underlying mechanism may be that FGF21 regulates leptin-STAT3 axis via Nrf-2 and SOCS3 pathway in activated HSCs.

Regional time-dependent changes in vasopressin V2 receptor expression in the rat kidney during water restriction

1998 ◽  
Vol 274 (5) ◽  
pp. F906-F913 ◽  
Author(s):  
Frank Park ◽  
George Koike ◽  
Allen W. Cowley
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Receptor Expression ◽  
Time Dependent ◽  
Dependent Manner ◽  
Water Restriction ◽  
Rt Pcr

Elevations of arginine vasopressin (AVP) binding to renal vasopressin V2 receptors (V2R) enhance water and urea reabsorption in the collecting duct epithelium. This study was designed to quantify the levels of V2R mRNA and protein within the distinct regions of the Sprague-Dawley rat kidney (i.e., the cortex and outer and inner medulla) during 24 and 48 h of water restriction. A competitive reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to quantify changes in the V2R mRNA, in which a deletion mutant RNA transcript was used to control for the efficiency of RT-PCR. Western blot analysis was utilized for the quantification of the V2R protein. The results showed that the steady-state levels of the V2R mRNA decreased in a time-dependent manner in the cortex and outer and inner medulla throughout 48 h of water restriction. Western blot analysis revealed that the V2R protein in the renal cortex decreased after the initial 24 h of water restriction and remained decreased at 48 h. In contrast, outer medullary V2R protein decreased significantly only after 48 h of water restriction, whereas no significant change in the inner medullary V2R protein was observed throughout the 48 h of water restriction. These results suggest that water restriction leads to a regional time-dependent downregulation of the V2R mRNA and protein within the rat kidney. The stability of the plasma membrane V2R protein within the inner medulla may allow for the optimization of urine concentration and minimize water loss during periods of water restriction.

scholarly journals Effects of Electroacupuncture on Ovarian Expression of the Androgen Receptor and Connexin 43 in Rats with Letrozole-Induced Polycystic Ovaries

2020 ◽  
Vol 2020 ◽  
pp. 1-13
Author(s):  
Ge Xu ◽  
Andong Zhang ◽  
Jiandang Liu ◽  
Xi Wang ◽  
Jiwei Feng ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Connexin 43 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Endocrine Dysfunction ◽  
Rt Pcr

Background. Polycystic ovarian syndrome (PCOS) occurs in women of reproductive age and is often characterized by reproductive and endocrine dysfunction. Androgens play a major role in PCOS, and previous studies reported abnormal expression of Connexin 43 (Cx43) in animal models of PCOS, suggesting an association of Cx43 with PCOS pathogenesis. Experimental and clinical evidence indicated that acupuncture may be a safe and effective approach for treating reproductive and endocrine disorders in women with PCOS. This study aimed to determine the effects of electroacupuncture (EA) on PCOS and its relationship with the expression of the androgen receptor (AR) and Cx43. Methods. In total, 30 female Sprague Dawley rats (6 weeks old) were randomly divided into three groups: control group, letrozole (LE) group, and LE + EA group. Rats were administered LE solution (1.0 mg/kg) for 21 consecutive days to induce PCOS. For the LE + EA group, additional EA treatment was conducted (2 Hz, 20 min/d) with “Guanyuan” (CV3) for 14 consecutive days. After hematoxylin-eosin staining, the ovarian structure was observed with an optical microscope, and serum levels of the following hormones were examined via enzyme-linked immunosorbent assay (ELISA): testosterone (T), estradiol (E2), sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH); luteinizing hormone (LH), insulin (INS), anti-Müllerian hormone (AMH), and inhibin B (INHB). Fasting blood glucose (FBG) levels were evaluated using glucose oxidase-peroxidase. Ovarian mRNA and protein expressions of AR and Cx43 were determined by real-time RT-PCR and Western blot analysis. Results. EA was found to restore the cyclicity and ovarian morphology in the PCOS rat model. Serum derived from the LE + EA group showed significant decreases in the levels of T, free androgen index (FAI), LH, LH/FSH ratio, AMH, INHB, and fasting serum insulin (FINS), and significant increases in the levels of E2, FSH, and SHBG. Western blot analysis showed a decreased protein expression of ovarian AR and Cx43; real-time RT-PCR showed reduced expression of ovarian mRNA levels of AR and Cx43. Conclusions. In conclusion, our results showed that EA can ease hyperandrogenism and polycystic ovary morphology in PCOS rats. Furthermore, EA counteracted the letrozole-induced upregulation of AR and Cx43. These results suggested that acupuncture can break the vicious cycle initiated by excessive androgen secretion and may be an effective treatment method for improving the reproductive and endocrine dysfunction caused by PCOS.

Visfatin facilitates gastric cancer malignancy by targeting snai1 via the NF-κB signaling

2021 ◽  
pp. 096032712110061
Author(s):  
D Cao ◽  
L Chu ◽  
Z Xu ◽  
J Gong ◽  
R Deng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Epithelial Mesenchymal Transition ◽  
Migration And Invasion ◽  
Mesenchymal Transition ◽  
Cell Migration And Invasion ◽  
Protein Levels

Background: Visfatin acts as an oncogenic factor in numerous tumors through a variety of cellular processes. Visfatin has been revealed to promote cell migration and invasion in gastric cancer (GC). Snai1 is a well-known regulator of EMT process in cancers. However, the relationship between visfatin and snai1 in GC remains unclear. The current study aimed to explore the role of visfatin in GC. Methods: The RT-qPCR and western blot analysis were used to measure RNA and protein levels, respectively. The cell migration and invasion were tested by Trans-well assays and western blot analysis. Results: Visfatin showed upregulation in GC cells. Additionally, Visfatin with increasing concentration facilitated epithelial-mesenchymal transition (EMT) process by increasing E-cadherin and reducing N-cadherin and Vimentin protein levels in GC cells. Moreover, endogenous overexpression and knockdown of visfatin promoted and inhibited migratory and invasive abilities of GC cells, respectively. Then, we found that snai1 protein level was positively regulated by visfatin in GC cells. In addition, visfatin activated the NF-κB signaling to modulate snai1 protein expression. Furthermore, the silencing of snai1 counteracted the promotive impact of visfatin on cell migration, invasion and EMT process in GC. Conclusion: Visfatin facilitates cell migration, invasion and EMT process by targeting snai1 via the NF-κB signaling, which provides a potential insight for the treatment of GC.

Abstract 2671: Thymosin Beta 4 Induced Oligodendrogenesis Is Associated With Upregulation Of Serum Response Factor

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Daniel C Morris ◽  
Benjamin Buller ◽  
Manoranjan Santra ◽  
Michael Chopp ◽  
Zheng Gang Zhang
Keyword(s):  
Western Blot ◽  
Progenitor Cells ◽  
Rat Model ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Embolic Stroke ◽  
Early Gene ◽  
Dependent Manner ◽  
Thymosin Beta 4 ◽  
Serum Response

Background: Thymosin beta 4 (Tβ4) is a G-actin sequestering peptide that improves neurological functional outcome when administered 24 hours after onset of stroke to a rat model of embolic stroke. Tβ4 increases the number of oligodendrocyte progenitor cells (OPCs) as well as mature oligodendrocytes (OLs). Mechanisms of Tβ4 induced oligodendrogenesis (OLG) remain unclear. Serum response growth factor (SRF) is a transcriptional factor which binds with ternary complex co-factors to primarily convey an immediate early gene response to influence and orchestrate neuronal migration and differentiation. Hypothesis: We tested the hypothesis that Tβ4 upregulates SRF with subsequent increase in the markers of OL differentiation. Results: We employed a mouse OPC line (N20.1) to investigate the mechanisms of Tβ4-induced OLG. The cells were plated at a density of 100,000 cells/ml and grown in the presence of 0, 12.5, 25 and 50 ng/ml of Tβ4 (RegeneRx Biopharmaceuticals, Inc.) for 14 days (n=3). Western blot analysis revealed that SRF was dose-dependently upregulated by a factor of 4. Quantitative real time PCR and Western blot analysis showed that Tβ4 treatment induced myelin basic protein (MBP) and 2’, 3’-cyclic nucleotide, 3’-phosphodiesterase (CNPase) expression in a dose-dependent manner by ∼2 fold, indicating the stimulation of OLG. In order to independently demonstrate that SRF promotes the differentiation of progenitor cells into mature oligodendrocytes, SRF was over expressed in the N20.1 cells using a plasmid encoding the SRF gene. After six days SRF over expressed N20.1 cells (n=3) demonstrated an increase of expression of MBP (26 ± 3%) and CNPase (23 ± 3%) when compared to cells transfected with an empty expression plasmid (n=3, MBP, 14 ± 3% and CNPase, 10 ± 4%, p<0.05). Conclusions: In this mouse model of OPCs, SRF was upregulated by Tβ4 and may be involved in Tβ4 induced OLG. Further in vivo investigation of SRF is warranted in our rat model of embolic stroke.

scholarly journals NHE1, NHE2, and NHE3 contribute to regulation of intracellular pH in murine duodenal epithelial cells

2000 ◽  
Vol 278 (2) ◽  
pp. G197-G206 ◽  
Author(s):  
J. Praetorius ◽  
D. Andreasen ◽  
B. L. Jensen ◽  
M. A. Ainsworth ◽  
U. G. Friis ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Intracellular Ph ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mrna Level ◽  
Rt Pcr ◽  
Acid Extrusion ◽  
Molecular Expression ◽  
Nhe Isoforms

Na+/H+-exchangers (NHE) mediate acid extrusion from duodenal epithelial cells, but the isoforms involved have not previously been determined. Thus we investigated 1) the contribution of Na+-dependent processes to acid extrusion, 2) sensitivity to Na+/H+ exchange inhibitors, and 3) molecular expression of NHE isoforms. By fluorescence spectroscopy the recovery of intracellular pH (pHi) was measured on suspensions of isolated acidified murine duodenal epithelial cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Expression of NHE isoforms was studied by RT-PCR and Western blot analysis. Reduction of extracellular Na+ concentration ([Na+]o) during pHirecovery decreased H+ efflux to minimally 12.5% of control with a relatively high apparent Michaelis constant for extracellular Na+. The Na+/H+exchange inhibitors ethylisopropylamiloride and amiloride inhibited H+ efflux maximally by 57 and 80%, respectively. NHE1, NHE2, and NHE3 were expressed at the mRNA level (RT-PCR) as well as at the protein level (Western blot analysis). On the basis of the effects of low [Na+]o and inhibitors we propose that acid extrusion in duodenal epithelial cells involves Na+/H+ exchange by isoforms NHE1, NHE2, and NHE3.

scholarly journals Integrin β3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway

2018 ◽  
Vol 49 (3) ◽  
pp. 985-997 ◽  
Author(s):  
Weisen Wang ◽  
Zhi Wang ◽  
Dingyuan Tian ◽  
Xi Zeng ◽  
Yangdong Liu ◽  
...  
Keyword(s):  
Western Blot ◽  
Notch Signaling ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Notch Signaling Pathway ◽  
Rt Pcr ◽  
Mesenchymal Transition ◽  
Integrin Β3 ◽  
Endothelial To Mesenchymal Transition ◽  
Tgf Β1

Background/Aims: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin β3 can modulate the EndMT, as well as its underlying mechanism. Methods: Integrin β3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. Results: Transforming growth factor (TGF)-β1 treatment or integrin β3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin β3 reversed these effects. Notch signaling was activated after TGF-β1 treatment of HUVECs. Knockdown of integrin β3 suppressed TGF-β1-induced Notch activation and expression of the Notch downstream target FSP-1. Conclusion: Integrin β3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.

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