scholarly journals TGF-β Signaling Cooperates with AT Motif-Binding Factor-1 for Repression of the α-Fetoprotein Promoter

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Nobuo Sakata ◽  
Satoshi Kaneko ◽  
Souichi Ikeno ◽  
Yutaka Miura ◽  
Hidekazu Nakabayashi ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Inhibitory Action ◽  
Fetal Liver ◽  
Direct Interaction ◽  
Detectable Level ◽  
Dependent Manner ◽  
Reporter System ◽  
Adult Liver ◽  
Binding Factor ◽  
Dose Dependent

α-Fetoprotein (AFP) is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver. On the other hand, hepatocellular carcinoma often shows high expression of AFP. Thus, AFP seems to be an oncogenic marker. In our present study, we investigated how TGF-β signaling cooperates with AT motif-binding factor-1 (ATBF1) to inhibit AFP transcription. Indeed, the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-β signaling. To further understand how TGF-β suppresses the transcription of the AFP gene, we analyzed the activity of the AFP promoter in the presence of TGF-β. We found that the TGF-β signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements (AT-motifs). Using a heterologous reporter system, both AT-motifs were required for transcriptional repression upon TGF-β stimulation. Furthermore, Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions. Since the N-terminal (ATBF1N) and C-terminal regions of ATBF1 (ATBF1C) lack the ability of DNA binding, both truncated mutants rescued the cooperative inhibitory action by the TGF-β signaling and ATBF1 in a dose-dependent manner. Taken together, these findings indicate that TGF-β signaling can act in concert with ATBF1 to suppress the activity of the AFP promoter through direct interaction of ATBF1 with Smads.

scholarly journals A Mechano-Activated Cell Reporter System as a Proxy for Flow-Dependent Endothelial Atheroprotection

2018 ◽  
Vol 23 (8) ◽  
pp. 869-876
Author(s):  
Bendix R. Slegtenhorst ◽  
Oscar R. Fajardo Ramirez ◽  
Yuzhi Zhang ◽  
Zahra Dhanerawala ◽  
Stefan G. Tullius ◽  
...  
Keyword(s):  
Vascular Endothelium ◽  
Critical Role ◽  
Green Fluorescence Protein ◽  
Dependent Manner ◽  
Reporter System ◽  
Health And Disease ◽  
Fluorescence Protein ◽  
Biomechanical Stimuli ◽  
Dose Dependent

The vascular endothelium plays a critical role in the health and disease of the cardiovascular system. Importantly, biomechanical stimuli generated by blood flow and sensed by the endothelium constitute important local inputs that are translated into transcriptional programs and functional endothelial phenotypes. Pulsatile, laminar flow, characteristic of regions in the vasculature that are resistant to atherosclerosis, evokes an atheroprotective endothelial phenotype. This atheroprotective phenotype is integrated by the transcription factor Kruppel-like factor-2 (KLF2), and therefore the expression of KLF2 can be used as a proxy for endothelial atheroprotection. Here, we report the generation and characterization of a cellular KLF2 reporter system, based on green fluorescence protein (GFP) expression driven by the human KLF2 promoter. This reporter is induced selectively by an atheroprotective shear stress waveform in human endothelial cells, is regulated by endogenous signaling events, and is activated by the pharmacological inducer of KLF2, simvastatin, in a dose-dependent manner. This reporter system can now be used to probe KLF2 signaling and for the discovery of a novel chemical-biological space capable of acting as the “pharmacomimetics of atheroprotective flow” on the vascular endothelium.

scholarly journals Point centromere activity requires an optimal level of centromeric noncoding RNA

2019 ◽  
Vol 116 (13) ◽  
pp. 6270-6279 ◽  
Author(s):  
Yick Hin Ling ◽  
Karen Wing Yee Yuen
Keyword(s):  
Noncoding Rna ◽  
Control Point ◽  
Optimal Level ◽  
Dependent Manner ◽  
Histone H2a ◽  
Protein Levels ◽  
Interference System ◽  
Binding Factor ◽  
In Trans ◽  
Dose Dependent

In budding yeast, which possesses simple point centromeres, we discovered that all of its centromeres express long noncoding RNAs (cenRNAs), especially in S phase. Induction of cenRNAs coincides with CENP-ACse4loading time and is dependent on DNA replication. Centromeric transcription is repressed by centromere-binding factor Cbf1 and histone H2A variant H2A.ZHtz1. Deletion ofCBF1andH2A.ZHTZ1results in an up-regulation of cenRNAs; an increased loss of a minichromosome; elevated aneuploidy; a down-regulation of the protein levels of centromeric proteins CENP-ACse4, CENP-A chaperone HJURPScm3, CENP-CMif2, SurvivinBir1, and INCENPSli15; and a reduced chromatin localization of CENP-ACse4, CENP-CMif2, and Aurora BIpl1. When the RNA interference system was introduced to knock down all cenRNAs from the endogenous chromosomes, but not the cenRNA from the circular minichromosome, an increase in minichromosome loss was still observed, suggesting that cenRNA functionsin transto regulate centromere activity. CenRNA knockdown partially alleviates minichromosome loss incbf1Δ,htz1Δ, andcbf1Δ htz1Δin a dose-dependent manner, demonstrating that cenRNA level is tightly regulated to epigenetically control point centromere function.

Study on Mechanisms of Telomerase Regulation in Arsenic Trioxide Inducing Apoptosis in Myelodysplasia Cell Line.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4706-4706
Author(s):  
Hongyan Tong ◽  
Jie Jin ◽  
Weilai Xu ◽  
Wenbin Qian ◽  
Maofang Lin
Keyword(s):  
Mrna Expression ◽  
Cell Line ◽  
Telomerase Activity ◽  
Time Dependent ◽  
Dependent Manner ◽  
Htert Gene ◽  
Binding Factor ◽  
Telomerase Regulation ◽  
Dose Dependent ◽  
Ttaggg Repeat

Abstract The telomerase activity can be down regulated by arsenic trioxide (As2O3), which is regarded as an apoptotic induction agent, is confirmed in many kinds of tumor cells. To investigate the mechanisms of telomerase regulation and to explore the correlation of As2O3 inducing apoptosis and telomerase regulation in MUTZ-1 cells, which are established as a high-risk myelodysplasia Cell line that derived from a MDS patient (FAB subtype refractory anemia with excess of blasts), a quantitative assessment of the telomerase activity by TRAP-ELISA and detection of the expression levels of hTERT, TRF1 (TTAGGG repeat binding factor 1), TRF2 (TTAGGG repeat binding factor 2), bcl-2, bax mRNA were performed, together with the assessment of the apoptosis by means of translocation of phosphatidylserine (PS) through flow cytometry assay. The results indicated that a typical apoptotic cell group distribution of DNA content was represented in the MUTZ-1 cells after being exposed to As2O3 at the range of concentration from 1μmol/L to 8μmol/L in a dose-dependent manner (r=0.736, P<0.001) and time-dependent manner (r=0.674, p<0.05), and the telomerase activity was down-regulated in a time-dependent manner (r=−0.976,P=0.024), and the expression level of hTERT mRNA in MUTZ-1 cells was represented in a dose-dependent manner (r=−0.892,P=0.042) and time-dependent manner (r=−1.000,P=0.04), after the cells were treated by As2O3 at the dosage as above. It was showed that a significant correlation between the decreased telomerase activity and the increased percentage of apoptotic cells in the treated cells (r=0.938,P=0.018), and there was a strong relationship between the telomerase activity and the mRNA expression of hTERT gene (r=0.783,P=0.022). However, As2O3 has no obvious effect on the expression level of TRF1 mRNA and TRF2 mRNA, which were regarded as two telomere-binding proteins. Further findings indicated that the inhibition of telomerase activity in MUTZ-1 cells was accompanied with down-regulated mRNA expression of bcl-2 gene (densitometry readings: 0.255±0.017 vs 0.466±0.069, P<0.05) and decreased ration of bcl-2/bax (densitometry reading ratios: 0.890±0.083 vs 0.546±0.014, P<0.05) at the dosage of 4μmol/L for 24 hours. These observations suggest that the apoptosis induced by As2O3 on MUTZ- 1 cells might be mediated through the inhibition of telomerase activity regulated by expression of hTERT gene, which implies that may be one of the mechanisms of As2O3 inducing apoptosis in MUTZ-1 cells.

The Zinc Finger Protein Gfi1 Controls TLR4-Mediated Inflammatory Response by Directly Antagonizing NF-κB Transcription Factor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 469-469
Author(s):  
Ehssan Sharif-Askari ◽  
Hui Zeng ◽  
Lothar Vassen ◽  
Christian Kosan ◽  
Cyrus Khandanpour ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Direct Interaction ◽  
Innate Immune ◽  
Dependent Manner ◽  
Negative Regulators ◽  
Tlr Signaling ◽  
Tnf Α ◽  
Dose Dependent ◽  
Lps Treatment ◽  
Stimulation Of

Abstract Inflammatory responses are complex and comprise multiple mediators including cytokines such as TNF-alpha (TNF-α) and IL-1beta. These cytokines are synthesized and secreted in response to signaling by plasma membrane receptors of the Toll-like receptor (TLR) family. A central downstream element of TLR-dependent signaling is the transcription factor NF-kappaB (NF-κB), which plays a pivotal role in controlling the proper sequence of events during an inflammatory response. In unstimulated cells, NF-κB is bound to inhibitory IkappaB (IκB) proteins and remains sequestered in the cytoplasm. Stimulation of TLRs triggers a signaling cascade that leads to phosphorylation and proteasomal degradation of IκB, resulting in the translocation of NF-κB to the nucleus, where it acts as a transcriptional activator of target genes. To keep the innate immune system under control, the TLR signaling cascade is under a tight control of many positive and negative regulators. We have previously shown that the transcription factor Growth Factor Independence 1 (Gfi1) represents a novel factor limiting the inflammatory immune response including TNF-α. Gfi1-deficient (Gfi1−/−) mice show a very strong systemic response to the TLR4 ligand and endotoxin LPS and die rapidly within 36 h with symptoms of septic shock. Here, we investigated the molecular mechanism of this exaggerated TNF-α production in the absence of Gfi1. It is known that endotoxin stimulation results in the activation of the transcription factor NF-κB through TLR4, leading to TNF-α production. This activation also resulted in rapid and de novo expression of Gfi1 in the nucleus in a time- and dose-dependent manner. The expression of Gfi1 was not due to feedback regulation from secreted TNF, since TNF-deficient macrophages were also able to upregulate Gfi1 mRNA following LPS stimulation. As expected, LPS stimulation of Gfi1−/− macrophages resulted in significantly higher levels of TNF-α mRNA, and secreted TNF-α cytokine. Strikingly and in contrast to most known negative regulators of TLRs, Gfi1 did not affect the activity or the expression levels of the cytoplasmic components of TLR signaling pathway. Additionally, NF-κB phosphorylation and nuclear translocation post- LPS treatment were intact in both Gfi1−/− and Gfi1+/+ macrophages. Immunoprecipitation analysis from cells endogenously expressing Gfi1 and NF-κB or over-expressing these two proteins post transfection, clearly revealed a direct interaction between Gfi1 and the p65 subunit of NF-κB. Immunofluorescence staining of macrophages post-LPS treatment confirmed direct interaction of these two proteins in the nucleus at the endogenous level. Gfi1 represses transcription by binding to DNA recognition sequences in target gene promoters. Thus, aiming to investigate the effect of Gfi1 expression on NF-κB nuclear signaling, we found that LPS treatment enhances NF-κB DNA binding activity in Gfi1−/− macrophages as compared to Gfi1+/+ cells. Furthermore, over expression of Gfi1 protein resulted in negative regulation of NF-κB mediated gene activation in a dose-dependent manner. Chromatin immune precipitation with anti-p65 antibodies from LPS stimulated Gfi1+/+ and Gfi1−/− macrophages revealed enhanced NF-κB promoter occupancy at the TNF gene in Gfi1−/− macrophages as compared to Gfi1+/+ cells. In conclusion, our findings reveal a novel function for Gfi1 in the innate immune response by directly antagonizing NF-κB function. This molecular perceptive of TNF-α regulation during inflammation may provide an attractive strategy for therapeutic intervention in chronic inflammatory diseases and certain cancers.

scholarly journals Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase

2007 ◽  
Vol 6 (9) ◽  
pp. 1618-1624 ◽  
Author(s):  
William Harold Witola ◽  
Choukri Ben Mamoun
Keyword(s):  
Transcriptional Repression ◽  
Morphological Changes ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Wild Type ◽  
Active Synthesis ◽  
Rapid Multiplication ◽  
Gene And Protein Expression ◽  
Transgenic Parasites ◽  
Dose Dependent

ABSTRACT During its intraerythrocytic life cycle, the malaria parasite Plasmodium falciparum undergoes dramatic metabolic and morphological changes and multiplies to produce up to 36 new daughter parasites. This rapid multiplication of the parasite requires an active synthesis of new membranes. The major component of these membranes, phosphatidylcholine, is synthesized via two metabolic routes, the CDP-choline pathway, which uses host choline as a precursor, and the plant-like serine decarboxylase-phosphoethanolamine methyltransferase (SDPM) pathway, which uses host serine as a precursor. Here we provide evidence indicating that the activity of the SDPM pathway is regulated by the CDP-choline precursor, choline. We show that the phosphoethanolamine methyltransferase, Pfpmt, a critical enzyme in the SDPM pathway, is down-regulated at the transcriptional level as well as targeted for degradation by the proteasome in the presence of choline. Transcript analysis revealed that PfPMT transcription is repressed by choline in a dose-dependent manner. Immunoblotting, pulse-chase experiments, and immunoprecipitation studies demonstrated that Pfpmt degradation occurs not only in wild-type but also in transgenic parasites constitutively expressing Pfpmt. The proteasome inhibitor bortezomib inhibited choline-mediated Pfpmt degradation. These data provide the first evidence for metabolite-mediated transcriptional and proteasomal regulation in Plasmodium and will set the stage for the use of this system for conditional gene and protein expression in this organism.

The Fibrinogen γ Chain Dodecapeptide Inhibits Agonist-induced Aggregation of Rabbit Platelets and Fibrinogen Binding to Rabbit Glycoprotein IIb-IIIa

1999 ◽  
Vol 82 (12) ◽  
pp. 1680-1686 ◽  
Author(s):  
Marian Packham ◽  
Desirée Taylor ◽  
Erik Yeo ◽  
Cynthia Gemmell ◽  
Sonali Patil ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Dependent Manner ◽  
Affinity Matrix ◽  
Human Fibrinogen ◽  
Fibrinogen Binding ◽  
Rabbit Platelets ◽  
Induced Aggregation ◽  
Dose Dependent

SummaryThe HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the γ chains and the RGD sequences in the Aα chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen γ chain sequence. Proteins of ∼135 kDa and ∼95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.

GLUCOCORTICOID EFFECTS ON VASCULAR CONNECTIVE TISSUE DURING REPAIR. IMPORTANCE OF DOSE LEVEL AND PRE- AND POST-INJURY TREATMENT

1977 ◽  
Vol 86 (2) ◽  
pp. 437-448 ◽  
Author(s):  
R. Mathorpe ◽  
C. Garbarsch ◽  
B. Kofod ◽  
I. Lorenzen
Keyword(s):  
Connective Tissue ◽  
Inhibitory Action ◽  
Amino Nitrogen ◽  
Dependent Manner ◽  
Intimal Thickening ◽  
Descending Thoracic Aorta ◽  
Post Injury ◽  
Collagen Protein ◽  
Injury Treatment ◽  
Dose Dependent

ABSTRACT Aorta of male albino rabbits were subjected to a single mechanical dilatation injury and the effects of different daily doses of prednisolone on the metabolism of collagen and glycosaminoglycans as well as on the content of alpha-amino nitrogen, RNA, DNA, water and fat and the histology of the descending thoracic aorta were analyzed 10 days after the injury. The effects of pre- and post-injury treatment with prednisolone were compared. Prednisolone inhibited the intimal thickening. This effect was enhanced by pre-injury treatment. Prednisolone also inhibited the biosynthesis of non-dialysable [14C]hydroxyproline-collagen, but increased the relative degradation of collagen in a dose dependent manner. The biosynthesis of glycosaminoglycans was decreased while prednisolone had no effect on the concentration and total amount of glycosaminoglycans, collagen, protein, RNA, DNA and fat. Finally the aortic content of water was decreased during treatment with prednisolone also in a dose dependent manner. It is concluded that the action of prednisolone on vascular collagen and water during repair is dose dependent and that the inhibitory action of prednisolone on the intimal thickening is enhanced by pre-injury treatment.

Effects of Naturally Occurring Polyols and Urea on Mitochondrial F0F1ATPase

2000 ◽  
Vol 55 (5-6) ◽  
pp. 392-398 ◽  
Author(s):  
Adriana dos Passos Lemos ◽  
Carlos E. Peres-Sampaio ◽  
Horácio Guimarães-Motta ◽  
Jerson L. Silva ◽  
José R. Meyer-Fernandes
Keyword(s):  
Atpase Activity ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Urea Denaturation ◽  
Naturally Occurring ◽  
Activation Level ◽  
Activation Rate ◽  
Initial Activation ◽  
Natural Inhibitor ◽  
Dose Dependent

We show that urea inhibits the ATPase activity of MgATP submitochondrial particles (MgATP-SMP) with Ki = 0.7 м, probably as a result of direct interaction with the structure of F0F1-ATPase. Counteracting compounds (sorbitol, mannitol or inositol), despite slightly (10-20% ) inhibiting the ATPase activity, also protect the F0F1 ATPase against denaturation by urea. However, this protection was only observed at low urea concentrations (less than 1.5 м ) , and in the presence of three polyols, the Ki for urea shift from 0.7 м to 1.2 м. Urea also increases the initial activation rate of latent MgATP-SMP in a dose-dependent-manner. However, when the particles (0.5 mg/ml) were preincubated in the presence of 1 м , 2 м or 3 м urea, a decrease in the activation level occurred after 1 h, 30 and 10 min, respectively. At high MgATP-SMP concentration (3 mg/ml) a decrease in activation was observed after 2 h, 1 h and 20 min, respectively. These data indicate that the effect of urea on the activation of MgATP-SMP depends on time, urea and protein concentrations. It was also observed that polyols suppress the activation of latent MgATP-SMP in a dose-dependent manner, and protect the particles against urea denaturation during activation. We suppose that a decrease in membrane mobility promoted by interactions of polyols with phospholipids around the F0F1 ATPase may also increase the compactation of protein structure, explaining the inhibition of natural inhibitor protein of ATPase (IF1) release and the activation of the enzyme.

scholarly journals Effects of a phorbol ester and clomiphene on protein phosphorylation and insulin secretion in rat pancreatic islets

1988 ◽  
Vol 249 (3) ◽  
pp. 825-830 ◽  
Author(s):  
S J Hughes ◽  
S J Ashcroft
Keyword(s):  
Insulin Release ◽  
Inhibitory Action ◽  
Glucose Oxidation ◽  
Subcellular Fractionation ◽  
Mean Value ◽  
Particulate Fraction ◽  
Dependent Manner ◽  
Phosphorylation State ◽  
Kinase C ◽  
Dose Dependent

The potentiation of glucose-stimulated insulin release induced by 100 nM-12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by clomiphene, an inhibitor of protein kinase C (PK C), in a dose-dependent manner. Clomiphene at concentrations up to 50 microM had a modest inhibitory action (27%) on insulin release stimulated by 10 mM-glucose alone, but had no effect on the potentiation of insulin release induced by forskolin. Islet PK C activity, associated with a particulate fraction, was stimulated maximally by 100 nM-TPA. This stimulation was blocked by clomiphene in a dose-dependent manner, with 50% inhibition at 30 microM. Incubation of intact islets with TPA after preincubation with [32P]Pi and 10 mM-glucose to label intracellular ATP resulted primarily in enhanced phosphorylation of a 37 kDa protein (mean value, +/- S.E.M., 36,700 +/- 600 Da; n = 7). This increased phosphorylation was blocked by the simultaneous inclusion of clomiphene. Subcellular fractionation revealed the presence of the 37 kDa phosphoprotein in a 24,000 g particulate fraction of islet homogenates. Neither clomiphene nor TPA affected the rate of glucose oxidation by islets. These results show that the phosphorylation state of a 37 kDa membrane protein parallels the modulation of insulin release induced by TPA and clomiphene and support a role for PK C in the insulin-secretory mechanism.

scholarly journals Antimicrobial activity of potato aspartic proteases (StAPs) involves membrane permeabilization

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2039-2047 ◽  
Author(s):  
Julieta R. Mendieta ◽  
Mariana R. Pagano ◽  
Fernando F. Muñoz ◽  
Gustavo R. Daleo ◽  
María G. Guevara
Keyword(s):  
Antimicrobial Activity ◽  
Direct Interaction ◽  
Antimicrobial Effect ◽  
Membrane Permeabilization ◽  
Dependent Manner ◽  
Abiotic And Biotic Stress ◽  
Green It ◽  
Aspartic Proteases ◽  
Sytox Green ◽  
Dose Dependent

Solanum tuberosum aspartic proteases (StAPs) with antimicrobial activity are induced after abiotic and biotic stress. In this study the ability of StAPs to produce a direct antimicrobial effect was investigated. Viability assays demonstrated that StAPs are able to kill spores of Fusarium solani and Phytophthora infestans in a dose-dependent manner. Localization experiments with FITC-labelled StAPs proved that the proteins interact directly with the surface of spores and hyphae of F. solani and P. infestans. Moreover, incubation of spores and hyphae with StAPs resulted in membrane permeabilization, as shown by the uptake of the fluorescent dye SYTOX Green. It is concluded that the antimicrobial effect of StAPs against F. solani and P. infestans is caused by a direct interaction with the microbial surfaces followed by membrane permeabilization.

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