scholarly journals Phosphorylation of α-dystrobrevin is essential for αkap accumulation and acetylcholine receptor stability

2020 ◽  
Vol 295 (31) ◽  
pp. 10677-10688
Author(s):  
Po-Ju Chen ◽  
Diego Zelada ◽  
Dina Cheryne Belhasan ◽  
Mohammed Akaaboune
Keyword(s):  
Acetylcholine Receptor ◽  
Muscle Cells ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tyrosine Residues ◽  
C Terminus ◽  
The Stability ◽  
High Level

The maintenance of a high density of the acetylcholine receptor (AChR) is the hallmark of the neuromuscular junction. Muscle-specific anchoring protein (αkap) encoded within the calcium/calmodulin-dependent protein kinase IIα (CAMK2A) gene is essential for the maintenance of AChR clusters both in vivo and in cultured muscle cells. The underlying mechanism by which αkap is maintained and regulated remains unknown. Here, using human cell lines, fluorescence microscopy, and pulldown and immunoblotting assays, we show that α-dystrobrevin (α-dbn), an intracellular component of the dystrophin glycoprotein complex, directly and robustly promotes the stability of αkap in a concentration-dependent manner. Mechanistically, we found that the phosphorylatable tyrosine residues of α-dbn are essential for the stability of α-dbn itself and its interaction with αkap, with substitution of three tyrosine residues in the α-dbn C terminus with phenylalanine compromising the αkap–α-dbn interaction and significantly reducing both αkap and α-dbn accumulation. Moreover, the αkap–α-dbn interaction was critical for αkap accumulation and stability. We also found that the absence of either αkap or α-dbn markedly reduces AChRα accumulation and that overexpression of α-dbn or αkap in cultured muscle cells promotes the formation of large agrin-induced AChR clusters. Collectively, these results indicate that the stability of αkap and α-dbn complex plays an important role in the maintenance of high-level expression of AChRs.

Role of CYP27A1 in progesterone metabolism in vitro and in vivo

2009 ◽  
Vol 297 (4) ◽  
pp. E949-E955 ◽  
Author(s):  
Geneviève Escher ◽  
Isabelle Vögeli ◽  
Robert Escher ◽  
Robert C. Tuckey ◽  
Sandra Erickson ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Human Kidney ◽  
Underlying Mechanism ◽  
Expression Cloning ◽  
Dependent Manner ◽  
Expression Library ◽  
Concentration Dependent Manner ◽  
Adrenodoxin Reductase

In the kidney, progesterone is inactivated to 20α-dihydro-progesterone (20α-DH-progesterone) to protect the mineralocorticoid receptor from progesterone excess. In an attempt to clone the enzyme with 20α-hydroxysteroid activity using expression cloning in CHOP cells and a human kidney expression library, serendipitously cDNA encoding CYP27A1 was isolated. Overexpression of CYP27A1 in CHOP cells decreased progesterone conversion to 20α-DH-progesterone in a dose-dependent manner, an effect enhanced by cotransfection with adrenodoxin and adrenodoxin reductase. Incubation of CHOP cells with 27-hydroxycholesterol, a product of CYP27A1, increased the ratio of progesterone to 20α-DH-progesterone in a concentration-dependent manner, indicating that the effect of CYP27A1 overexpression was mediated by 27-hydroxycholesterol. To analyze whether these observations are relevant in vivo, progesterone and 20α-DH-progesterone were measured by gas chromatography-mass spectometry in 24-h urine of CYP27A1 gene knockout (ko) mice and their control wild-type and heterozygote littermates. In CYP27A1 ko mice, urinary progesterone concentrations were decreased, 20α-DH-progesterone increased, and the progesterone-to-20α-DH-progesterone ratio decreased threefold ( P < 0.001). Thus CYP27A1 modulates progesterone concentrations. The underlying mechanism is inhibition of 20α-hydroxysteroid dehydrogenase by 27-hydroxycholesterol.

scholarly journals DAB2IP inhibits p53 ubiquitin-mediated degradation by competitively binding to GRP75 and suppresses tumor malignancy in colon cancer

2021 ◽  
Author(s):  
Jie Shen ◽  
Shengjie Feng ◽  
Jiao Deng ◽  
Qingwen Huang ◽  
Dayong Zhao ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Animal Experiments ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Wild Type ◽  
Vitro Assay ◽  
Cell Invasiveness ◽  
The Stability

Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibition role in many tumors. However, the underlying mechanism is still uncertain. Our study shows that DAB2IP is positively associated with a better prognosis in colon cancer patients with wild-type TP53 expression. In vitro assay shows that DAB2IP elicits potent tumor-suppressive effects on inhibiting cell invasiveness, colony formation and promoting cell apoptosis in wild-type TP53 colon cancer cell lines. Subsequently, DAB2IP is demonstrated to up-regulate the stability of wild-type TP53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we unveil that DAB2IP and p53 could both interact with the ubiquitin ligase-related protein, GRP75. Mechanistically, DAB2IP could competitively bind with GRP75, thus reducing GRP75-mediated p53 ubiquitination and degradation. Finally, animal experiments also reveal that DAB2IP inhibits the tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, which provides a new insight in DAB2IP-induced tumor suppression and provides a novel molecular aspect of the p53 pathway.

4969Enhanced gap junction coupling organised and terminated acute ventricular fibrillation in ex-vivo perfused hearts

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
B S Handa ◽  
X Li ◽  
C Roney ◽  
D Pitcher ◽  
R A Chowdhury ◽  
...  
Keyword(s):  
Ventricular Fibrillation ◽  
Ex Vivo ◽  
Electrical Coupling ◽  
Underlying Mechanism ◽  
Dominant Frequency ◽  
Diffuse Fibrosis ◽  
Dependent Manner ◽  
Sprague Dawley ◽  
Concentration Dependent Manner

Abstract Background The underlying mechanism of ventricular fibrillation (VF) remains unclear. There are both experimental and clinical data to support the existence of rotational drivers (RDs), though other opposing studies suggest that VF is the result of disorganized myocardial activation. Abnormal electrical coupling between cardiomyocytes through gap junctions (GJ) has been considered an important factor in the genesis and maintenance of VF and pre-treatment with GJ couplers, rotigaptide (RTG), has been shown to reduce VF inducibility. Purpose We hypothesized that the degree of GJ coupling determines the underlying mechanism of VF, and that changes in GJ coupling can shift or modify the predominant mechanism of fibrillation along the spectrum between disorganised activity and organised drivers. We proposed that increased organisation of VF is critical to its termination. Methods Thirty Sprague-Dawley rat hearts were explanted, perfused ex-vivo and acute VF was induced with burst pacing and 30μM pinacidil. Optical mapping of transmembrane potential was performed at baseline and the effects of GJ coupling on VF dynamics were studied in an acute VF model by perfusing with increasing concentrations of a GJ uncoupler; carbenoxolone (0–50μM, CBX, n=10) or a GJ coupling-enhancer; RTG (0–80nM, n=10). A chronic diffuse fibrosis model (n=10) was generated with 4 weeks of in-vivo angiotensin infusion (500nm/kg/min). Fibrillation dynamics were quantified using phase analysis, phase singularity (PS) tracking and our novel method of global fibrillation organisation quantification, frequency dominance index (FDI), which is a power ratio of highest amplitude dominant frequency in the frequency spectrum. Results RTG increased average rotations per RD (Baseline: 2.86±0.10 vs 80nM: 5.66±0.43, p<0.001) whilst CBX caused a reduction (Baseline: 3.77±0.39 vs 50μM: 0.26±0.26, p<0.001). Maximum rotations for a RD increased with RTG (5.4±0.45 vs 48.20±12.32, p<0.001) and decreased with CBX (8.0±1.3 vs 0.3±0.3, p<0.001). Proportion of time PSs were detected in VF increased with RTG (0.44±0.06 vs 0.93±0.02, p<0.001) and decreased with CBX (0.61±0.9 vs 0.03±0.02, p<0.001). RTG reduced meander of longest duration RD (20.6±1.68 vs 11.51±0.77 pixels, p<0.001) for PS >5 rotations. FDI increased with RTG (0.53±0.04 vs 0.78±0.3, p<0.001) and decreased with CBX (0.60±0.05 vs 0.17±0.03, p<0.001). In the diffuse fibrosis group, in comparison to baseline RTG 80nM increased FDI (0.35 vs 0.65, p<0.001) and terminated VF in 40% of hearts. Conclusion The degree of GJ coupling is a key determinant of the underlying mechanism of VF. RTG organised fibrillation and stabilised RDs in a concentration-dependent manner whilst CBX disorganised VF. Enhancing GJ coupling with RTG in diseased hearts with fibrosis can terminate VF and may be a potential therapeutic target in acute VF. Acknowledgement/Funding BHF Programme Grant PG/16/17/32069

scholarly journals Hypotensive and Vasorelaxant Effects of Sericin-Derived Oligopeptides in Rats

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Amnart Onsa-ard ◽  
Dawan Shimbhu ◽  
Jiraporn Tocharus ◽  
Manote Sutheerawattananonda ◽  
Rungusa Pantan ◽  
...  
Keyword(s):  
Barium Chloride ◽  
Underlying Mechanism ◽  
Hypotensive Effect ◽  
Hypotensive Activity ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Relaxant Effect ◽  
Rat Thoracic Aorta

Sericin-derived oligopeptides obtained from silk cocoons were investigated for the in vivo hypotensive effect and investigated for the underlying mechanism involved in vasodilation in isolated rat thoracic aorta. In normotensive anesthetized rats, oligopeptides induced an immediate and transient hypotensive activity. In rat aortic rings, oligopeptides induced a concentration-dependent vasorelaxation in vessels precontracted with both KCl and phenylephrine (PE) with endothelium-intact or endothelium-denuded rings. In endothelium-intact rings, pretreatment with Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 µM), an inhibitor of the NO synthase (NOS) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 µM), a selective inhibitor of the guanylyl cyclase enzyme, significantly reduced the relaxant effect of oligopeptides. However, indomethacin, an inhibitor of the cyclooxygenase, had no effect on oligopeptides-induced relaxation. In addition, pretreatment with tetraethylammonium (TEA, 5 mM) reduced the maximal relaxant effect induced by oligopeptides. By contrast, relaxation was not affected by 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 µM), or barium chloride (BaCl2, 1 mM). In depolarization Ca2+-free solution, oligopeptides inhibited calcium chloride- (CaCl2-) induced contraction in endothelium-denuded rings in a concentration-dependent manner. Nevertheless, oligopeptides attenuated transient contractions in Ca2+-free medium containing EGTA (1 mM) induced by 1 µM PE, but they were not affected by 20 mM caffeine. It is obvious that potent vasodilation effect of oligopeptides is mediated through both the endothelium and the vascular smooth muscle.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

scholarly journals Influence of icariin on inflammation, apoptosis, invasion, and tumor immunity in cervical cancer by reducing the TLR4/MyD88/NF-κB and Wnt/β-catenin pathways

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chunyang Li ◽  
Shuangqing Yang ◽  
Huaqing Ma ◽  
Mengjia Ruan ◽  
Luyan Fang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Underlying Mechanism ◽  
Tissue Growth ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Antitumor Effects ◽  
Siha Cells ◽  
Cervical Cancer Cells

Abstract Background Cervical cancer is a type of the most common gynecology tumor in women of the whole world. Accumulating data have shown that icariin (ICA), a natural compound, has anti-cancer activity in different cancers, including cervical cancer. The study aimed to reveal the antitumor effects and the possible underlying mechanism of ICA in U14 tumor-bearing mice and SiHa cells. Methods The antitumor effects of ICA were investigated in vivo and in vitro. The expression of TLR4/MyD88/NF-κB and Wnt/β-catenin signaling pathways were evaluated. Results We found that ICA significantly suppressed tumor tissue growth and SiHa cells viability in a dose-dependent manner. Also, ICA enhanced the anti-tumor humoral immunity in vivo. Moreover, ICA significantly improved the composition of the microbiota in mice models. Additionally, the results clarified that ICA significantly inhibited the migration, invasion capacity, and expression levels of TGF-β1, TNF-α, IL-6, IL-17A, IL-10 in SiHa cells. Meanwhile, ICA was revealed to promote the apoptosis of cervical cancer cells by down-regulating Ki67, survivin, Bcl-2, c-Myc, and up-regulating P16, P53, Bax levels in vivo and in vitro. For the part of mechanism exploration, we showed that ICA inhibits the inflammation, proliferation, migration, and invasion, as well as promotes apoptosis and immunity in cervical cancer through impairment of TLR4/MyD88/NF-κB and Wnt/β-catenin pathways. Conclusions Taken together, ICA could be a potential supplementary agent for cervical cancer treatment.

scholarly journals Modulatory Effects of Osthole on Lipopolysaccharides-Induced Inflammation in Caco-2 Cell Monolayer and Co-Cultures with THP-1 and THP-1-Derived Macrophages

Nutrients ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 123
Author(s):  
Natalia K. Kordulewska ◽  
Justyna Topa ◽  
Małgorzata Tańska ◽  
Anna Cieślińska ◽  
Ewa Fiedorowicz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inflammatory Reaction ◽  
Wide Spectrum ◽  
Cell Monolayer ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Tnf Α

Lipopolysaccharydes (LPS) are responsible for the intestinal inflammatory reaction, as they may disrupt tight junctions and induce cytokines (CKs) secretion. Osthole has a wide spectrum of pharmacological effects, thus its anti-inflammatory potential in the LPS-treated Caco-2 cell line as well as in Caco-2/THP-1 and Caco-2/macrophages co-cultures was investigated. In brief, Caco-2 cells and co-cultures were incubated with LPS to induce an inflammatory reaction, after which osthole (150–450 ng/mL) was applied to reduce this effect. After 24 h, the level of secreted CKs and changes in gene expression were examined. LPS significantly increased the levels of IL-1β, -6, -8, and TNF-α, while osthole reduced this effect in a concentration-dependent manner, with the most significant decrease when a 450 ng/mL dose was applied (p < 0.0001). A similar trend was observed in changes in gene expression, with the significant osthole efficiency at a concentration of 450 ng/μL for IL1R1 and COX-2 (p < 0.01) and 300 ng/μL for NF-κB (p < 0.001). Osthole increased Caco-2 monolayer permeability, thus if it would ever be considered as a potential drug for minimizing intestinal inflammatory symptoms, its safety should be confirmed in extended in vitro and in vivo studies.

scholarly journals RASSF1A inhibits PDGFB-driven malignant phenotypes of nasopharyngeal carcinoma cells in a YAP1-dependent manner

2020 ◽  
Vol 11 (10) ◽  
Author(s):  
Ying-Ying Liang ◽  
Xu-Bin Deng ◽  
Xian-Tao Lin ◽  
Li-Li Jiang ◽  
Xiao-Ting Huang ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Underlying Mechanism ◽  
Carcinoma Cells ◽  
Dependent Manner ◽  
Transcriptional Coactivator ◽  
Actin Remodeling ◽  
Aggressive Tumor ◽  
Subunit B

Abstract Nasopharyngeal carcinoma (NPC) is a highly aggressive tumor characterized by distant metastasis. Deletion or down-regulation of the tumor suppressor protein ras-association domain family protein1 isoform A (RASSF1A) has been confirmed to be a key event in NPC progression; however, little is known about the effects or underlying mechanism of RASSF1A on the malignant phenotype. In the present study, we observed that RASSF1A expression inhibited the malignant phenotypes of NPC cells. Stable silencing of RASSF1A in NPC cell lines induced self-renewal properties and tumorigenicity in vivo/in vitro and the acquisition of an invasive phenotype in vitro. Mechanistically, RASSF1A inactivated Yes-associated Protein 1 (YAP1), a transcriptional coactivator, through actin remodeling, which further contributed to Platelet Derived Growth Factor Subunit B (PDGFB) transcription inhibition. Treatment with ectopic PDGFB partially increased the malignancy of NPC cells with transient knockdown of YAP1. Collectively, these findings suggest that RASSF1A inhibits malignant phenotypes by repressing PDGFB expression in a YAP1-dependent manner. PDGFB may serve as a potential interest of therapeutic regulators in patients with metastatic NPC.

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2648-2656 ◽  
Author(s):  
Juan A. Rosado ◽  
Else M. Y. Meijer ◽  
Karly Hamulyak ◽  
Irena Novakova ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

MondoA Drives B-ALL Malignancy through Enhanced Adaptation to Metabolic Stress

Blood ◽  
2021 ◽  
Author(s):  
Alexandra Sipol ◽  
Erik Hameister ◽  
Busheng Xue ◽  
Julia Hofstetter ◽  
Maxim Barenboim ◽  
...  
Keyword(s):  
Stress Responses ◽  
Lymphoblastic Leukemia ◽  
Metabolic Stress ◽  
Tca Cycle ◽  
Underlying Mechanism ◽  
Patient Data ◽  
Data Sets ◽  
Dependent Manner

Cancer cells are in most instances characterized by rapid proliferation and uncontrolled cell division. Hence, they must adapt to proliferation-induced metabolic stress through intrinsic or acquired anti-metabolic stress responses to maintain homeostasis and survival. One mechanism to achieve this is to reprogram gene expression in a metabolism-dependent manner. MondoA (also known as MLXIP), a member of the MYC interactome, has been described as an example of such a metabolic sensor. However, the role of MondoA in malignancy is not fully understood and the underlying mechanism in metabolic responses remains elusive. By assessing patient data sets we found that MondoA overexpression is associated with a worse survival in pediatric common acute lymphoblastic leukemia (B-ALL). Using CRISPR/Cas9 and RNA interference approaches, we observed that MondoA depletion reduces transformational capacity of B-ALL cells in vitro and dramatically inhibits malignant potential in an in vivo mouse model. Interestingly, reduced expression of MondoA in patient data sets correlated with enrichment in metabolic pathways. The loss of MondoA correlated with increased tricarboxylic acid (TCA) cycle activity. Mechanistically, MondoA senses metabolic stress in B-ALL cells by restricting oxidative phosphorylation through reduced PDH activity. Glutamine starvation conditions greatly enhance this effect and highlight the inability to mitigate metabolic stress upon loss of MondoA in B-ALL. Our findings give a novel insight into the function of MondoA in pediatric B-ALL and support the notion that MondoA inhibition in this entity offers a therapeutic opportunity and should be further explored.

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