bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Insights into Tachyphylaxis Phenomenon in the Angiotensin II Type 1 Receptor
Authors: Duarte Diego Angelo , Parreiras‐e‐Silva Lucas Tabajara , de Oliveira Eduardo Brandt , Bouvier Michel , da Costa‐Neto Claudio Miguel 
Published: June 2021

Tachyphylaxis is a phenomenon characterized as a progressive decrease in drug response after prolonged or repetitive administration; however, the precise mechanism behind such phenomenon remains unclear. Here, we assessed the molecular bases of tachyphylaxis using the Angiotensin II (AngII) type 1 receptor (AT1R) and two AngII analogs, named #001 and #002, which function as non‐tachyphylactic agonists. The affinity of both analogs for the AT1R was obtained by radioligand competition binding assays, and they were pharmacologically characterized by prolonged and short stimulation using bioluminescence resonance energy transfer (BRET) assays in Human Embryonic Kidney (HEK) 293T cells expressing the AT1R. Analogs #001 and #002 presented low affinity for the AT1R as compared with AngII, but behaved as full agonists for Gq protein activation, intracellular calcium mobilization, as well as concerning to β‐arrestin recruitment, with potencies similar to those of AngII. We also showed that the activation of Gq protein and the translocation of β‐arrestins to the plasma membrane triggered by the analogs were rapidly reversed either by washout or addition of the antagonist Losartan, whereas AngII induced a sustained response even after washout or addition of Losartan. Furthermore, the internalization of the AT1R induced by analogs #001 or #002 showed a faster recycling and to be more sensitive to washout or addition of Losartan than when induced by AngII. Our results suggest that the molecular bases of AngII tachyphylaxis are linked to distinct kinetics of sustained signaling, which we believe could be due to a higher dissociation rate of the analogs. According to our hypothesis, the non‐tachyphylactic AngII analogs activate the AT1R in a fast but transient manner, consequently leading the AT1R to be quickly recycled to the plasma membrane, becoming available to a new cycle of activation. On the other hand, the sustained activation of AT1R by AngII and the low dissociation rate of AngII seem to lead to a sustained internalization of the AT1R in endosomes, retarding their recycling to the plasma membrane. We believe that understanding the molecular mechanisms involved in tachyphylaxis may ultimately contribute to improve the efficiency of drugs and therapies.Support or Funding InformationFAPESP (process numbers 2014/09893‐0 and 2012/20148‐0)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Saturated fat‐enriched diet decreases SIRT1 expression in the mouse hippocampus ‐ The SIRTain effects of saturated fat in the brain
Authors: Marwarha Gurdeep , Ghribi Othman 
Published: June 2021

A multitude of prospective cohort epidemiological studies have posited a direct positive correlation between saturated fat‐enriched diets and an increased risk for cognitive impairment, deficits in learning and memory, leading to enhanced susceptibility for the development and progression of neurodegenerative disorders such as Alzheimer's disease (AD). Our previous studies have shown that saturated‐fat enriched diet cause an increase in BACE1 expression and activity leading to an increase in Amyloid‐beta burden in the brains of the C57BL/6J wild‐type mice (1–3). The histone deacetylase and the sirtuin SIRT1, is a master regulator of molecular processes that regulate lifespan and aging, the primary risk factor for AD. A plethora of laboratory studies have demonstrated the indispensable role of SIRT1 in regulating and maintaining synaptic integrity as well as synaptic plasticity at the molecular level and cognitive processes that underlie learning and memory. Herein, we show that feeding nine‐month old C57BL/6J wild‐type mice, a palmitate‐enriched diet for three months, elicits a profound attenuation of SIRT1 expression in the hippocampal region of the brain. Exogenous palmitate treatment of human SH‐SY5Y neuroblastoma cells also results in an attenuation of SIRT1 expression. We further elucidated and delineated the underlying signaling and molecular mechanisms involved and discovered a deleterious role of the palmitate‐evoked Endoplasmic Reticulum (ER) stress‐induced transcription factor C/EBP Homologous Protein (CHOP) in mediating the palmitate effects on SIRT1 expression. Homozygous Chop−/− knock‐out mice fed a palmitate‐enriched diet did not exhibit the same degree of loss of basal SIRT1 expression in the brain compared to their C57BL/6J wild‐type littermates. Also, RNA‐interference mediated knock‐down of CHOP expression in SH‐SY5Y neuroblastoma cells rescued the palmitate‐induced deficits in SIRT1 expression. Further insight into the mechanisms unveiled the role of CHOP in transcriptional repression of SIRT1 expression executed through the negative regulation of the transcription factor C/EBPα. Our study unveils SIRT1 as a unique molecular target that is negatively regulated by palmitate‐enriched diets. Further studies are warranted to determine the potential biochemical and physiological impact of this palmitate‐induced attenuation of SIRT1 expression, a histone deacetylase that plays an indispensable role in the regulation of cognitive processes and aging at the molecular level.Support or Funding InformationThis work was supported by a Grant from the NIH (R01AG045264) to Dr. Othman Ghribi. The authors declare that they have no competing interests.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium‐induced apoptosis in cultured, primary corneal endothelial cells from monkey
Authors: Nakajima Emi , Gleason Ellen K. , Walkup Ryan D. , Shearer Thomas R. , Azuma Mitsuyoshi 
Published: June 2021

Apoptosis plays a key role in the loss of corneal endothelial cells (CEC). Losses can occur during corneal storage and after transplantation. Unlike the epithelium, CEC do not regenerate. CEC stretch to compensate for dead cells. This reduces cell density, allows influx of water, and promotes formation of opacities. Several studies confirm that staurosporine and mitomycin cause apoptosis in CEC. However, molecular mechanisms responsible for apoptosis in CEC have not been well studied. The purpose of the present study was to determine if CEC apoptosis is initiated by calcium uptake.To increase calcium levels, cultured primary CEC from monkey were treated with either A23187 (A23) or thapsigargin (TG). The ionophore A23 allows influx of Ca2+ across cell membranes, while TG inhibits the outward directed Ca2+‐ATPase pump in endoplasmic reticulum (ER).We found that both A23 and TG caused: increased cytosolic calcium levels, up‐regulation of ER stress markers BiP and CHOP, and positive staining for TUNEL. Apoptosis was further confirmed by positive immunostaining for activated caspase‐3. The known CEC maker/calcium binding protein S100 was decreased after A23 treatment but not after TG. (Further testing is needed as to why only A23 down‐regulates S100). We suggest that apoptosis in CEC occurs when elevated cytosolic calcium stresses the ER, the stressed ER release calcium, and the calcium flows into the mitochondrial matrix to trigger the apoptotic response. The unfolded protein response (UPR) may also contribute since Ca‐mediated ER stress is known to cause CEC degeneration.Support or Funding InformationDr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are employees of, Senju Pharmaceutical Co., Ltd.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In‐Silico Comparative Structural Analysis of Human and Murine Cyclooxygenase‐2 (Cox‐2)
Authors: Hari Saran Krishnan , Boluwaduro Adeyemi , Gautham Sakthi , Villanueva James Amador 
Published: June 2021

Cyclooxygenase 2 (COX2) is a major enzymatic mediator of inflammation that catalyzes the formation of a host of prostaglandins, leukotrienes and thromboxanes. Inflammation is a principal component of a vast range of pathologic processes, ranging from simple infections to malignant neoplasms. Since this physiologic response is sometimes more damaging than beneficial to the human body, curbing its excesses by controlling the activity of COX2 is of great clinical importance. In‐silico studies have utilized murine, bovine, or other forms of COX2, as the human COX‐2 crystal structure has only been very recently elucidated. Using molecular docking and alignment studies, we found differences in the bonding interactions of murine COX2, and a recently resolved human COX2 crystal structure with their substrate, arachidonic acid, as well as piceatannol. Piceatannol is a metabolite of resveratrol, which is found in red wine. Our experiments show that although the sequence and structures of human and murine COX2 are largely similar with variation of 12%, there are important differences in the way their binding sites interact with ligands, with respect to binding energies and amino acid involvement. These differences may become significant in the search and design of small molecules that can function to inhibit the activity of COX‐2. Clinically, this would translate to better control over the deleterious effects of inflammation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing Structure‐Activity Relationships to Design Non ‐ Oxime Reactivators
Authors: Cadieux C. Linn , Canter Zachary , Martin Kevin , Hepperle Michael , Cerasoli Douglas M. 
Published: June 2021

Organophosphorus nerve agents are highly toxiccompounds that pose a threat worldwide. These compounds induce toxicity bycovalently binding to the active site serine of acetylcholinesterase, whichresults in inhibition of the native enzyme function. Without functionalacetylcholinesterase, levels of the neurotransmitter acetylcholine inneuromuscular junctions rise quickly, causing overstimulation of the nervoussystem, which can quickly culminate in death if not treated. Current treatmentsrely on small molecules that interact with inhibited enzyme to disrupt thecovalently bound phosphorus moiety at the active site. The most effectivemolecules incorporate a pyridinium oxime which acts via direct nucleophilicattack on the phosphorus to achieve reactivation of the enzyme. These compoundshave limited effectiveness because the charged portion of the molecule does notallow them to cross into the central nervous system where acetylcholinesteraseinhibition is most harmful. We recently characterized a small moleculereactivator (4‐amino‐2‐((diethylamino)methyl)phenol [ADOC]) that does not utilizean oxime but is capable of reactivating nerve agent‐inhibited enzyme as well asor better than current treatments; these results have been used to inform thedesign of additional novel compounds. This study describes the in vitro characterization of these novelcompounds as reactivators of phosphonylated human acetylcholinesterase.Support or Funding Information*Thisresearch was supported in part by an appointment to the Postgraduate ResearchParticipation Program at the U.S. Army Medical Research Institute of ChemicalDefense administered by the Oak Ridge Institute for Science and Educationthrough an interagency agreement between the U.S. Department of Energy and theU. S. Army Medical Research and Materiel Command.Thisresearch was supported by the Defense Threat Reduction Agency – Joint Scienceand Technology Office, Medical S&T Division.The views expressedin this abstract are those of the author(s) and do not reflect the officialpolicy of the Department of Army, Department of Defense, or the U.S.Government.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The structural diversity and the biological meaning of antibody
Authors: HIFUMI EMI , AKIYOSHI YUKO , UDA TAIZO 
Published: June 2021

Along with the development of antibody drugs and catalytic antibodies, the structural diversity issue has attracted much attention. Structural diversity(or structural heterogeneity) is generated caused by the modification of different charges, different molecular sizes, and/or modified amino acid residues. Namely, an antibody takes multi‐molecular forms in the solution. It is desirable that the antibody and/or the subunits must have a defined structure for practical use. Regarding with the difficult issue on the structural diversity of antibody, we found the interesting phenomena in which copper ion can convert the multi‐molecular forms of antibodies to mono‐molecular form (1). The ion hugely contributed to the enrichment of the dimer‐form and the homogenation of the differently charged full‐length of the light chain. The role of copper ion must be significant for preparing a single, defined, not multiple, isoform structure.In addition, we found that the constant region domain (CL) of the antibody light chain also plays an important role in the structural heterogeneity of the light chain (2), which was clarified by expressing the constant domain. Considering the chemical features of the full length light chain (VL+CL) and CL, note that the CL domain can influence to generation of the structural diversity of the VL domain. This finding is important, because, even at the present, the function of CL is still unknown. It is considered that the generation of the structural diversity of a protein may influence on the binding ability as well as the specificity.In the presentation, the authors will discuss not only the reason why antibodies take such structural diversity but also their biological meanings.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of CNTM on high‐fat diet‐induced obesity in C57BL/6J mice
Authors: Cho Bong‐Yeon , Choi Sun‐Il , Jung Tae‐Dong , Choi Seung‐Hyun , Sim Wan‐Sup , Han Xionggao , Ra Moon‐Jin , Kim Sun‐Young , Kang Il‐Jun , Han Kyoung Chan , Kim Young‐Cheul , Lee Ok‐Hwan 
Published: June 2021

Cirsium setidens Nakai, a wild perennial herb, grows mainly in Gangwon province, Korea, and it has been reported to contain bioactive ingredients with various medicinal activities including treat edema, bleeding and hemoptysis. In our previous studies have demonstrated that C. setidens nakai ethanolic extract had an anti‐adipogenic effect during the adipogenesis of 3T3‐L1 cells and beneficial effect on high‐fat diet (HFD)‐induced obesity in C57BL/6J mice. The purpose of this study describes a preliminary evaluation of the anti‐obesity activity on Cirsium setidens Nakai ethanolic extract containing test material (CNTM). Also, this study was to evaluate the effects of CNTM high‐fat diet‐induced obesity in C57BL/6J mice. To study for anti‐obesity agents effectively, we first examined the effect of CNTM on body weight gain and relative weight of the adipose tissue. In addition, we investigated the beneficial effect of CNTM on high‐fat diet (HFD)‐induced obesity in C57BL/6J mice. C57BL/6J mice were fed a high‐fat diet to induce obesity and were orally administered CNTM (160, 320 mg/kg) for 8 weeks. CNTM‐treated groups were reduced body weight, adipose tissue weight and improved serum lipid profiles and adiponectin protein expression in obese C57BL/6J mice fed a high‐fat diet. These results suggested that CNTM may have an anti‐obesity effect through the regulation of transcription factors related to adipogenesis, lipogenesis, and lipolysis. metabolism., hypertension, and diabetes. This study suggests that CNTM are potential candidates as anti‐obesity agents for treatment of obesity as non‐toxic natural resources.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of High‐throughput Assays for Testing of Potential Inhibitors of the Oncogenic G12C Mutant of KRas
Authors: Vertessy Beata G , Nyiri Kinga , Steger Anett , Koppany Gergely 
Published: June 2021

Objective and BackgroundWe aim at creating and optimizing in vitro assay methods to follow binding and potential covalent linkage of small drug‐like compounds to the G12C mutant of KRas. Oncogenic mutations of Ras proteins are found in numerous cancers. The glycine 12‐position in KRas is known to harbor several oncogenic mutations, such as cysteine, aspartate and valine. All of these mutations efficiently prevent GAP binding to mutant KRas thereby resulting in a constitutively active GTP‐bound KRas conformation that drives oncogenic processes. High‐throughput assays can facilitate identification of novel drugs that specifically target the GDP‐bound inactive KRas mutant and lock it in this inactive conformation, such as eg the recently reported ARS‐853 (1).MethodsKRas wild type and G12C was expressed and purified. Assays for thermal stability of proteins and their complexes were run on 96‐well plates using Thermofluor in a real‐time PCR instrument. Fluorescent‐based assays for thiol reactivity and (mant) GTP/GDP exchange were run on 384‐well or 96‐well plates, respectively, using a plate reader equipped with kinetics module.ResultsThermofluor assays showed that a distinct and well‐reproducible upward shift in the thermal melting point of KRas G12C upon binding to ARS‐853. The thiol‐reactivity assay was optimized with regards to protein concentration, since we found only about 25% of the expected signal was observable. The GTP/GDP exchange assay indicated that evaluation of the kinetic parameters of the exchange assay are essential to characterize functional effects of ARS‐853 that are not revealed in simple equilibrium end‐point titrations.ConclusionsCombination of assays focusing on protein stability, thiol reactivity and nucleotide‐exchange resukt in a complex test system that can distinguish drug‐like compounds that bind to KRas with or without functional effect. This test system is proposed for testing compound libraries.Support or Funding InformationSupported by the National Research, Development and Innovation Office, Hungary, project number NVKP‐16‐1‐2016‐0020This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autophagy Regulates Bile Acid Metabolism via a NRF2‐FXR Signaling Axis
Authors: Khambu Bilon , Yan Shengmin , Huda Nazmul , Liu Gang , Hong Honghai , Chen Xiaoyun , Yin Xiao‐Ming 
Published: June 2021

Autophagy is a vital intracellular degradation and recycling process which is required for hepatic metabolic homeostasis. Deficiency of autophagy in the liver causes severe injury that may be causatively related to disturbed metabolism. However, it is not clear how autophagy mechanistically contributes to hepatic metabolism and whether the disturbed hepatic metabolism could be a contributing factor for hepatic injury. Here we found that hepatic deletion of Atg7 or Atg5 resulted in dysregulated bile acid (BA) metabolism, with a downregulation FXR functionality. Reduced FXR resulted in intrahepatic cholestasis due to reduced expression of Bsep, a key bile acid transporter at the apical surface of hepatocytes. Further studies indicated that an excessively high level of NRF2 could transcriptionally repress the expression of FXR. Pharmacologically or genetic activation of FXR in autophagy‐deficient liver corrected the BA metabolism and hence alleviated the cholestatic liver injury. Thus, excessive activation of Nrf2 mediated FXR repression is responsible for the dysregulation of BA metabolism and hence the cholestatic liver injury. This exciting new finding suggests that autophagy regulates hepatic metabolism beyond nutrients breakdown by controlling the NRF2‐FXR signaling components.Support or Funding InformationDepartment of Pathology and Laboratory Medicine, Indiana University School of MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inflammatory cell death‐related proteins as potential biomarkers in Takotsubo syndrome
Authors: Borodzicz Sonia , Czarzasta Katarzyna , Wojciechowska Małgorzata , Puchalska Liana , Główczyńska Renata , Kołodzińska Agnieszka , Opolski Grzegorz , Cudnoch‐Jędrzejewska Agnieszka 
Published: June 2021

BackgroundTakotsubo syndrome (TS), triggered by emotional or physical stress, is prevalent in postmenopausal women and is characterized by an acute and usually reversible left ventricular (LV) wall motion abnormalities. Histopathological analyses of TS animal models revealed presence of single cardiomyocyte cell death with features of inflammatory reaction, however the type of cell death in TS and its impact on the course of the disease has not been explained so far.PurposeThe aim of the study was to evaluate the plasma concentration and release dynamics of proteins associated with inflammatory cell death (pyroptosis, necroptosis) and apoptosis in female rat model of TS.Methods9‐weeks old Sprague Dawley rats were ovariectomized and after 3 weeks received an intraperitoneal injection with a single dose of 150 mg/kg isoprenaline (TO group). The control rats were ovariectomized and received an injection of 0.9% NaCl (CO group). Echocardiography and blood collection was performed 6 (TO6h), 12 (TO12h), 24 (TO24h), 72 (TO72h) hours, and 10 (TO10d) days after ISO or NaCl administration. The plasma concentrations of cell death‐related proteins including: interleukin 1 beta (IL1b) and caspase 1 (casp1) involved in pyroptosis, caspase 3 (casp3) in apoptosis, mixed lineage kinase domain like pseudokinase (MLKL), receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) and calcium/calmodulin‐dependent protein kinase type II delta chain (CAMK2d) in necroptosis were evaluated.ResultsThe echocardiographic examination in TO group revealed several types of left ventricular wall motion abnormalities, including basal, mid‐basal and apical akinesia, as well as a mixed variant. In comparison to the CO group, the median plasma concentration of IL1b was significantly higher in the TO6h (p<0.01), TO12h (p<0.01), TO24h (p<0.05) and TO72h (p<0.001) series. Moreover, when compared to the CO group, the median plasma concentration of CAMK2d was significantly higher in TO6h (p<0.001), TO12h (p<0.001), TO24h (p<0.001), TO72h (p<0.001) and TO10d (p<0.05) series. No significant differences in mean concentrations of casp1, casp3, MLKL and RIPK1 between CO and TO groups were found.ConclusionsSignificant variability of concentration and release dynamics of inflammatory cell death‐related proteins, including pyroptosis and necroptosis, indicates their potential role in pathogenesis of TS and suggests the potential use of them as biomarkers in TS.Support or Funding InformationThe research is funded by “Diamond Grant” from the Polish Ministry of Science and Higher Education (project No. DI2015 003045).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.