bluuebunny/crossref_metadata_2025_split huggingface dataset:
Postconditioning Effect of PDE5 inhibitor, Sildenafil in Normal and Diabetic Rabbits following Myocardial Ischemia/Reperfusion injury.
Authors: Ockaili Ramzi , Samidurai Arun , Filippone Scott M. , Cain Chad K. , Das Anindita , Kukreja Rakesh C. 
Published: June 2021

BackgroundSeveral studies have demonstrated cardioprotective effects of the phosphodiesterase 5 inhibitor, sildenafil (SILD), in the setting of myocardial ischemia and reperfusion (MI/R) in mice and rabbits. However, it is unclear whether sildenafil exerts postconditioning effect in normal and diabetic rabbits.Methods and ResultsTo induce diabetes, alloxan monohydrate (125 mg/kg, i.v.) was administered in New Zealand white male rabbits (n=10; ~3 kg, 4 months old). Blood glucose level increased to 342±21 mg/dL following 2 weeks of alloxan administration. Balloon occluders were implanted on the top of the coronary artery and 7 days later, conscious rabbits were subjected to 45‐min ischemia (I) and 3 days of reperfusion (R) by inflating/deflating the balloon. Saline (as control) or Sildenafil (0.71 or 1.4 mg/kg; i.v.) was infused for 60 min starting 5 min before reperfusion. Myocardial infarct size was determined by triphenyltetrazolium chloride staining and troponin I (cTnI) was measured using ELISA method in the plasma. The result showed that sildenafil (both concentrations) significantly reduced myocardial infarct size (Figure A) and peak plasma levels of cardiac troponin I. Similarly, sildenafil reduced infarct size in diabetic rabbits (DM) following MI/R (Figure C). There were no differences in hemodynamics during MI/R or myocardial area‐at‐risk (AAR) (Figure B and D).ConclusionSildenafil exerts significant postconditioning effect in normal and diabetic rabbits of conscious I/R model that is potentially is free of confounding factors associated with open‐chest preparations.Support or Funding InformationNIHRO1HL134366 (AD & RCK), RO1HL118808 (RCK), RO1HL133167 (RCK)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bilirubin, a novel endocrine hormone with fat burning properties
Authors: Hinds Terry D. , Gordon Darren M. , Combs Shauna D. , Stec David E. 
Published: June 2021

Plasma bilirubin levels have been shown to be inversely related to obesity, diabetes, and cardiovascular events. Numerous clinical and population studies have demonstrated that increased serum bilirubin levels protect against metabolic diseases such as obesity and diabetes. Bilirubin is a potent antioxidant, and the beneficial actions of moderate increases in plasma bilirubin have been thought to be due to the antioxidant effects of the bile pigment. In the present study, we found that bilirubin has a new function as an endocrine hormone that can signal to the fat burning nuclear receptor, peroxisome proliferator‐activated receptor α (PPARα). The bilirubin‐PPARα mediated effects cause a significant decrease in lipid accumulation in adipose and liver of mice. Bilirubin is conjugated in the liver by UDP‐glucuronosyltransferase 1‐1 (UGT1A1) protein and then excreted into bile. Furthermore, we found that blockade of the UGT1A1 by either anti‐sense morpholinos or by genetic mutation caused significantly raised plasma unconjugated bilirubin levels which also heightened PPARα transcriptional activity in liver and adipose. Ultimately, increasing plasma unconjugated bilirubin decrease adiposity and lower blood glucose levels. These data indicate a new function for bilirubin as an endocrine hormone that can be regulated in the liver to control adiposity and insulin resistance in peripheral tissues. Targeting of UGT1A1 in the liver may offer future implications in the treatment of obesity and type II diabetes.Support or Funding InformationThis research was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K01HL125445 (T.D.H.) and PO1HL‐51971 (D.E.S) and National Institute of General Medical Sciences P20GM‐104357 (D.E.S.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Excessive βeta‐adrenergic receptor stimulation induces cardiomyocyte necroptosis via a RIP3‐dependent pathway
Authors: Wu Penglong , Wang Yibin , Wang Xuejun 
Published: June 2021

Background/HypothesisExcessive β‐adrenergic receptor activation causes maladaptive cardiac remodeling in humans with myocardial infarction. Cardiomyocyte apoptosis and necrosis are both considered as the main modes of cell death in β‐adrenergic receptor agonist isoproterenol (ISO)‐induced myocardial injury but the mechanism underlying necrosis remains elusive. Necroptosis is a newly discovered form of regulated cell death that exhibits morphological characteristics of necrosis but is regulated by the receptor interacting protein kinase (RIP) 3‐dependant pathway. Necroptosis has been suggested to play a role in the pathogenesis of common cardiovascular diseases. During necroptosis, the formation of necroptosomes consisting of proteins including RIP3 and RIP1 is critical. More recently, Ca2+/calmodulin‐dependent kinase II (CaMKII), which can be phosphorylated and thereby activated by β‐adrenergic receptor activation, was also reported to be involved in cardiomyocyte necroptosis by directly binding to and being phosphorylated by RIP3. However, whether ISO‐induced cardiomyocyte death involves necroptosis requires further investigation. Hence, we sought to test the hypothesis that RIP3‐depedent cardiomyocyte necroptosis contributes to ISO‐induced cardiac injury.Methods and ResultsYoung adult mixed‐sex wild type (RIP3+/+) or RIP3 global knockout (RIP3−/−) C57BL/6 mice were subject to two consecutive subcutaneous injections of ISO (85mg/kg body weight/injection, 24h interval) or vehicle, myocardial tissues were collected at 24h after the 2nd ISO injection. Evan's blue dye (EBD) was administered (100ug/g body weight, i.p.) at 18h before tissues collection for the EBD‐based detection of increased cardiomyocyte membrane permeability, a hallmark of necrosis. In RIP3+/+ mice, the ISO treatment significantly increased myocardial protein expression of RIP3 (both in the soluble and insoluble fractions), RIP1, RIP3 bund RIP1 (as detected by immunoprecipitation), and phosphorylated CaMKII and resulted in increased EDB uptake by a large number of cardiomyocytes (p<0.01). By contrast, the elevation of RIP1 and the number of EBD‐positive cardiomyocytes, but not phosphorylated CaMKII, were significantly less in the RIP3−/− mice subject to the same ISO treatment (p<0.01).ConclusionInduction of myocardial injury by excessive β‐adrenergic receptor activation involves cardiomyocyte necroptosis via a RIP3‐dependent but CaMKII‐independent pathway.Support or Funding InformationThis work is supported in part by grants from the NIH [HL072166, HL085629, and HL131667] and the National Natural Science Foundation of China [81570278]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ascorbate Rescues Peroxide Induced Insulin Resistance in Skeletal Muscle
Authors: Eccardt Amanda , Mattathil Lyn , Bell Thomas , Patel Rishi , Mannino Mark , Fisher Jonathan 
Published: June 2021

Ascorbate plays a key antioxidant role in protection of cells from damage by reactive oxygen species, which have been implicated in causing metabolic dysfunction such as diabetes. Thus, we hypothesized that ascorbate could aid in rescue from insulin resistance brought on by exposure to reactive oxygen species. In addition, we hypothesized that the peroxidase mimetic Fe(III)tetrakis(4‐benzoic acid)porphyrin (FeTBAP) could act synergistically with ascorbate in defense against oxidative stress. In order to measure the protective effect of ascorbate, we pretreated C2C12 myotubes with ascorbate. We then exposed the myotubes to glucose oxidase (GO), an enzyme that utilizes glucose to produce hydrogen peroxide, thus mimicking a state of oxidative stress prior to exposure to insulin. Data were quantified via western blot analysis monitoring phosphorylated AKT and total AKT. In order to assay the ability of FeTBAP in conjunction with ascorbate to protect against insulin resistance, C2C12 myotubes and isolated soleus were first pretreated with ascorbate and FeTBAP and then exposed to glucose oxidase followed by insulin. We found that ascorbate rescues insulin resistance brought on by exposure to reactive oxygen species. In addition, preliminary data from C2C12 myotubes and soleus illustrated a trend in which FeTBAP in combination with ascorbate protected against insulin resistance. Taken together, these data demonstrate a functional role of ascorbate in which the antioxidant on its own and in conjunction with FeTBAP rescues cells from peroxide induced insulin resistance.Support or Funding InformationThis project was supported by United States Public Health Service award R15DK102122 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Promoting ubiquitination‐mediated degradation of Orai1 protein by high glucose in glomerular mesangial cells
Authors: Chaudhari Sarika , Hui Jiang , Zou Shubiao , Huang Linjing , Ma Rong 
Published: June 2021

Glomerular mesangial cells (MCs) are an important target of metabolic abnormalities in a diabetic environment. The Orai1‐mediated store‐operated calcium entry (SOCE) is associated with many physiological processes in a variety of cells, including MCs. However, whether SOCE in MCs is involved in diabetic kidney disease is not clear. High glucose (HG) is the principal cause of MC pathogenesis in diabetes and altered MC function by HG is central to the pathogenesis of progressive diabetic glomerulopathy. The present study was carried out to determine if HG treatment altered the protein content of Orai1 and the protein‐mediated SOCE in MCs. Western blot was conducted to estimate abundance of Orai1 protein and Fura‐2 fluorescence ratiometry was used to analyze SOCE. We found that treatment of rat MCs with HG (25 mM) for time periods ranging from 2 hours to 24 hours decreased the abundance of Orai1 protein. A significant decrease was observed at the time point of 8 hours, which sustained at least for additional 16 hours. Consistently, HG treatment for 8 hours significantly reduced SOCE. HG treatment for the same time periods did not alter the level of Orai1 transcript. In the presence of cycloheximide, a protein synthesis inhibitor, the HG effects on the level of Orai1 protein still existed, suggesting posttranslational mechanisms involved. Furthermore, both MG132 (the ubiquitin‐proteasome inhibitor) and NH4Cl (the lysosomal pathway inhibitor) significantly attenuated the HG‐induced reduction of Orai1 protein abundance. Moreover, HG treatment for 8 hours stimulated ubiquitination of Orai1 protein. We further found that HG treatment elevated the level of cellular hydrogen peroxide (H2O2) in a time‐dependent manner. Treatment of cells with PEG‐catalase significantly blunted the HG‐induced reduction of Orai1 protein abundance. In addition, H2O2 itself also significantly decreased the abundance of Orai1 protein and increased the level of ubiquitinated Orai1. Taking together, these results suggest that HG treatment for a short‐term decreased abundance of Orai1 protein in MCs by promoting its degradation through the ubiquitination‐proteosome and –lysosome mechanisms. This HG‐stimulated posttranslational regulation of Orai1 protein was mediated by H2O2.Support or Funding Information1) NIH/NIDDK: R56 DK108761; 2) NIH/NIDDK: 1RO1DK115424‐01; 3) AHA Southwest Affiliate: 16GRNT27780043; 4) The Harry S. Moss Heart Trust.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Splicing Variation of TTN Novex Isoforms Across Species and RBM20 Does Not Regulate Splicing of Novex Isoforms in Cardiac Muscle.
Authors: Chen Zhilong , Guo Wei 
Published: June 2021

TTN is a major disease‐causing gene in cardiac muscle. TTN contains 363 exons in human encoding various sizes of TTN protein due to alternative splicing regulated mainly by RNA binding motif 20 (RBM20). Three isoforms of TTN protein are produced by alternative exon usages of exon 45 (Novex 1), exon 46 (Novex 2) and exon 48 (Novex 3). It is unknown how these exons are alternatively used in Novex isoforms across species and whether Novex isoforms are associated with heart disease. In this study, we compared alternative exon usage between species with the mVISTA online tool, and found that exon 45 is conserved across all species, but exons 46 and 48 only found in some species. Interestingly, we found a conserved region between exons 47 and 48 across species which has never been characterized before. RT‐PCR and DNA sequencing confirmed that this is a new exon named as 48′ in Novex 3. In addition, with primer pairs for Novex 1, we found two new splicing forms, which one has intron 44 retention and the other has intron 45 retention. With Novex 2 primer pairs, we found that Novex 2 is lack of expression in pig, mouse and rat. A new truncated isoform with intron 46 retention is mainly expressed in human and frog heart, and another new truncated isoform with exon 45 and 46 are co‐expressed predominantly in chicken and frog heart. Using RBM20 knockout heart, we revealed that RBM20 doesn't regulate splicing of Novex isoforms. Finally, we examined the expression levels of Novex isoforms in human heart with cardiomyopathies, and we didn't find any difference. These results suggest that exon usage of Novex isoforms varies across species which is not associated with cardiomyopathies. Hence, their functional role in the heart need be further studied in the future, which is beyond the scope of this study.Support or Funding InformationThis work was supported by the NIFA‐USDA 1009266, National Institutes of Health NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of Wyoming.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the importance of pseudoknot formation to HTLV‐1 pro‐pol ‐1 programmed ribosomal frameshift stimulation.
Authors: Cooper‐Sansone Andrew , Williams Marcus , Chadeayne Devon , Contreras Uriah , Dailey Leandrew , Joe Natalie , Mylroie Elena , Mouzakis Kathryn 
Published: June 2021

The Human T‐cell Lymphotropic Virus Type 1 (HTLV‐1) encodes two ‐1 programmed ribosomal frameshift (PRF) sites, which allow for the translation of enzymes critical to viral replication. The frequency, or frameshift efficiency, of this event is also thought to be important in the viral life cycle. Each PRF site includes a heptanucleotide “slippery” sequence, a spacer, and a downstream structure. The downstream structure in the HTLV‐1 pro‐pol frameshift site is predicted to be a pseudoknot. The importance of this structure to the efficiency of frameshifting has not yet been established. To improve our understanding of the role this structure has in frameshift stimulation, we designed frameshift site variants that disrupt the pseudoknot structure. The frameshift efficiency for these sites was then measured using an in vitro dual‐luciferase assay. Preliminary data suggests that the removal or mutation of sequences important to pseudoknot formation significantly reduce the HTLV‐1 pro‐pol frameshift efficiency. These findings are significant because they suggest that the HTLV‐1 pro‐pol frameshift site pseudoknot structure plays a criticial role in frameshift stimulation.Support or Funding InformationIH NIGMS SCORE SC2 Award (1SC2GM121197‐01), Research Corporation for Science Advancement Cottrell Scholar Award (23983), Fort Lewis College Undergraduate Scholarly and Creative Activities AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanistic interrogation of the entry‐ and exit‐channel arms of eIF3
Authors: Aitken Colin Echeverría , Beznoskova Petra , Dong Jinsheng , Yourik Paul , Valasek Leos , Hinnebusch Alan G. , Lorsch Jon R. 
Published: June 2021

Eukaryotic translation initiation factor 3 (eIF3) is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC. We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning. We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel. Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional and Post‐Transcriptional Regulation of mtlA in Vibrio cholerae
Authors: Wang Jessica , Zhang Mark 
Published: June 2021

Vibrio cholerae, the bacterium that causes cholera, performs a complex regulation of genes encoding carbon‐source transporters in order to survive in both aquatic reservoirs and the human small intestine. One such transporter is MtlA, which operates within the phosphotransferase system (PTS), a highly‐conserved system for carbohydrate uptake across bacteria. MtlA concomitantly phosphorylates and transports mannitol, the six‐carbon sugar alcohol produced by algae, into the cell. In addition to mtlA, the mtl locus contains mtlS and mtlR, which encode two major regulators of MtlA. mtlS encodes a 120‐nucleotide small regulatory RNA (sRNA) that posttranscriptionally regulates MtlA synthesis by binding to the mtlA mRNA 5′ untranslated region. Our objective is to identify regulators of mtlS expression. We have constructed a V. cholerae strain harboring a chromosomal mtlS‐lacZ transcriptional fusion. Using a V. cholerae genomic library, we identified putative regulators of mtlS expression. The second regulator of mtlA, encoded by mtlR, showed repressive effects in Escherichia coli, but had not been well characterized in V. cholerae. We studied how MtlR regulates MtlA activity in V. cholerae grown in a variety of carbon sources. Biofilm assays demonstrated that mtlR overexpression results in a greater than 50% decrease in biofilm formation (p<0.001 by unpaired two‐tailed t‐test), an activity known to be positively affected by MtlA. qRT‐PCR showed that a ΔmtlR mutant has higher mtlA RNA levels than a wild‐type control when grown in glucose‐supplemented minimal medium (p<0.0001 by unpaired two‐tailed t‐test), and even higher mtlA RNA levels when grown in maltose‐ and mannose‐ supplemented minimal medium (p<0.01 by unpaired two‐tailed t‐test). Western blotting analysis reflected these increases; MtlA protein levels were more elevated in ΔmtlR compared to wild‐type when grown in glucose, maltose, and mannose. The ΔmtlR mutant was partially complemented, as demonstrated by qRT‐PCR (p<0.01 by unpaired two‐tailed ttest) and western blotting analysis. This shows that the observed increases in MtlA were a direct effect of the gene product of mtlR. These results contribute to a model in which MtlR acts as a transcriptional repressor of mtlA in non‐mannitol conditions, working synergistically with MtlS to regulate mtlA.Support or Funding InformationNIH R15 AI090606This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of the PIP2 binding sites on ENaC
Authors: Archer Crystal R. , Chen Yinghua , Stockand Alexis M. , Stockand James D. 
Published: June 2021

Hypertension is a major contributor to cardiovascular disease. Much of the details about the molecular mechanisms that regulate blood pressure, though, are obscure. Activity of the Epithelial Na+ Channel (ENaC) is rate limiting for Na+ reabsorption in the distal nephron. Consequently, ENaC activity fine‐tunes plasma volume and blood pressure. ENaC activity is facilitated by plasma membrane‐bound phosphatidylinositol 4,5‐bisphosphate (PIP2). Phospholipase C (PLC)‐mediated depletion of PIP2 reduces ENaC activity. Specific PIP2 binding sites within ENaC, however, remain as yet undefined. We hypothesize that ENaC has multiple PIP2 binding sites within its beta and gamma subunits. In this study, we investigate the direct biochemical binding of PIP2 to specific sites within ENaC subunits by biochemical and electrophysiology methods. We designed synthetic peptides that correspond with regions in the N‐termini of ENaC subunits that are enriched in basic residues. Binding affinities of these channel peptides are tested with fluorescent labeled PIP2 analogs using biophysical methods like microscale thermophoresis. To verify these findings, pulldown of transfected CHO cell lysates using ENaC antibodies followed by dot blot analysis is used to compare the binding of PIP2 to wild type ENaC, with the binding of ENaC containing mutations in the proposed binding sites. To simultaneously analyze the functional importance of these proposed PIP2 sites, we also compare macroscopic currents of wild type and mutant ENaC in responses to activation of PLC. We expect these results to improve our understanding of the regulation of ENaC by PIP2 as we aim to define the specific binding sites and the binding affinity of PIP2 for ENaC subunits.Support or Funding InformationNIH/T32 HL007446This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.