bluuebunny/crossref_metadata_2025_split huggingface dataset:
Month‐long AICAR Treatment Reverses Age‐related mTOR Pathway Hyperactivity
Authors: Wright Nathan A. , Wilcox Shalene H. , Thomson David M. 
Published: June 2021

Sarcopenia is a condition defined by a loss of muscle mass with aging. It is associated with changes in many metabolic pathways. One of these is the mechanistic target of rapamycin (mTOR) pathway, which is chronically activated in aged skeletal muscle under basal conditions. This mTOR hyperactivity is thought to deactivate autophagy, leading to accumulation of defective cell machinery thereby potentially contributing to age‐related muscle dysfunction. AMP‐activated protein kinase (AMPK) acts as an inhibitor of the mTOR pathway. AMPK activation is generally suppressed in sarcopenic muscle, which may contribute to the age‐related increase in mTOR activity. The purpose of this study was to determine if chronic AICAR treatment could reverse mTOR hyperactivation in sarcopenic muscle. To do this, young adult (YA; 5 mo.) and old (O; 23 mo.) C57Bl/6 mice were injected with either AICAR (0.3 – 0.5 mg/g body weight) or saline for one month (31 days). Twenty‐four hours after the final AICAR injection, muscle was harvested and phosphorylation of mTOR‐related signaling components was measured. Sestrin2 protein, which promotes AMPK activity, was decreased in O vs. YA muscles, but was not affected by AICAR. Phosphorylation of both p70S6 kinase (S6k) and ribosomal protein S6 (rpS6) was significantly (p ≤ 0.05) elevated in saline‐treated O vs. YA muscles, similar to previous findings. S6k and rpS6 phosphorylation was unaffected by AICAR in YA muscles, but was decreased by AICAR in O muscles. Eukaryotic elongation factor 2 (eEF2) phosphorylation tended to be elevated in O vs. YA saline‐treated muscles, consistent with our previous results in rats. Since increased S6k activity would promote decreased eEF2 phosphorylation, any age‐related increase in eEF2 phosphorylation would be due to other factors besides S6k. In conclusion, chronic AICAR treatment in old mice reverses the age‐related change in resting mTOR pathway signaling that is observed in O skeletal muscle. This suggests that AMPK activation is a potential strategy for rescuing autophagy and other processes adversely affected by mTOR hyperactivation.Support or Funding InformationThis work was supported by an internal Gerontology Program Grant from Brigham Young University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of High Mobility Group Box 1 to Nicotine‐Induced Podocyte Injury
Authors: Boini Krishna M , Patibandla Sai , Singh Gurinder Bir , Puchchakayala Goverdhan , Koka Saisudha 
Published: June 2021

High mobility group box 1 protein (HMGB1), a nuclear DNA binding protein is released under pathological conditions and locally act as one of the potent damage‐associated molecular patterns (DAMPs) to produce tissue injury and chronic inflammation. However, it remains unknown whether HMGB1 is implicated in nicotine‐induced podocyte injury. In the present study, we found that nicotine dose‐dependently increased the production of HMGB1 in cultured podocytes, and that HMGB1 binder and inhibitor, glycyrrhizin (Gly) completely blocked its release induced by nicotine. Immunofluorescence and western blot analysis showed that nicotine treatment significantly decreased the podocin expression compared to control cells. However, prior treatment with Gly attenuated the nicotine‐induced podocin reduction. Furthermore, western blot analysis showed that nicotine treatment significantly increased the TLR4 expression compared to control cells but not in Gly treated cells. In addition, prior treatment with TLR4 siRNA transfection significantly attenuated the nicotine‐induced podocin reduction in podocytes. Based on these results, it is concluded that HMGB1 is one of important mediators of nicotine‐induced podocyte injury. HMGB1 may be a therapeutic target for treatment or prevention of glomerulosclerosis associated with smoking.Support or Funding InformationNational Institutes of Health (DK104031)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fructooligosaccharides induce tight junction assembly in intestinal epithelial cells via AMPK activation
Authors: Ariyadamrongkwan Jutharat , Wongkrasant Preedajit , Muanprasat Chatchai 
Published: June 2021

Several intestinal and metabolic diseases including inflammatory bowel diseases (IBD), malnutrition, necrotizing enterocolitis and diabetes mellitus (DM) involve impairment of intestinal tight junction assembly. AMP‐activating protein kinase (AMPK) is an energy sensor that enhances tight junction assembly. Fructooligosaccharides (FOS) are well known prebiotics protecting against intestinal and metabolic diseases with unclear mechanisms. This study aimed to investigates effect of FOS on AMPK and tight junction assembly in intestinal epithelial cells (IEC). Using western blot analyses, it was found that FOS with concentrations from 0.001 to 10 mg/ml induced AMPK activation on T84 cells. FOS at concentration of 0.1 mg/ml showed the greatest effect on AMPK activation and it was used in the subsequent experiments. Calcium‐switch assays and fluorescein isothiocyanate (FITC) leakage experiments were then performed. The results revealed that FOS significantly increased percentage of TER at 15 minute, 1 hour, 6 hour, 12 hour and 24 hour post‐treatment. Interestingly, STO‐609, a CaMKKβ inhibitor, suppressed the effect of FOS on increasing TER and reducing FITC leakage. These results indicate that FOS increased tight junction assembly via CaMKKβ‐AMPK activation.Support or Funding InformationThis work is supported by Tonkla Ramathibodi program and Thailand Research Fund and Mahidol University (BRG5980008)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Capacitance in Biochemical Networks: Sequential Reaction Delays
Authors: Bassingthwaighte James B 
Published: June 2021

In an enzymatic reaction network there are four unavoidable, generally neglected, sources of delay: (1) diffusion of substrate S to the enzyme, (2) the rate k1 of binding S to the enzyme E, (3) the retention in the form of the ES complex as S is converted to product P, (4) “volume delay”, the transient delay for concentrations to reach the next steady state after each change in a flux, and (5)flux delay with reversible reactions. Michaelis‐Menten forward flux is the product of the maximal flux, Vmax, times the fractional occupancy of the active site, [S]/(Km +[S]), where Vmax= Etot • k2 , (Etot = enzyme concentration; k2 = rate of product release). This accounts for neither binding/unbinding nor the capacitance of the ES complex. We examine these delays for an unbranched unidirectional reaction sequence A→B→C→D→E, ignoring diffusion delay. The delays are nearly exponential single lags, each with time constant = exchanged mass/flux, e.g. t = ES/(k2 • ES) = 1/k2 . We consider four versions of this reaction set (available at www.physiome.org/Models): #423 forward first order reactions, #424 Michaelis‐Menten forward reaction, #425 M‐M coupled with a correcting time lag of 1/k2 , compensating virtually precisely for the lack of capacitance, and #426, the full enzyme binding and release kinetics. The programs include verification tests, and are open source, human readable differential equations in JSim's MML. Time constants for lags are: for binding substrate, 1/k1; for retention in ES complex 1/k2 ; for the “volume delay”, (Vol ml)/(Clearance in ml/sec) while going from one steady state to the next. A fifth model, #427, “flux reversal”, shows that with simultaneous forward and backward fluxes in an enzyme sequence such as A→B←→C←→D→E, there is increased capacitance or retention in the B←→D block of reactions that is dependent on the relative dissociation constants, Kd's, of the enzyme for binding substrate versus product. Such delays, and resultant flux limitations and increased susceptibility to system instabilities need to be accounted for in characterizing systems especially when branched or feedback‐controlled. Unidirectional M‐M models can be made temporally accurate by adding capacitance‐mimicking lags in series. In the full kinetic models the delays are fully accounted for by equations describing the kinetics directly, including the reversibility delays.Support or Funding Information(Supported by NIH/NHLBI U01‐HL122199)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibitory Effects of Novel Oridonin Analog CYD0617‐2 on Hepatic Stellate Cell Activation through NF‐κB and Stat3 Pathways
Authors: Wang Xiaofu , Cummins Claire , Gu Yanping , Vaishnavi Krishnamurthy , Zhou Jia , Radhakrishnan Ravi 
Published: June 2021

IntroductionInhibition of activated hepatic stellate cell (HSC) proliferation is recognized as a promising therapeutic strategy for liver fibrosis. Previously, we reported that natural compound oridonin exhibits potent anti‐hepatic fibrosis activity, and that NF‐κB and Stat3 signaling contribute to HSC activation. In this study, we determined the anti‐fibrotic effects of a novel oridonin analog CYD0617‐2 in activated HSCs and the underlying mechanism.MethodsThe activated human and rat HSC lines LX‐2 and HSC‐T6 were treated with CYD0617‐2. Cell viability was measured by alamarBlue assay. Apoptosis was determined by Cell death ELISA. Cellular proteins were analyzed with Western blot. Cytokines were determined with ELISA.ResultsCYD0617‐2 treatment significantly inhibited proliferation and induced apoptosis in both cell lines in a dose‐dependent manner approximately 10 fold more potent than the parent compound oridonin. CYD0617‐2 stimulated apoptotic proteins p21, p27, and cleaved‐PARP, while suppressing extracellular matrix proteins fibronectin and laminin. CYD0617‐2 blocked LPS‐induced NF‐κB p65 nuclear translocation and DNA binding activity, prevented LPS‐induced NF‐κB inhibitory protein IκBα phosphorylation and degradation, and suppressed LPS‐stimulated IKKα/β phosphorylation. LPS‐induced NF‐κB cytokines IL‐6, MCP‐1, and IL‐1β were attenuated by CYD0617‐2. The endogenous and LPS‐induced NF‐κB p65 S536 phosphorylation was inhibited by CYD0617‐2. Importantly, NF‐κB chemical inhibitor Bay‐11‐0782 was found to inhibit proliferation and promote apoptosis in both cell lines. Moreover, CYD0617‐2 attenuated Stat3 activation by suppressing pStat3/T705 phosphorylation and target gene c‐myc.ConclusionThe potent anti‐hepatic fibrogenetic effect of CYD0617‐2 may be through suppression of IKK/IκBα/NF‐κB and Stat3 pathway.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BRCT domains contain an intrinsic post‐translational modification (PTM) recognition code that affects it stability
Authors: Davis Jamaine 
Published: June 2021

BRCA1 C‐terminal (BRCT) domains are protein binding modules that relay signals throughout various cellular pathways. The human genome encodes 23 BRCT‐domain containing proteins that are commonly found in tandem and have been characterized as phosphoprotein binding domains with functions throughout the DNA damage response. Emerging evidence suggests these domains bind other post‐translationally modified (PTM) substrates although the details are not defined. Recently, the BRCT domains from BRCA1 were shown to bind an asymmetrically dimethylated arginine (ADMA) p300 substrate to regulate the transcription of the p21 in response to DNA damage. Based on this observation, we hypothesize that the methyl‐specific binding site for the ADMA interaction is distinct from the phospho‐substrate recognition interface. To test this hypothesis, we conducted peptide binding and thermal stability experiments to demonstrate the change in stability upon binding phospho‐versus methylated substrates. Our results show that phosphorylated substrates increase the stability of BRCA1's BRCT domains. Doubly phosphorylated substrates showed the greatest increase in stability while the methylated substrate demonstrated a decrease in stability. To define the BRCT‐ADMA binding interface, we set out to determine the three‐dimensional structure. High‐throughput co‐crystallization of the BRCA1 BRCT protein with a ADMA peptide derived from the p300 substrate identified one condition for crystal growth optimization. In silico modeling of the BRCA1 BRCT structure with the ADMA molecule has highlighted two potential binding sites for the ADMA substrate, which is not conserved throughout the BRCT domain family. Our results indicate that while BRCT domains share a conserved mode of binding phosphorylated substrates, which increases their stability, and their ability to binding methylated substrates may not be conserved. These finding will be critical in understanding how BRCT domains can accommodate binding various modified substrates and regulating signals throughout the DNA damage response.Support or Funding InformationNCI K22 CA166544‐03This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Ribosome Recycling Factor in Translational Coupling as a Ribosome Releasing Factor
Authors: Inokuchi Yoshio , Quaglia Fabio , Kaji Hideko , Kaji Akira 
Published: June 2021

The objective of this presentation is to study the in vivo actions of ribosome recycling factor (RRF) and compare them with those found in vitro. RRF is known to catalyze three separate reactions: release of tRNA and mRNA from the post‐termination complex (PoTC), and splitting of the ribosome of the PoTC. To study the role of RRF in translational coupling in vivo, we used E. coli harboring temperature sensitive (ts) RRF and assayed by following downstream reading of translationally coupled ORF. At the non‐permissive temperature, ribosomes remain on the termination codon of the junction sequence of coupled ORFs and translate downstream ORF without the Shine‐Dalgarno (SD) sequence. The readings were in all three frames due to thermal frameshift at the termination codon. Introduction of sequence complementary to the 3′‐terminal of 16S rRNA into the region surrounding the junction sequence increased downstream reading. When upstream ORF was short, translation of the downstream reading was abolished, suggesting that the ribosomes released by RRF are moving toward the SD sequence of the upstream ORF. The thermal frame shift at the stop codon was also stopped by the upstream SD sequence. Our data suggest that the ribosome‐bound mRNA may take a secondary structure around the junction sequence. For in vitro studies, we used mRNA that incorporated different radioactively labeled amino acids based on the frameshift at the junction sequence, UAAUG, of two translationally coupled ORFs. In the absence of RRF, the ribosome stayed on the mRNA and translated in frame with the termination codon UAA. In the presence of RRF, amino acid incorporation occurred in frame with the start codon AUG. This suggests that RRF releases the ribosome from UAA and the released ribosome binds to AUG and begins translation. With the use of tethered, unsplittable ribosomes (Ribo‐T) in the in vitro system, we showed that complete ribosomal splitting is not required for the action of RRF in translational coupling. Therefore, the main role of RRF in translational coupling appears to be the release of ribosomes from mRNA.Support or Funding InformationSupported and funded by Creative Biomedical Research Institute (CBRI) in Philadelphia, Pennsylvania.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overcoming the Rapid Metabolism of the Promising Anticancer Natural Product Rooperol
Authors: Jemal Mauricio A , Schwartz Zachary T , Zhao Zhenze , Rodebaugh Mary , Christy Quinn M , Shelton Spencer , Du Liqin , Kerwin Sean M 
Published: June 2021

Rooperol [1,5‐bis(3′,4′‐dihydroxyphenyl)pent‐1‐en‐4‐yne] is a naturally occurring bis‐catechol that was originally isolated from the African Potato (Hypoxis hemerocallidea). Rooperol shows promising anti‐cancer activity in vitro, but it is rapidly and completely metabolized to inactive phase‐II metabolites in vivo. To address this problem, analogues of rooperol have been designed in which the linker separating the catechol groups is varied or in which some or all of the catechol groups are replaced with more metabolically stable isosteres in order to explore the role of the bis‐catechol groups and the effect of catechol replacement on anticancer activity. Here we report the synthesis and cancer cell cytotoxicity of these rooperol analogs. Based upon the activity of these compounds in a cell growth inhibition assay with HeLa cells, we conclude that the bis‐catechol nature of rooperol is not an essential feature for its cytotoxicity and that an analog of rooperol that is predicted to be more resistant to metabolism yet which retains much of the anti‐cancer activity of the natural product can be identified.Support or Funding InformationWilliam and Ella Owens Medical Research Foundation and Texas State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Molecular Dynamics Simulations to Evaluate Ligand Binding of L, L‐diaminopimelate Aminotransferase, an Enzyme Involved in Protein and Peptidoglycan Biosynthesis
Authors: Adams Lily , Babbitt Gregory , Hudson André 
Published: June 2021

A common protocol, Site Directed Mutagenesis (SDM) experiments facilitate the alterations to a gene sequence to study downstream effects on its product. In addition, inhibitor/antagonistic studies investigate the potential of common ligands to inhibit or inactivate enzymes. Both approaches have seen minor updates in recent years, but methods to streamline these processes are highly desired. For example, a rate‐limiting step in SDM lies in choosing key nucleotides that will subsequently affect enzymatic activity. To facilitate SDM studies and subsequent analyses to save time and resources, a comprehensive and comparative molecular dynamics (MD) software package – DROIDS (Detecting Relative Outlier Impacts in Dynamic Simulations) 2.0 – has been applied. Here, we use a novel aminotransferase, L, L‐diaminopimelate aminotransferase (DapL) as a test to propose a method of augmenting SDM and related experiments using MD simulations. DapL is involved in peptidoglycan and protein metabolism. As such, the essentiality of the dapL gene in eubacteria is currently a key question yet to be answered. Information to supplement knowledge of this enzyme will aid in evaluating its potential as a narrow spectrum antibiotic target. We have used DROIDS 2.0 to investigate the impact of different point mutations and putative antagonistic ligands on the activity of the DapL enzyme, identifying those that most significantly impact its dynamic behavior. In addition, the DROIDS 2.0 protocol was taken further to predict mutations that lessen binding affinity to chosen inhibitors, which may have the potential to guide the rational design of inhibitory ligands. Comparative dynamics in DROIDS 2.0 can be visualized (Figure 1) in a variety of statistical plots, colored structures, and colored movies of protein movement, enhancing interpretation and communication to a broad audience.Support or Funding InformationNational Institutes of Health (NIH) award (R15GM120653)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Full‐Time Physioxic Cell Culture and Handling Improves MSC Proliferation Over Hypoxic Pre‐Conditioning in Vitro
Authors: Henn Alicia D. , Darou Shannon , Alm Kersti , Yerden Randy 
Published: June 2021

Cells cultured and handled in traditional room air experience a dramatic shift in oxygen levels when administered in vivo. It has been reported that hypoxic pre‐conditioning of mesenchymal stem/stromal cells (MSC) in culture can change cell function, increasing cell survival in vivo after administration, changing cell migration and modulating cytokine and exosome secretion patterns. These treatments place cells in low oxygen atmospheres for lengths of time from 15 minutes to 36 hours, sometimes with a period of reoxygenation in room air conditions before administration, risking oxidative stress. Here, we compare hypoxic pre‐conditioning to full‐time physioxic conditions with the null hypothesis that in vitro culture conditions make no difference to MSC morphology and proliferation rates after the cells are introduced to physioxic conditions as if administered. MSC cultured and handled under completely controlled conditions in the Xvivo System were split into two groups: (1) cells cultured under full‐time physioxia (5%O2), and (2) cells cultured under supraphysioxic room air incubator conditions (18%O2) and then pre‐conditioned in low oxygen (1% O2) conditions for 24 hours before reoxygenation at supraphysioxic room air incubator oxygen (18% O2) for 24 hours. The cells were then exposed to venous blood oxygen levels (5% O2) as if injected in vivo. Using an immersion oxygen probe, we recorded vessel headspace and pericellular medium oxygen levels under each regimen. Using the PHI Holomonitor M4, we recorded holographic time‐lapse images of the cells for changes in cell morphology or proliferation rates. In looking at cell culture oxygen change kinetics, we found that sharp changes in atmospheric oxygen levels were followed by much slower oxygen changes in the vessel headspace, and pericellular oxygen. MSC were actually at low oxygen conditions for far less time than the pre‐conditioning period. Hypoxic pre‐conditioning after incubation at 18%O2 increased the numbers of cells that failed to divide at physioxic oxygen levels, disproving the null hypothesis. We concluded that full‐time physioxic conditions for cell handling as well as incubation may be more conducive to MSC proliferation than hypoxic pre‐conditioning.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.