bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Characterization of Allograft Inflammatory Factor 1‐Like (Aif1L) in Skeletal Muscle
Authors: Smith Anastasia , Waddell David 
Published: June 2021

Skeletal muscle wasting, or atrophy, results from decreased protein synthesis and an associated increase in protein degradation. This shift towards a decrease in protein content of the cell can result from various physiological conditions, including immobilization, denervation, and aging. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has been found to be upregulated during almost all conditions of muscle wasting and is believed to play an important role in targeting proteins for degradation. However, preliminary data described in this study provides new evidence that MuRF1's role in the skeletal muscle atrophy cascade is likely more complex and it appears that MuRF1 is necessary for full transcriptional activation of a small subset of neurogenic atrophy‐induced genes, including allograft inflammatory factor 1‐like (Aif1L). Aif1L has been identified as a novel skeletal muscle gene that is upregulated in wild‐type mice, while MuRF1‐null mice show blunted upregulation in response to denervation. Aif1L is predicated to contain an EF‐hand domain, which can act as a calcium ion binding domain. Furthermore, Aif1L has also recently been implicated in regulating actomyosin contractility and filopodial extensions and may play a similar role in muscle cells. To confirm that Aif1L in expressed in muscle cells, qPCR was used to assess the expression levels in cultured muscle cells, while the cDNA of Aif1L was cloned from the C2C12 mouse myoblast cell line and fused with a green fluorescence protein (GFP) tag. C2C12 cells were then transfected with the Aif1L‐GFP expression plasmid in order to localize the Aif1L protein in proliferating myoblast cells. C2C12 cells transiently overexpressing Aif1L fused to GFP showed punctate fluorescence localized to the perinuclear region of the cytoplasm. The results presented here in combination with additional research into the transcriptional regulation and function of Aif1L will help contribute new insight into the molecular genetic mechanisms of skeletal muscle atrophy and may provide a more complete understanding of how MuRF1 may regulate the neurogenic atrophy cascade during muscle wasting.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BMP‐7 inhibits Pyroptosis in Vascular Smooth Muscle Cells in ATH
Authors: Garner Kaley , Singla Dinender 
Published: June 2021

Atherosclerosis (ATH) is an inflammation‐mediated disease in which lipids, immune cells, and vascular cells contribute to the formation of plaque along vessel walls. Cell death by apoptosis and necrosis have been demonstrated in ATH; however, pyroptosis, an inflammation‐mediated cell death, has not yet been fully elucidated. We hypothesized that pyroptosis of vascular smooth muscle cells contributes to ATH progression. We further investigated whether administration of bone morphogenetic protein‐7 (BMP‐7) can inhibit these effects and demonstrate therapeutic potential. ApoE−/− mice (11±1 week old) were divided into three groups: Sham, PLCA, PLCA+BMP‐7 (200μg/kg; i.v). ATH was generated using our established partial ligation of the left carotid artery (PLCA) model and immunohistochemical staining and western blot analysis of arterial tissue was performed. ATH generated in PLCA mice demonstrated an upregulation in Toll‐like receptor‐4 (TLR‐4) and NLRP3 inflammasome components (NLRP3 and Caspase‐1), indicating the initiation and activation of pyroptosis in vascular smooth muscle (α‐SMA) cells. Further, inflammasome cleavage of pro‐IL‐1β and pro‐IL‐18 to mature forms released through cell membrane pores mediated by Caspase‐11 were investigated. IHC staining of arterial tissue demonstrated a significant increase in IL‐1β and IL‐18 expression following PLCA (p<0.05), which was significantly improved upon treatment with BMP‐7. Western blot analysis of arterial tissue supported these findings, indicating a significant upregulation in pyroptosis initiation via TLR‐4, inflammasome activation (Caspase‐1 and NLRP3) and release of pro‐inflammatory factors, IL‐1β and IL‐18 (p<0.05). Overall, this study indicates that pyroptosis occurs in the vascular smooth muscle cells in ATH and that BMP‐7 administration attenuates pyroptosis significantly.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Necroptosis Resulting from Activation of a RIP3‐dependent Pathway Contributes to Cardiomyocyte Death Induced by Isoproterenol
Authors: Wu Penglong , Wang Yibin , Liu Jinbao , 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 undefined. 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 (85 mg/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 NIH grants HL072166, HL085629, and HL131667This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of Proximal Tubule Cell Endocytic Capacity by Nephrotic Levels of Albumin
Authors: Long Kimberly R , Rbaibi Youssef , Weisz Ora A 
Published: June 2021

The glomerulus and proximal tubule (PT) of the kidney coordinate to prevent the excretion of albumin and other plasma proteins in the urine. Disruption of the glomerular filtration barrier in nephrotic disease overwhelms the endocytic capacity of the PT, leading to proteinuria. Nephrotic levels of albumin are cytotoxic to the PT and trigger cell signaling and adaptive responses. Recent studies have suggested that fatty acids carried by albumin cause mitochondrial oxidative damage that contribute to PT toxicity. However, fundamental questions about the PT response to albumin remain unanswered. Discordant results using different PT cell models, treatment conditions, and outcome measures have further complicated our understanding of how PT cells respond to albumin and its metabolic load in different disease conditions. We used a recently developed cell culture model that recapitulates key in vivo features of PT function to examine how PT cells adapt their endocytic capacity in response to nephrotic levels of albumin. We treated opossum kidney cells cultured under orbital shear stress with physiologic or nephrotic levels of apically‐added Fraction V bovine serum albumin (BSA), fatty acid‐free or palmitate‐loaded BSA, or human serum and monitored the cells for oxidative damage, megalin expression levels, and endocytic capacity. None of these parameters were altered by incubation with fatty acid‐free BSA, however, incubation with 20 mg/mL Fraction V BSA resulted in a rapid and reproducible decrease in endocytic capacity (within 3 h) followed by increased oxidative stress and decreased megalin expression measured after overnight treatment. Palmitate‐loaded BSA and human serum elicited very different profiles of cellular changes (relative to Fraction V BSA and each other) over a 3 – 48 h incubation period. These data suggest that PT cells initiate different adaptive signals in response to the composition of the metabolic load on endocytosed albumin. Our results have implications for the development of therapeutic strategies to mitigate disease‐dependent proteinuric tubular injury.Support or Funding InformationNIH R01‐DK101484; NIH R01‐DK100357; NIH P30‐DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circulating Oligomeric State and Circadian Regulation of CTRP3
Authors: Trogen Greta , Shipley Madison M , peterson Jonathan 
Published: June 2021

Adipose tissue secretes many important biologically active proteins called adipokines. A subset of adipokines, called C1q tumor necrosis factor (TNF) related proteins (CTRPs), play a key role in metabolism, inflammation, and cell signaling. C1q TNF Related Protein 3 (CTRP3) increases hepatic fatty acid oxidation, decreases inflammation, and aids in cardiovascular recovery following a myocardial infarction. However, the mechanisms behind CTRP3's protective effects on organ systems are unknown. This exploratory study aims to analyze the circulating oligomeric state of CTRP3 and the circadian regulation of CTRP3 to help understand the role of CTRP3 in preventing disease.METHODSThe oligomeric state of CTRP3 was analyzed in serum separated by size exclusion filtration collected from non‐fasting high‐fat fed hyperglycemic mice as well as low‐fat fed normoglycemic mice. For analysis of the circadian regulation of CTRP3, serum samples were collected from mice at 4 different times throughout the day (early & late dark cycle and early & late light cycle). Circulating CTRP3 levels were then analyzed by immunoblot analysis.RESULTSIn both high fat (HF) and low fat (LF) fed mice CTRP3 was found to circulate as oligomers above 300kDa as well as between 100 and 300kDa, with no detectable amount of CTRP3 observed below the 100 kDa cutoff. Interestingly, although there was no difference in the total amount of CTRP3 between the HF and LF fed mice, the proportion of CTRP3 above the 300 kDa cutoff was higher in the HF fed mice whereas the majority of CTRP3 in the LF fed mice was found between 100–300 kDa cutoffs. Additionally, we found that serum CTRP3 levels varied greatly throughout a 24‐hour time‐period within each mouse, but no consensus circadian pattern was observed.CONCLUSIONIn vitro mammalian produced recombinant CTRP3 protein exists as trimer, hexamer, and high molecule weight oligomer. This is the first study to indicate that CTRP3 circulates in different oligomeric states in vivo, and this is also the first study to observe a difference in the oligomeric state of CTRP3 related to metabolic state. Combined these findings indicate that oligomeric state of CTRP3 may be more metabolically relevant than total amount of circulating CTRP3. In addition, our finding of a high variability of CTRP3 within the same mouse at different times throughout the day indicates that is not regulated by circadian rhythms but is susceptible to variability due to some unknown regulatory factor. Combined, these findings have identified novel unknown aspects of CTRP3 which require further research to understand the role of CTRP3 in human health and disease.Support or Funding InformationNIH‐R15DK105496This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing Binding Interactions and Elucidating 3D Structure of Aptamer‐Based Biosensors
Authors: Armstrong Lindsay , Bonham Andrew J 
Published: June 2021

Electrochemical DNA biosensors, whose signaling is based on signal change induced by the conformational dynamics of DNA aptamers upon target binding, have found success as sensitive and rapid methods to detect a wide variety of proteins, toxins, antibodies, and heavy metals. However, the precise mechanisms by which these surface‐bound DNA molecules change conformation upon target binding, thus changing the dynamics of an appended redox‐active tag and generating a measurable signal, is poorly understood. Here, we have used gel mobility shift assays and dimethyl sulfate footprinting fragment analysis to investigate 3D structural interactions of DNA aptamer biosensors in the presence and absence of binding targets. These have allowed us to better characterize the individual basepair interactions involved in binding targets, as well as offer clues to the dynamic three‐dimensional folded structures of these biosensors. These results will help inform the field of biosensors and aptamers on strategies for future optimization.Support or Funding InformationThis work is supported by internal grant funding from the Metropolitan State University of Denver.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using a Fluorescent Unnatural Amino Acid to Characterize the Role of Conformational Dynamics in High Fidelity DNA Replication
Authors: Dangerfield Tyler 
Published: June 2021

The DNA polymerase of bacteriophage T7 is a model enzyme for understanding high fidelity DNA replication. Crystal structures of the open, binary (E‐DNA) complex and the closed, nucleotide bound ternary (E‐DNA‐dNTP) complex reveal large structural changes as nucleotide binds and the fingers domain closes around the correct base pair. However, the contribution of this structural transition to DNA replication fidelity has been greatly disputed, and its measurement has been further complicated by the difficulty in site specific fluorescent labeling of the T7 DNA polymerase using traditional cysteine – maleimide coupling chemistry. The work presented here determines the contribution of the motions of the fingers domain of the T7 DNA polymerase to its high DNA replication fidelity, through direct measurement using a fluorescent unnatural amino acid incorporated site‐specifically in the fingers domain of the enzyme. Each step in the pathway of correct nucleotide incorporation was measured using stopped flow and quench flow techniques, and data from all experiments were globally fit in Kintek Explorer to derive rate constants for each step in the pathway. Our data support a model where the conformational change preceding chemistry is not rate‐limiting but is still the major determinant of fidelity, and provides new information on post‐chemistry conformational dynamics of the enzyme and their contribution towards DNA replication fidelity.Support or Funding InformationThe Welch Foundation (F‐1684)This 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 LMAN1 (Ergic‐53) in the trafficking of GABAARs
Authors: Fu Yanlin 
Published: June 2021

LMAN1, also called as ERGIC 53, is known as a cargo receptor that gathers mainly in the tubulovesicular cluster, also referred as ER‐to‐Golgi intermediate compartment (ERGIC), and recycles between ER and Golgi in a COPII and COPI dependent pathways (Hauri, Kappeler et al. 2000). Its C terminus contains a diphenylalanine motif that binds to COPII coat and facilitates the anterograde transport and a dilysine motif that binds to COPI coat and mediates the retrograde transport (Khoriaty, Vasievich et al. 2012). LMAN1 recognizes and binds to the high mannose structure on the glycoprotein through its luminal carbohydrate recognition domain (CRD) (Zheng, Liu et al. 2010). LMAN1 is the known specific cargo receptor for factor V and factor VIII, cathepsin C, cathepsin Z and α1‐antitrypsin (Hauri, Kappeler et al. 2000, Zheng, Liu et al. 2010).Our lab's initial mass spec data shows that LMAN1 is one of the molecules that interact with GABAARs. We hypothesize that LMAN1 is a trafficking factor for GABAARS receptors. We have several new findings: firstly, endogenous LMAN1 interacts with at least α1 subunits in HEK293T cells overexpressed with WT GABAARs. This interaction is not dependent on the glycosylation status of α1 subunits. And the carbohydrate recognition domain (CRD) of LMAN1 is critical in the interaction between α1 subunits and LMAN1. Secondly, knocking‐down of LMAN1 in mouse hypothalamic GnRH neuronal (GT17) cells negatively affected the surface expression level of endogenous α1 and β3 subunits. We conclude that LMAN1 is also a trafficking molecule for multi‐subunits transmembrane GABAARs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Chemical Compounds that Inhibit the Function of Initiation Factors I and III from Pseudomonas aeruginosa
Authors: Cantu Natalie M , Hu Yanmei , Bullard James M 
Published: June 2021

BackgroundPseudomonas aeruginosa is a bacterium that is known for its increasing rate of antibiotic resistance. As an essential metabolic process, protein synthesis is a target for the development of new antibiotics. Initiation of protein synthesis is stimulated by three initiation factors (IF), in which two of them (IF1 and IF3) act to facilitate formation of an active 30S initiation complex and are released from the 30S initiation complex as a result of the addition of the 50S ribosomal subunit to form the 70S ribosomal complex.Methods and ResultsWe previously developed a protein synthesis system in the form of the aminoacylation/translation (A/T) assay which was used to screen for inhibitors of protein synthesis. This system allows the polyU mRNA dependent synthesis of poly‐Phe peptides and in the presence of low concentrations of IF1 and IF3 the activity is enhanced. Titration of either IF1 or IF3 into the A/T assay results in reduced activity. Treating IF1 and IF3 as inhibitors of activity of the A/T assay allows the IC50 for each to be determined (127 and 10.9 μM, respectively). The effect is additive and addition of both results in increased inhibition of protein synthesis. Holding IF3 at 10 μM, and titrating IF1 into the assay decreases the IC50 of IF1 to 6.6 μM. This assay can be used to detect inhibitory effects of the function of IF1 and IF3. If either IF1 or IF3 is inhibited the activity of the A/T assay is restored. Development of this assay into a screening platform to detect compounds with the ability to inhibit the function of IF1 or IF3 allowed the screening of a synthetic compound library from ChemDiv, Inc. containing over 2000 distinct compounds. A number of inhibitory compounds were confirmed and are currently being characterized for the ability to inhibit both the enzymatic activity (IC50) of the initiation factors and growth (MIC) of a panel of problematic pathogens in culture. They are also being analyzed to determine the potential toxicity to human cell cultures.ConclusionIF1 and IF3 at elevated concentrations block the activity of the P. aeruginosa protein synthesis assay. In this format they are being screened to identify chemical compounds that inhibit their function to interact with the 30S initiation complex and thus restore the ability of the A/T assay to carry out protein synthesis. This assay allows us to specifically identify inhibitors of the initiation phase of protein synthesis in P. aeruginosa and may lead to identification of potential broad spectrum antibacterials.Support or Funding InformationNational Institutes of Health (grant number: 1SC3GM098173)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification of Palmitoyl Protein Thioesterase and Acyl Protein Thioesterase for use in In Vitro Depalmitoylation Reactions
Authors: Esoe Dave‐Preston , Erickson Jeffery A , Hamann Michael J 
Published: June 2021

Palmitoylation refers to the attachment of palmitate to protein cysteine residues and assists with protein localization to the plasma membrane. Like protein phosphorylation, palmitoylation is often a dynamic process, and it involves the attachment of the palmitate by palmitoyl transferases and removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Currently, the techniques to study palmitoylation are difficult to perform and involve the use of harsh chemical reagents like hydroxylamine (HAM) to depalmitate proteins, often leading to destruction of the proteins being studied. Recombinant, purified PPT/APTs could be used as an alternative reagent to depalmitate proteins and also to investigate their individual specificities in depalmitoylation reactions. To purify APTs and PPTs, the coding sequences of human variants were PCR amplified, cloned and expressed to produce either GST or MBP‐tagged fusion proteins. Expressed proteins have been analyzed using SDS‐PAGE and bands of expected molecular mass have been identified. Enzyme activity is currently being tested with the substrate 4‐methylumbelliferyl‐6‐thiopalmitoyl‐b‐glucoside (MU‐6S‐palm‐bGlc) in a fluorogenic enzyme assay, with the goal of eventually using the fusion proteins in depalmitolyation reactions involving palmitoylated protein substrates.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.