bluuebunny/crossref_metadata_2025_split huggingface dataset:
Omega‐3 polyunsaturated fatty acids protect endothelial adhesion of monocytes through FFA4 in monocytes
Authors: Lee Seung‐Jin , Im Dong‐Soon 
Published: June 2021

A G protein‐coupled receptor (GPCR) named free fatty acid receptor 4 (FFA4, also known as GPR120) was found to act as a GPCR for omega‐3 polyunsaturated fatty acids (n−3 PUFA). Recently, FFA4 is known to participate in the progression of atherosclerosis, however, the underlying mechanisms are unclear. Thus, this study investigated the molecular mechanisms responsible for monocyte adhesion to the vascular endothelium, which is a key early event in macrophage foam cell formation. In ex vivo study, LPS‐induced the primary monocyte adhesion to the vascular endothelial cells were significantly attenuated by pretreatment of EPA and DHA, a representative n−3 PUFA. This EPA and DHA‐induced inhibition were blunted by primary monocytes isolated from FFA4 (−/−) mice. In in vitro study, The LPS‐induced monocyte adhesion to the endothelial cells and adhesion molecule, Mac‐1 expression were significantly attenuated by pretreatment of EPA and DHA in monocytes. This EPA and DHA‐induced inhibition was blunted by pretreatment of AH7614 (a FFA4 antagonist) and by transfection of FFA4 siRNA, suggesting a pivotal role of FFA4 on LPS‐induced monocyte adhesion to the endothelial cells and Mac‐1 expression. In addition, in promoter activity analysis and chromatin immunoprecipitation assays to identify transcription factors involved in EPA and DHA inhibited LPS‐induced Mac‐1 expression, both AP‐1 and NF‐κB played central roles to increase Mac‐1 transcription in LPS‐treated monocytes. The results of this study suggest that n‐3 PUFA protect monocyte adhesion to vascular endothelial cells and Mac‐1 expression via signaling pathways involving AP‐1 and NF‐κB by activating FFA4 in monocytes, leading to increased foam cell formation in the development of atherosclerosis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resolvin D1 interferes with several of the required steps for angiogenesis
Authors: Fatima Farheen , Garrett Joshua 
Published: June 2021

New growth of blood vessels, angiogenesis, is essential in cancer development since the proliferation, as well as metastatic spread, of cancer cells depends on an adequate supply of oxygen and nutrients and the removal of waste products. Endothelial cells, which line the inside of the entire vasculature, play an important role in angiogenesis. Specifically, endothelial cells detach from an existing blood vessel by down‐regulating their junctional cell‐cell adhesion protein complexes, they migrate away from the existing blood vessel, and they proliferate, all in an attempt to form a new blood vessel. Resolvin D1 (RvD1) is an autocoid molecule which possesses the ability to strengthen endothelial cell‐cell adhesion leading us to hypothesize that RvD1 may inhibit some of necessary steps for angiogenesis to occur. Here we show that treatment of endothelial monolayers with RvD1 increases the amount of junctional vascular endothelial‐cadherin (VE‐Cad) and slows endothelial cell migration. We also found that RvD1 slightly decreased endothelial cell proliferation. Taken together, we found that treatment of the endothelium with RvD1 inhibits several of the essential steps required for angiogenesis to occur. This suggests that RvD1 may be a useful therapeutic in the treatment cancer as it may decrease angiogenesis, preventing cancer cell proliferation and metastasis.Support or Funding InformationThis work was supported in part through the Hartwick College Faculty research grants program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pioglitazone Improves the Maternal High Fructose Diet‐Impaired Glycolysis and Oxidative Phosphorylation of Astrocytes
Authors: Wu Kay Li‐Hui , Wu Chih‐Wei 
Published: June 2021

Accumulating reports suggest that cell metabolism in the offspring is disturbed by maternal nutritional insults, resulting in the development of metabolic diseases in the offspring. Our previous study further indicated that the spatial learning and memory is impaired in female offspring by maternal high fructose diet (HFD). However, the underlying mechanisms are largely unknown. Astrocytes, a non‐neuronal cell type in the brain known for metabolic support to neurons, elevate glycolysis and oxidative phosphorylation rates in response to energy demand arising from the high neuronal activity. In this study, we dissected the profiles of glycolysis and oxidative phosphorylation of hippocampal astrocytes obtained from female offspring of maternal regular diet (ND) and of maternal HFD. The results indicated that maternal HFD impaired astrocytic glycolysis, oxygen consumption rate and the activities of electron transport chain. Moreover, upregulation of p85 subunit of phosphatidylinositide 3‐kinases (PI3K), down‐regulation of glucose transporter 1 (GLUT1) and a decrease of glucose uptake were detected in the maternal HFD‐programmed astrocytes. An insulin sensitizer, pioglitazone, treatment effectively improved the impaired glycolysis and oxidative phosphorylation and enhanced the astrocytic ATP content. Moreover, the suppressions of GLUT1 and glucose uptake in the HFD group were reversed to the ND level. Together, these pieces of evidence suggested that maternal HFD during gestation and lactation impairs the astrocytic energy metabolism that can be normalized by promoting insulin signaling.Support or Funding InformationThis work was supported by grants from CMPRG8F0062 and CMRPG8E0263 to KLHW from the Chang Gung Memorial Hospital, Taiwan, Republic of China.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serum albumin as a model protein in designing an undergraduate laboratory course in biochemistry
Authors: BROWNING SARAH E , ODUNUGA ODUTAYO O 
Published: June 2021

A previous work on the design of an undergraduate laboratory course in biochemistry using serum albumin as model protein, presented at the EB 2017 conference was widely accepted. Based on comments and suggestions from faculty colleagues, we are extending the project. Extensions to the course include using fluorescence spectroscopy to monitor and determine the kinetic parameters of chemically induced unfolding of serum albumin. Serum albumin has desirable properties that make the protein a suitable model for the laboratory course design. The protein is highly abundant in blood plasma or serum; it is very soluble and stable in a wide range of buffer, pH and salt conditions. The structure of albumin makes it an ideal candidate for students to study protein structure‐function relationship using spectroscopic methods such as fluorescence, circular dichroism, infrared and UV‐Visible spectroscopies. Of particular note is the robustness of the course in terms of cost‐effectiveness, skill‐sets and pedagogical aims. All the data presented herein were generated and analyzed by students under the guidance of the professor. This is a classic example of using faculty research as a teaching tool.Support or Funding InformationWelch Foundation Grant # AN‐0008This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Protein Arginine Methyltransferase 5‐mediated Symmetric Dimethylation of Histone H4R3 as a Target of Endocrine Disruptors and its Molecular Consequences
Authors: Ghosh Krishna , Chatterjee Biji , Kanade Santosh R 
Published: June 2021

Protein arginine methyltransferase 5 (PRMT5) is a type II PRMT that catalyzes the symmetric dimethylation of arginine residues of many histone and non‐histone proteins. The resultant symmetrically dimethylated arginine on histone H4 (H4R3me2s) is a critical histone mark found to be associated with repressed genes. Endocrine disruptors (EDs) are chemicals whose environmental and occupational exposure significantly affects the endocrine homeostasis and have been associated with health hazards. However, the underlying mechanisms including epigenetic alterations are not well understood. In the present study, we show that PRMT5/H4R3me2s is a target of four common environmental EDs (bisphenol A, 4‐nonylphenol, di‐2‐ethylhexyl phthalate, and cadmium) resulting in cellular proliferation in vitro. Exposure of these four EDs to the MCF‐7 or HEK293 cells exhibited a significant increase of both PRMT5 expression and global level of H4R3me2s. The western blotting and chromatin immunoprecipitation analysis suggest these chemicals coincidently increased the level of specificity protein 1 (SP1) and nuclear factor Y alpha (NFYA) and their enrichment in the PRMT5 proximal promoter. Interestingly, all the EDs exerted dose‐dependent inhibitory action on p21CDKN1A expression while cyclin D1 expression was increased. Finally, we demonstrated an enhanced selective enrichment of H4R3me2s at the proximal promoter of p21 but not cyclin D1. We propose this significant alteration in the p21 promoter might have contributed in increased recruitment of DNMT3A and HDAC1 and subsequent hypermethylation of p21 promoter CpG island and deacetylation of the promoter histones. As PRMT5 is an oncogene often overexpressed in many cancers, elevation of its activity and function by these exogenous EDs could potentially initiate cancer through epigenetic mechanisms as observed in this study.Support or Funding InformationThe authors are thankful to SERB, Central University of Kerala, and KSCSTE for financial support for the experiments.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Insights into Di‐acetyllysine Histone Recognition by the BRPF1 Bromodomain
Authors: Obi Juliet 
Published: June 2021

Bromodomains are often found in chromatin‐modifying complexes, whose activity can lead to aberrant expression of genes that drive certain diseases in humans, including cancer, neurological disorders and inflammation. The bromodomain‐PHD finger protein 1 (BRPF1) is a part of the MOZ (monocytic leukemic zinc‐finger protein) HAT (histone acetyltransferase) complex implicated in chromosomal translocations known to contribute to the development of acute myeloid leukemia (AML). BRPF1 contains a unique combination of post‐translational modification (PTM) reader domains, including two plant homeodomain (PHD) fingers, a bromodomain, and a proline‐tryptophan‐tryptophan‐proline (PWWP) domain, through which it is known to modulate the HAT activity of MOZ. We hypothesized that the BRPF1 bromodomain can recognize histone ligands with multiple acetyllysine marks, and that the recognition can be modulated by PTMs adjacent to these marks. We carried out isothermal titration calorimetry (ITC), analytical ultracentrifugation (AU), and site‐directed mutagenesis experiments with the purified BRPF1 bromodomain to investigate its binding affinity to different di‐acetyllysine histone ligands. Our data reveal the BRPF1 bromodomain is able to bind several histone ligands containing di‐acetyllysine, and modifications adjacent to the acetyllysine modifications alter the binding affinity. Notably, our AU data show that the BRPF1 bromodomain binds to mono‐ and di‐acetyllysine ligands as a monomer. Our studies will further elucidate the role of the bromodomain in recruiting BRPF1 to histones, and subsequently assembling and activating the MOZ HAT complex, which has been implicated in acute myeloid leukemia.Support or Funding InformationNational Institutes of Health, National Institute of General Medical Sciences grant number R15GM104865 to KCGThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ribosomal ambiguity (ram) mutations promote 30S domain closure and thereby increase miscoding
Authors: Hoffer Eric D , Maehigashi Tatsuya , Fredrick Kurt , Dunham Christine M. 
Published: June 2021

During protein synthesis, ribosomes select correct aminoacyl‐tRNAs by monitoring the nucleotide pairing between the codon on the mRNA and the anticodon of the tRNA in the decoding center. Despite the large pool of near‐cognate tRNAs, the ribosome is highly accurate with an error rate of ~10−3 – 10−5. The process of decoding by the ribosome is thought to be governed by a conformational transition in the ribosome from open to closed that occurs upon codon‐anticodon pairing within the A site. Ribosomal ambiguity (ram) mutations increase miscoding and map to both ribosomal proteins and rRNA in disparate regions, yet precisely how these mutations act has remained unclear. In this study, we solved crystal structures of Thermus thermophilus 70S ribosomes harboring 16S rRNA ram mutations G299A and G347U in the absence of A‐site tRNA (A‐tRNA) and in the presence of a near‐cognate anticodon stem‐loop. In the absence of an A‐tRNA, each of the mutant ribosomes exhibits a partially closed state. In the 70S‐G347U structure, the 30S shoulder is rotated inward and intersubunit bridge B8 is disrupted. In the 70S‐G299A structure, the 30S shoulder is rotated inward and decoding nucleotide G530 flips into the anti conformation. Both of these mutant ribosomes adopt the fully closed conformation in the presence of near‐cognate A‐tRNA, just as they do with cognate A‐tRNA. Thus, these ram mutations act by promoting the open to closed transition, albeit in somewhat distinct ways. This work reveals the functional importance of 30S shoulder movement for productive aminoacylated‐tRNA incorporation in the A site of the ribosome.Support or Funding InformationThis work was supported in part by the National Institutes of Health [grant numbers GM093278 to CMD, GM072528 to KF]. The X‐ray crystallography datasets were collected at the at the NE‐CAT beamlines (funded by the NIGMS from the NIH (P30 GM124165)), using a Pilatus detector (RR029205) and an Eiger detector (OD021527), and at the SER‐CAT beamlines (funded by its member institutions, and NIH equipment grants RR25528 and RR028976). This research used resources of the APS, a US Department of Energy Office of Science User Facility operated by Argonne National Laboratory under Contract DE‐AC02‐06CH11357 (NE‐CAT) and W‐31‐109‐Eng‐38 (SER‐CAT). Dr. Dunham is a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Characterization of Rosetta Designed Amino Acyl‐tRNA Synthetase Active Sites for Genetic Code Expansion
Authors: Beyer Jenna N. , Hosseinzadeh Parisa , Mehl Ryan A. , Cooley Richard B. 
Published: June 2021

The site‐specific incorporation of non‐standard and novel amino acid structures enabled by Genetic Code Expansion (GCE) provides access to new avenues of protein research allowed by engineering diverse functionality into proteins. At its core, GCE requires the engineering of an orthogonal amino acyl‐tRNA synthetase/tRNA cognate pair that can function with high catalytic efficiency for a non‐canonical amino acid (ncAA). While the GCE community has generated many new amino acyl‐tRNA synthetase/tRNA pairs, these pairs have kinetics that are orders of magnitude worse than the natural translational components. A major challenge for the GCE field is identifying which amino acyl‐tRNA synthetase residues to target for engineering improved amino acyl‐tRNA synthetase/tRNA pairs.Here we use Rosetta modeling to identify mutations in the amino acyl‐tRNA synthetase active site that will improve the efficiency of the nitroTyr amino acyl‐tRNA synthetase/tRNA pairs. Rosetta guided mutations were successful in generating improved GCE function in vivo.Crystal structures were obtained for the Rosetta improved amino acyl‐tRNA synthetases to understand the structural basis for the enhanced activity as part of a comprehensive kinetic and structural analysis of these improved synthetases. Here, we used the 3‐nitro‐tyrosine amino acyl‐tRNA synthetase/tRNA pair as a model system for synthetase improvement and structural characterization.We observe that the Rosetta‐improved synthetases allow a more planar 3‐nitro‐tyrosine (nitroTyr) amino acid upon binding, which is predicted to contribute to their increased activity. This Rosetta guided approach provides a third generation of more efficient nitroTyr amino acyl‐tRNA synthetase/tRNA pairs that will accelerate the study of this oxidative stress post‐translational modification and its role in human disease.We demonstrate that Rosetta successfully identified key residues for amino acyl‐tRNA synthetase/tRNA pair optimization. This strategy can be utilized to systematically improve the efficiency of all ncAA amino acyl‐tRNA synthetase/tRNA pairs, resulting in more robust GCE technologies and facilitating new applications.Support or Funding InformationNIH Grant #R01‐GM114653‐01, Oregon State University Summer Undergraduate Research Experience (SURE) in Science ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards a New Aminoacylation Assay for Alanyl‐tRNA Synthetases
Authors: Duplessis Kyle , Heath Jacob , McCarthy Ian , Chihade Joseph 
Published: June 2021

Aminoacyl‐tRNA synthetases are crucial enzymes in protein biosynthesis, ensuring the fidelity of the genetic code by charging transfer RNAs with the appropriate amino acid. Aminoacylation is typically measured using radioactively labeled substrates, using assays that are non‐continuous, relatively expensive, and time consuming. Recently, a new assay1 was developed for tyrosyl‐tRNA synthetases in which the formation of AMP during the aminoacylation reaction is coupled to production of NADH which can be measured spectrophotometrically. To improve sensitivity, the aminoacyl‐tRNA product was regenerated through hydrolysis with a second enzymatic activity, allowing detection of aminoacylation even at low concentrations of tRNA. We have extended this assay to the alanyl‐tRNA synthetase system. Three different aminoacylation and tRNA regeneration schemes have been developed – mischarging with serine, followed by hydrolysis with the intrinsic editing domain; mischarging with glycine, followed by hydrolysis with D‐aminoacyl‐tRNA deacylase (DTD); and charging with alanine, followed by hydrolysis with a variant of DTD.Support or Funding InformationCarleton College and the Towsley FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pathogenic Mutations in a Human Mitochondrial Enzyme Affect Protein Stability
Authors: Kennicott Hannah , McCurdy Clara , Makori Jessica , Chihade Joseph 
Published: June 2021

Mutations in human mitochondrial alanyl tRNA‐synthetase have been correlated with at least two different pathologies, infantile cardiomyopathy and leukodystrophy. We have used a bacterial expression system to study the effects of these mutations on protein stability. All mutations tested lead to reduced amounts of full‐length soluble enzyme. Several render the protein insoluble, while others lead to proteolysis into soluble or insoluble fragments. Correlations between protein stability and disease state will be presented. We are currently investigating whether the addition of solubility tags will reverse these effects and facilitate the purification of full‐length mutants.Support or Funding InformationCarleton College and the Towsley FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of N‐Terminal Acetylation on the Membrane Binding Behavior of Alpha Synuclein
Authors: Bond Judith Elizabeth , Sarver Jessica 
Published: June 2021

Alpha Synuclein (asyn) is a neuronal protein that acts as a hallmark of Parkinson's Disease through fibril formation that later form Lewy bodies in the brain. The function of asyn is still relatively unclear, however it is known to bind to membranes. It is of interest to study how membrane binding is affected by N‐terminal acetylation of asyn. N‐terminal acetylation is a form of post‐translational modification. A co‐transformation in the presence of NatB in E coli was carried out, to enable the acetylated N‐terminus. In order to reduce protein modification for purification methods, such as Nickel Column Chromatography, Fast Pressure Liquid Chromatography (FPLC) was utilized. The protein was eluted from the column based on electrostatic interactions among the column, protein, and Tris‐NaCl. The fractions of interest were analyzed using an SDS‐PAGE gel, to validate the presence of the purified protein. Following purification, membrane binding behaviors will be analyzed using Electron Paramagnetic Resonance (EPR), by measuring the signal of an unpaired electron, which is introduced through site‐directed spin labeling. The spin label has a nitroxide group, which provides a three distinct sharp peaks in the EPR spectra. Through the binding of the asyn with large unilamellar vesicles (LUVs) the spectra will demonstrate a reduced peak sharpness, which indicates binding. The changes in the EPR spectra will provide insight into how the post‐translational modifications affect the ability of asyn to bind to membranes, as well as the greater impact on protein structure.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.