bluuebunny/crossref_metadata_2025_split huggingface dataset:
Unique enzyme specificity of three human phospholipases A2 toward phospholipids containing sn‐2 omega‐3 and omega‐6 fatty acids
Authors: Hayashi Daiki , Mouchlis Varnavs D. , Dennis Edward A 
Published: June 2021

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyzes the sn‐2 acyl‐chain of membrane phospholipids producing lyso‐phospholipids and free fatty acids. The products of PLA2 have various functions, especially omega‐3 and omega‐6 fatty acids which are precursor of various eicosanoids and related metabolites involved in numerous pro‐inflammatory and pro‐resolving cellular responses. The PLA2 superfamily is composed of six different types of enzymes, each with distinct structural features. Among them, the cytosolic PLA2 (cPLA2), calcium‐independent PLA2 (iPLA2), and secreted PLA2 (sPLA2) are well studied. cPLA2 is the main provider of free arachidonic acid (AA), an omega‐6 fatty acid involved in the inflammatory cascade. Several knockout mice studies demonstrated the contribution of iPLA2 to the metabolism of the omega‐3 fatty acid docosahexaenoic acid (DHA) in brain (J. Lipid Res. 2010, 51, 3166–3173). sPLA2 is known for its antibacterial action and involvement in atherosclerosis. Previous studies in our laboratory determined the substrate specificity of all three PLA2s toward a variety of membrane phospholipids (J. Am. Chem. Society 2018 140, 3285–3291). However, the specificity of these enzymes toward phospholipids containing omega‐3 and omega‐6 fatty acids at the sn‐2 position has been unclear. In this study, we have employed a lipidomics‐based LC‐MS/MS assay, which measures the lysophospholipid product to measure the activity of PLA2, and to investigate the sn‐2 acyl‐chain specificity in various phospholipids. We have also developed a lipidomics‐based GC‐MS assay which allow us to measure free fatty acids in mixtures of phospholipid substrates. Although cPLA2 showed relatively high activity toward the omega‐3 eicosapentaenoic acid (EPA), it prefers AA. iPLA2 showed a distinct preference for EPA over AA and a much lesser activity toward phospholipids containing DHA. Unlike the other two PLA2s, sPLA2 showed a remarkable preference for DHA among the omega‐3 and omega‐6 fatty acids. Importantly, similar sn‐2 acyl‐chain specificity was obtained in mixtures of phospholipids. This study investigated the preference of three human PLA2 enzymes for the sn‐2 omega‐3 and omega‐6 fatty acids for the first time. Our findings will help us to understand the difference of function of PLA2 enzymes at the cellular level and their implications in inflammatory diseases.Support or Funding InformationSupported by NIH grant GM20501This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perilipin 5 and Rab 18 Occupy Similar Cellular Pools
Authors: Hughes Erin K , Tansey John T 
Published: June 2021

Type II diabetes mellitus and non‐alcoholic fatty liver disease are two examples of metabolic diseases that are contributing to the emerging health crisis in the west. Key to understanding these diseases and their progression is an understanding of neutral lipid metabolism. Perilipins are a class of five conserved proteins that are key regulators of lipid metabolism and are potentially involved in lipid trafficking within cells. The most recently discovered member of the family is perilipin 5, which is expressed most strongly in tissues that are highly oxidative such as heart, oxidative muscle, and fasting liver. Perilipin 5 is predominantly located on the surface of lipid storage droplets, but has also been found in the cytoplasm, nucleus, and the endoplasmic reticulum and mitochondria. We hypothesized that perilipin 5 moves between these cellular pools via interactions with other proteins. Furthermore, we hypothesized that interactions with Rabs were necessary for this trafficking to occur. CHO cells ectopically expressing perilipin 5 and endogenously expressing Rab18 were assayed using western blotting. These proteins were found in the same cellular pools. Immunoprecipitations are being used to assay for interactions between perilipin 5, Rab18, and Rab32. Immunofluorescence microscopy indicates that perilipin 5 and Rabs colocalize to the same cellular pools. Collectively these data will provide a more detailed picture of perilipin 5 in the cell.Support or Funding InformationOtterbein University Student Research FundThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatocyte‐Specific Deletion of eNOS Impairs Mitochondrial Function and Exacerbates Hepatic Steatosis
Authors: Cunningham Rory P. , Moore Mary P. , Meers Grace M. , Nguyen Nhu , Boccardi Luigi , Jepkemoi Vivien , Takahashi Takamune , Rector R. Scott 
Published: June 2021

IntroductionWhole body loss of endothelial nitric oxide synthase (eNOS) worsens hepatic mitochondrial function and exacerbates nonalcoholic fatty liver disease (NAFLD) development and progression. However, the precise role of eNOS in hepatocytes in the contribution to NAFLD has not been established. Here, we examine the effects of in vivo hepatocyte‐specific ablation of eNOS on hepatic steatosis and NAFLD susceptibility.MethodsHepatocyte‐specific eNOS knockout (eNOShep−/−) mice were generated by crossing homozygous eNOS floxed (eNOSfl/fl) mice on a C57/BL6J background with albumin‐Cre recombinase transgenic mice. At 10 weeks of age, male eNOSfl/fl and eNOShep−/− mice (n = 12–14) were fed a control diet (10% fat) for 16 weeks.ResultsBody weight, body fat, and food intake did not differ between eNOSfl/fl and eNOShep−/− mice. eNOShep−/− mice developed elevated histological hepatic steatosis and increased fasting serum insulin (~30%) compared to eNOSfl/fl mice (p<0.05), along with trends for elevations in serum liver enzymes AST and ALT (p<0.17). Elevated hepatic steatosis in eNOShep−/− was related to significant reductions (−50%) in hepatic mitochondrial fatty acid oxidation and ADP‐stimulated and maximal uncoupled hepatic mitochondrial respiration (p<0.05). However, hepatic citrate synthase activity, a marker of mitochondrial mass, was significantly elevated in eNOShep−/− mice compared to eNOSfl/fl mice (p<0.05), while protein content of oxidative phosphorylation complexes and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) did not differ between groups. Furthermore, eNOShep−/− mice presented with significant reductions (p<0.05) in key proteins involved in mitophagy and autophagy including PTEN‐induced kinase 1 (PINK1) and phosphorylated Unc‐51 Like Autophagy Activating Kinase 1 (pUlk1/Ulk1) and tended to have significant reductions in BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) compared to eNOSfl/fl mice.ConclusionHere we demonstrate for the first time that hepatocyte‐specific deletion of eNOS impairs hepatic mitochondrial function, reduces markers of autophagy/mitophagy, and exacerbates hepatic steatosis development. Further studies are necessary to determine the mechanisms by which deletion of eNOS regulates mitochondrial homeostasis.Support or Funding InformationVA Merit Grant I01BX003271‐01 (R.S.R.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transfer RNA post‐transcriptional modifications of Bacillus subtilis
Authors: Psihountas Christina , Jora Manasses , Limbach Patrick A 
Published: June 2021

Transfer Ribonucleic Acids (tRNAs) have more posttranscriptional modifications to nucleosides than other species of RNA.1 It has been found that when bacteria are analyzed at different stages of a growth curve, changes in these nucleoside modification identities or amounts exist.2 Similarly, nucleoside modification profiles are dependent on growth media.3 Here we are examining whether domestication of Bacillus subtilis lead to differences in nucleoside modification profiles for tRNAs. We have compared tRNA modifications from two different strains: (i) a domesticated lab strain 168, the most widely studied strain, and (ii) strain NCIB 3610, a Marburg strain isolate. NCIB 3610 is reported in literature to be “wilder” than strain 168.4 We are using strain 168 as the B. subtilis model against which we can evaluate how domestication may impact the types and quantities of tRNA modifications. Our experimental approach is to use liquid chromatography tandem mass spectrometry (LC‐MS/MS). Once nucleosides are identified and quantified, we will then compare those results to the genetic variations arising between the domesticated and undomesticated strains to understand how such modification changes may arise.Support or Funding InformationThe National Science Foundation (CHE 1507357)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reconstituting human P‐glycoprotein (MDR1) into membrane nanodiscs
Authors: Farokhnia Roxana , Olivera Maisa C. , Okwuone Dakota D.D. , Jensen Madeline K. , Edgar Jennifer M. , Wise John G. , Vogel Pia D. 
Published: June 2021

P‐glycoprotein (P‐gp) is a multidrug resistance protein found in the membrane of many cells. As an ATP powered pump, P‐gp exports toxins and other hydrophobic compounds outward across the plasma membrane. This trait becomes problematic when overexpression of P‐gp occurs in cancer cells and causes chemotherapeutics to be pumped out of the cancer cells, rendering therapy ineffective. For biochemical and/or biophysical assessment of P‐glycoprotein, the protein is removed from the membrane by detergents and purified into detergent micelles using affinity chromatography. Although P‐gp is active when dissolved into micelles, the detergent environment has been shown to affect its biological activity. To avoid this issue, native‐like membrane nanodiscs have been introduced and used successfully over the last several years. Nanodiscs typically contain the P‐glycoprotein surrounded by lipids and enclosed within membrane scaffold proteins (MSP) which create an amphipathic belt around the membrane disc for water solubility. This more native‐like environment within the nanodiscs allows access to both the nucleotide and drug binding domains of the inserted proteins. In order to effectively assemble proteins into nanodiscs, specific ratios of protein to MSP to lipid are needed. In our lab, different ratios of these components have been used to optimize the assembly of the mouse cysteine‐less variant of P‐gp (MDR3CL) into nanodiscs. For the human P‐gp (MDR1) preliminary assembly successes were achieved at a 1:10:500 ratio (P‐gp: MSP: phosphatidylcholine). However, the yield of P‐gp assembled into nanodiscs was very low (about 30%). Here we report our efforts to optimize the nanodisc assembly with the goal of increasing P‐gp reconstitution yield and specific ATP hydrolysis activity. We will use the nanodisc reconstituted P‐glycoprotein in our efforts to investigate efficacy and mechanism of inhibition of novel P‐glycoprotein inhibitors discovered in our lab.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Hydrogen Peroxide on Hydrolysis of Proteins
Authors: Ailes Isaiah , Tohidi David , Ngo Luke , Keenan Kelly 
Published: June 2021

Hydrogen peroxide (H2O2) is the active ingredient in over‐the‐counter whitening strips for teeth. It has previously been shown to damage certain proteins by making them more susceptible to hydrolysis by proteases. The goal of this project was to characterize the hydrolysis of various proteins with H2O2. Albumin protein was mixed with different concentrations of H2O2 for one hour. The protease trypsin was added and portions were removed and immediately mixed with trichloroacetic acid to precipitate either unhydrolyzed proteins or proteins that were minimally hydrolyzed. After centrifugation, the supernatant, which contains small protein fragments that were released by hydrolysis, was collected and; the total amount was measured using the Lowry assay. The results indicate that even without the protease, there is significant hydrolysis with peroxide and none when the proteins were mixed with water. The amount of hydrolysis correlated with the amount of peroxide. In addition, the amount of hydrolysis in the presence of H2O2 with trypsin greatly exceeded that when only trypsin was present. It was important in this method to inactivate peroxide and efficacy of different metals were tested. This effect was further characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). These results suggest that hydrogen peroxide; even at the concentrations used in whitening strips, can damage protein by promoting hydrolysis. This work was supported by Research and Professional Development Grant from Stockton 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.
Structural insight into pre‐mRNA splicing catalyzed by the spliceosome
Authors: Zhao Rui , Li Xueni , Liu Shiheng , Zhou Hong 
Published: June 2021

Pre‐mRNA splicing is catalyzed by the spliceosome, a huge protein‐RNA complex composed of the U1, U2, U4, U5, U6 snRNPs and many non‐snRNP related proteins. U1 snRNP plays a critical role in 5′ splice site recognition and is a frequent target of alternative splicing factors. These factors transiently associate with human U1 snRNP and are not amenable for structural studies, while their Saccharomyces cerevisiae (yeast) homologs are stable components of U1 snRNP. We have recently determined the structure of yeast U1 snRNP at 3.6Å resolution using cryoEM. The structure reveals common features as well as important differences from the human U1 snRNP. It provides atomic models of nearly all essential domains of U1 snRNA, all core proteins, and five auxiliary proteins. The structure offers a framework to integrate a wealth of existing genetic and biochemical data regarding the structure and function of yeast U1 snRNP and the mechanism of 5′ ss recognition. In addition, the yeast U1 snRNP structure and biochemical analyses based on the structure provided intriguing insight into the structure and function of these auxiliary human U1 snRNP proteins in alternative splicing in higher eukaryotes.The spliceosome undergoes dramatic changes signified by the E, A, Pre‐B, B, Bact, B*, C, C*, P, and ILS complexes in a splicing cycle. CryoEM structures of B, Bact, C, C*, and ILS revealed mechanisms of 5′ ss recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release, all related to the post‐catalytic P complex. We have recently determined the cryoEM structure of the yeast P complex at 3.3Å resolution, revealing that the 3′ ss recognition is driven by the interaction between 3′ and 5′ ss, likely facilitated by a stem‐like structure formed in the intronic regions between the branch site and 3′ ss. The structure shows that a new protein(s) becomes stably associated with the core components of the P complex around Prp8 and Prp22, securing the 3′ exon and potentially regulating the activity of Prp22. The structure demonstrates that Prp22 binds 15–21 nucleotides downstream of the exon‐exon junction, enabling it to pull the 3′ intron‐exon and ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading and exon release, respectively. By providing insights into the 3′ ss recognition, exon ligation, and the action of RNA helicase Prp22, the P complex structure fills a major gap in our understanding of the splicing cycle.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of lymphotoxin‐αβ on antigen‐specific T cells is required for dendritic cell function
Authors: deLuca Leslie Summers , McCarthy Douglas D , Cosovic Bojana , Ward Lesley , Lo Calvin , Scheu Stefanie , Pfeffer Klaus , Gommerman Jennifer 
Published: June 2021

The lymphotoxin (LT) pathway has been shown to be important in T cell responses and DC homeostasis/activation. LTαβ is a TNF family member upregulated on activated T cells and its receptor, LTβR, is constitutively expressed on DC. Elucidating the role of LTβR signaling in DC function is complicated by the expression of LTβR on both lymphoid stromal cells and DC. Here we have used two methods for inhibiting LTβR signaling. In the first we use an in vivo adoptive transfer system where the expression of the LTβR ligands is manipulated only on the Ag‐specific T cells that interact with and condition Ag‐bearing DC. Using this approach we demonstrate a novel requirement for the LTαβ for optimal DC conditioning during an immune response against protein antigen.In the second system, we have manipulated the expression of LTβR on DC using a mixed bone marrow chimera approach. Here we report a requirement for DC‐intrinsic LTβR signaling for the optimal upregulation of CD86. In addition, DC‐intrinsic LTβR signaling influences DC accumulation in the inflamed LN. Together these data identify a fundamental role for DC‐intrinsic LTβR signaling during T cell immune responses. Funding from CIHR (JLG and LSD) and MS Society of Canada (LSD) supported this research.
Efficient delivery of vector DNA to dendritic cells using silica‐nanoparticles
Authors: Messmer Davorka , Huang Chien‐Tze , Seible Daniel , Yang Jian , Saenz Rebecca , Esener Sadik , Trogler William 
Published: June 2021

Objective: Optimize plasmid DNA delivery to DCs using silica‐nanoparticles (NPs) for tumor immunotherapy.Methods: Human monocyte‐derived DCs were generated from PBMCs by culturing adherent cells in GM‐CSF and IL‐4 for 5 to 7 days. SiO2 nanoparticles were generated using polymer beads as template. The surface was functionalized with amino silane or amino phosphoric acid and the polymer core was removed by calcination or organic solvents.Results: We sought to determine the optimal parameters in terms of size and surface modification of silica‐NPs with regard to DNA adsorption. DNA could be adsorbed to silica‐NPs of different sizes and surface modifications including amine groups and poly‐L‐lysine (pLL) lead to increased DNA adsorption to the NPs. NPs of 45, 100, and 200 nm diameter were effectively taken up by DCs and no obvious preference was observed. Interestingly pLL‐modified NPs were not taken up by DCs. Confocal microscopy indicated that NPs were also present in the nucleus of the DC which showed high viability for at least 7 days. To determine whether the NPs can be used to deliver DNA into DCs and lead to gene expression of the transgene, immature DCs were exposed to NP‐DNA complexes containing gfp‐plasmid and analyzed for Gfp expression by flow cytometry. 45% of the DCs expressed GFP at 48h.Conclusion: SiO2‐nanoparticles of 80 and 120 nm are more efficient than electroporation to introduce a transgene into DCs in vitro and possibly in vivo for DNA vaccines.
15‐deoxy‐Δ 12,14 ‐prostglandin J2 (15d‐PGJ2) increases oxidative stress and differentially upregulates hemeoxygenase‐1 (HO‐1) in malignant B cells
Authors: Bancos Simona , Bernstein Steven , Baglole Carolyn , Phipps Richard P. 
Published: June 2021

Background: 15‐deoxy Δ‐ 12,14 ‐prostaglandin J2 (15d‐PGJ2) is an endogenous ligand of peroxisome proliferator‐activator receptorγ (PPARγ ). Treatment of some B cell lymphomas with 15d‐PGJ2 increases oxidative stress and induces apoptosis. HO‐1 is an oxidative stress‐inducible enzyme but its expression in human B cell lymphomas is unknown. We hypothesized that PPAR ligands induce HO‐1 in malignant B cells.Methods: Ramos and Granta B cell lymphoma cells and 8226 multiple myeloma cells were treated with 15d‐PGJ2. HO‐1 protein expression was assessed by western blot. Markers of oxidative stress included mitochondrial superoxide production and generation of reactive oxygen species (ROS).Results: Treatment of Ramos cells with 15d‐PGJ2 strongly increased ROS and mitochondrial superoxide production and significantly reduced cell viability. Granta and 8226 cells had a similar response to 15d‐PGJ2 treatment but they exhibited a concentration‐ and time‐dependent increase of HO‐1 expression. Interestingly, Ramos B cells did not up‐regulate HO‐1.Conclusions: The PPARγ ligand 15d‐PGJ2 induces cell death in some B malignant cell lines and differentially regulates the induction of HO‐1. This suggests that malignant B cells have different anti‐oxidant defense mechanisms.Research supported by DE011390, ES01247, The Leukemia and Lymphoma Society and the Lymphoma Research Foundation.
Characteristic changes in plasma phosphatidylcholine species in humans depleted of choline
Authors: Costa Kerry‐Ann da , Miller Jameson , Giddings Morgan C. , Cui Zheng , Zeisel Steven H. 
Published: June 2021

Phosphatidylcholine (PtdCho) is an essential membrane phospholipid synthesized from choline via the CDP‐choline pathway. PtdCho is also formed via sequential methylation of phosphatidylethanolamine (PE) using PE methyltransferase (PEMT). In the latter case, choline contributes its methyl group to PtdCho via betaine. PtdCho produced from the CDP‐choline pathway mainly contains medium chain, saturated fatty acid species such as 16:0/18:0, while those from the PEMT pathway preferentially have long chain polyunsaturated species such as 22:6. We investigated whether there are changes in PtdCho species that are characteristic of choline deficiency. Adult men were fed a diet containing 550 mg choline/d for 10 d. They were then fed a choline deficient diet for up to 42 d. Choline was returned to the diet of subjects who developed clinical symptoms of deficiency. At the end of each phase, plasma was collected and analyzed using gas chromatography/mass spectrometry. Changes in PtdCho mass 758 (16:0/18:2) and 760 (16:0/18:1) appear to be a signature of choline status in that the higher the ratio, the less likely the subject will deplete on a low choline diet. Principal component analysis identified other variations in PtdCho profiles that correlate with clinical choline status. Thus, PtdCho profiling may be a useful indicator of choline status.This research is supported by grants from NIH‐ DK55865, DK56350 and RR00046.