bluuebunny/crossref_metadata_2025_split huggingface dataset:
Palmitoylation and Phosphorylation–Impact on NHE1 Transport
Authors: Bakker Clare , Hovde Moriah , Foster James , Wallert Mark , Provost Joseph 
Published: June 2021

The sodium hydrogen exchanged isoform 1 (NHE1) has two key roles. First is to regulate intracellular pH (pHi) by exchanging an intracellular proton for an extracellular sodium. The extended intracellular carboxyl terminus binds several proteins that regulate NHE1 transport and act as a scaffold for proteins on the leading edge of a migrating cell. The regulation and interaction on the C‐terminus (amino acids 503–815) is complex and not fully understood. Several regulatory events are linked to multiple phosphorylation sites by kinases including AKT, ERK1/2, ROCK and several other protein kinases. Interacting proteins include isoforms 1 and 2 of calcineurin B homologous protein (CHP1 and CHP2) and 14‐3‐3. We have recently discovered that NHE1 is also reversibly lapidated. Palmitoylation is a unique post‐translational fatty acid modification (S‐acetylation) in that it is reversible and aids in binding specificity, folding, aggregation and clustering. The influence of both phosphorylation and palmitoylationsites upon NHE1 activity could influence both intracellular pH homeostasis and cellular behavior. To determine how both post translational modifications regulate NHE1 transport activity, we have determined the change in intracellular pH (pHi) after exposure to a known activator of NHE1 transport, lysophosphatidic acid (LPA), in the absence and presence of 2‐bromopalmitate (2BP) and specific inhibitors of protein kinases known to phosphorylate NHE1. Preincubation of cells with 0.1 mM 2BP inhibits NHE1, resulting in a significant reduction in LPA‐stimulated pHi. Inhibition of MEK, ERK1/2, AKT and ROCK all inhibit NHE1 transport by >50%. Assessing steady state and transport rate after acid load recovery of cells, with combinations of palmitoylation and protein kinase inhibitors, will help elucidate the relationship between the two post translational events in fibroblasts.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High exogenous NO inhibits proliferation and induces apoptosis by targeting PI3K/AKT/mTOR and MEK/ERK pathways in hepatocellular carcinoma cells
Authors: Liu Ling , Chen Jingjing , Cao Mengyao , Huang Zile , Li Ruifang , Wang Jiangang 
Published: June 2021

PABA/NO (O2‐ {2,4‐dinitro‐5‐[4‐(Nmethylamino)benzoyloxy]phenyl} 1‐(N,N‐dimethylamino) diazen‐1‐ium‐1,2‐diolate) has N‐methyl‐p‐ aminobenzoic acid bound via its carboxyl oxygen as a 5‐substituent on the 2,4‐dinitrophenyl ring, a diazeniumdiolate‐based NO‐donor prodrug through releasing exogenous nitric oxide at high concentrations to induce apoptosis in many tumor cell lines. In the present study, the effects of PABA/NO on hepatocellular carcinoma proliferation and apoptosis induction were investigated both in vitro and in vivo experiments. PABA/NO dramatically inhibited the cell growth and significantly induced apoptosis in a concentration‐dependent manner, accompanied by down‐regulation of Bcl‐2, up‐regulation of Bax, and activation of cleaved‐caspase‐9 and cleaved‐caspase‐3. PABA/NO attenuated the PI3K/AKT/mTOR pathway through decreasing phosphorylation of PI3K p85 at Tyr458, AKT at Ser473, mTOR at Ser2448 and p70S6K at Thr389 in SMMC7721 cells. In addition, LY294002 as a specific PI3 kinase inhibitor can aggravate PABA/NO‐induced cell apoptosis. Furthermore, PABA/NO repressed the MEK/ERK pathway through decrease phosphorylation of MEK at Ser217/221, ERK at Thr202/Tyr204, and p90RSK at Ser380 in PABA/NO‐treated cells. ERK inhibitor U0126 could greatly intensify the apoptotic effects on PABA/NO‐treated cells compared to PABA/NO single treatment. Additionally, H22 tumor‐bearing mice experiments demonstrated that PABA/NO exerted good antitumor effects via reducing tumor volume, tumor weight and decreasing the expression of CD34. Furthermore, PABA/NO treatment strongly inhibited the phosphorylation of PI3K/AKT/mTOR and MEK/ERK signaling pathways in H22 hepatocellular carcinoma tissues. Collectively, these data clearly demonstrated PABA/NO ‐induced apoptosis through inhibition of PI3K/Akt/mTOR and MEK/ERK pathway in hepatocellular carcinoma cells.Support or Funding InformationThis work was financially supported by the National Natural Science Foundation of China (No.81502627) and the Young Backbone Teachers Assistance Scheme of Henan Province Colleges and Universities (No.2016GGJS‐065)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Compartment‐specific neuron activity‐dependent redox dynamics
Authors: Tantama Mathew 
Published: June 2021

Reactive oxygen species (ROS), redox active proteins, and molecules such as glutathione are central to the propagation of redox signals and oxidative stress. Propagation can occur between cells in the tissue environment, and it can also occur across organelles and cellular compartments within a single cell. However, simultaneous measurements of compartment‐specific redox dynamics at the single‐cell level have remained a challenge. We are therefore developing genetically‐encoded red fluorescent redox sensors that can be used together with currently available green fluorescent redox sensors in multiplexed live‐cell imaging experiments. Our protein engineering strategy employs Förster‐type resonance energy transfer (FRET) between the redox‐sensitive green fluorescent protein (roGFP) as the energy donor tethered to various red fluorescent proteins acceptors. This FRET‐based design generates a spectral relay which will ideally shift the green emission from roGFP to orange and red emission wavelengths. We have validated a set of second‐generation “rogRFP” sensors, and we have measured activity‐dependent redox changes in the cytosol and mitochondria of individual neurons that differ in compartment‐specific phenotypes. Our experiments with cultured primary mouse hippocampal neurons demonstrates that our sensors will be effective tools for studying compartment‐specific redox dynamics and signaling.Support or Funding InformationNIH/NINDS R21 NS106319This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of Bacterial Ribosome 30S and 50S Subunit Recognition and Modification by M. tuberculosis TlyA
Authors: Laughlin Zane Timothy , Conn Graeme 
Published: June 2021

RNA (rRNA) modification is important for correct ribosome assembly, can alter ribosome function, and can confer resistance to clinically important ribosome‐targeting antibiotics. While the activity of most ribosome‐targeting antibiotics is blocked by rRNA methylation, the tuberactinomycin antibiotic capreomycin requires ribose 2′‐OH methylation at C1409 of the 16S rRNA within the small ribosome subunit (30S) and C1920 of the 23S rRNA within the large ribosome subunit (50S). TlyA is the 2′‐O‐methyltransferase that incorporates both modifications using S‐adenosyl‐L‐methionine (SAM) as a methyl group donor. The X‐ray crystal structure of the C‐terminal domain (CTD) of TlyA showed that the domain adopts a Class I methyltransferase fold, while homology modeling suggests the N‐terminal domain (NTD) adopts an S4 ribosomal protein fold. Additionally, the TlyA CTD structural studies revealed that the short interdomain linker was able to adopt two different conformations and was unexpectedly critical for SAM binding within the CTD. This project will test the resulting hypothesis that the interdomain linker acts a “molecular switch,” altering the interaction between the NTD and the CTD to control TlyA activity upon correct recognition of its two structurally distinct substrates. Using a sequence alignment and structure‐guided mutagenesis approach coupled with fluorescence polarization (FP) TlyA‐30S binding and methyltransferase activity assays, the role of the NTD and individual residues in this domain in 30S and 50S binding will be defined. Using complementary studies of TlyA protein dynamics (using HDX‐MS) and structure (using X‐ray crystallography and cryo‐EM), the long‐term goal of this project is to determine TlyA's molecular mechanism of site‐specific ribosome subunit recognition and modification. Defining these molecular details will help complete the picture of TlyA activity and its role in bacterial drug sensitivity or resistance, as well as furthering our general understanding of rRNA modification processes that are prevalent in all domains of life.Support or Funding InformationThis work was supported in part by the Graduate Division of Biological and Biomedical Sciences (GDBBS) of the Laney Graduate School (LGS) of Emory University, NIH grant R01‐AI088025, and the Antibiotic Resistance and Therapeutic Discovery Training Program (ARTDTP) T32‐AI 106699.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Splicing of Perilipin 5
Authors: Jordan Spencer P , Tansey John T 
Published: June 2021

Included among the most common diseases in the U.S., are metabolic diseases such as type II diabetes mellitus and nonalcoholic fatty liver disease. Understanding these diseases requires an understanding of neutral lipid storage and metabolism. Perilipins are a family of five conserved proteins that function in the regulation of neutral lipid metabolism. Of the perilipins, perilipin 5 is the most recently discovered and studied protein in the family. Perilipin 5 is a major regulator of lipolysis and is expressed in highly oxidative tissue such as heart, oxidative muscle, and fasting liver. It is primarily found localized to the surfaces of lipid storage droplets, but it can also be found in the cytosol. Alternative splicing has been observed in perilipins 1 and 3, resulting in several other proteins with various functions as these proteins lack domains which have the functions of the full‐length protein. We have identified a truncated form of perilipin 5 we have termed perilipin 5b using western blotting. Reverse transcriptase PCR indicate that this truncation results from a read through of one of several different introns, most likely the intron between introns 7 and 8. cDNA BLAST searches also provide evidence of this splice variant. Collectively, these data suggest that like other members of the family, perilipin 5 undergoes differential splicing resulting in shortened protein products with short, unique carboxy terminal tails.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
REDD1 upregulation via AP‐1 alleviates oxidative stress‐mediated liver injury
Authors: Ki Sung Hwan , Cho Sam Seok , Kim Jae Kwang , Cho Il Je 
Published: June 2021

Regulated in development and DNA damage responses 1 (REDD1) is an inducible gene in response to various stresses, which functions as a negative regulator of the mammalian target of rapamycin protein kinase in complex 1. In the present study, we identified the role of REDD1 under the oxidative stress‐mediated hepatocyte injury and its regulatory mechanism. REDD1 protein was increased in H2O2 or tert‐butylhydroperoxide (t‐BHP)‐treated hepatocytes· H2O2 also elevated REDD1 mRNA levels. This event was inhibited by antioxidants such as diphenyleneiodonium chloride, N‐acetyl‐L‐cysteine, or butylated hydroxy anisole. Interestingly, we found that H2O2‐mediated REDD1 induction was transcriptionally regulated by activator protein‐1 (AP‐1), and that overexpression of c‐Jun increased REDD1 protein levels and REDD1 promoter‐driven luciferase activity. Deletion of the putative AP‐1 binding site in proximal region of the human REDD1 promoter significantly abolished REDD1 transactivation by c‐Jun. A NF‐E2‐related factor 2 activator, tert‐butylhydroquinone treatment also elevated REDD1 levels, but it was independent on NF‐E2‐related factor 2 activation. Furthermore, we observed that REDD1 overexpression attenuated H2O2 or t‐BHP‐derived reactive oxygen species formation as well as cytotoxicity. Conversely, siRNA against REDD1 aggravated t‐BHP‐induced reactive oxygen species generation and cell death. In addition, we showed that REDD1 was induced by in vitro or in vivo ischemia/reperfusion model. Our results demonstrate that REDD1 induction by oxidative stress is mainly transcriptionally regulated by AP‐1, and protects oxidative stress‐mediated hepatocyte injury. These findings suggest REDD1 as a novel molecule that reduced susceptibility to oxidant‐induced liver injury.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A high‐throughput microfluidic microphysiological system (PREDICT‐96) to recapitulate hepatocyte function in dynamic flow conditions
Authors: Tan Kelly , Keegan Philip , Rogers Miles , Lu Mingjian , Gosset James , Charest Joseph , Bale Shyam Sundhar 
Published: June 2021

Microphysiological systems (MPSs) are dynamic cell culture platforms that incorporate mechanical and chemical cues to recapitulate organ‐specific functions and architecture. Recent advances in microfabrication technologies and processes have enabled the development of enhanced in vitro models that capture human physiology more accurately while increasing throughput and reducing material‐based technical artifacts. A common candidate for MPS development is liver due to its crucial role in drug metabolism, inflammation and human disease. While several liver‐on‐chip models exist, few studies have combined long term culture with dynamic inputs, such as media flow, in a high‐throughput format. Herein, we have developed an oxygen permeable, high‐throughput (96‐well form factor), thermoplastic MPS (PREDICT‐96 array) integrated with an on‐board, ultra‐low volume, recirculating pumping system (PREDICT‐96 pump) to facilitate media flow in the system; collectively, these technologies comprise the PREDICT‐96 platform. To demonstrate usability of the PREDICT‐96 array, we introduce PHHs using standard handling procedures for multi‐well plates and maintain viable culture for up to 14 days. By incorporating media flow to mimic in vivo mass transport, we observe a robust increase in metabolic and secretory functions of PHHs when compared to static culture. The PHHs demonstrate reproducible baseline metabolic activity and secretion markers which underscores the utility of the system for drug screening applications. Furthermore, long‐term culture with fluid flow allows for the periodic introduction of media components (e.g., fatty acids, cytokines) and resolve cellular responses to chronic stimuli over time. The low‐volume footprint of the pump and small media volume in the MPS allow for the interrogation of hepatic responses incorporating secretion feedback to a stimulus, which is essential for disease model development and drug interrogation. We envision future development of this liver model to incorporate key primary hepatic cells, multi‐cellular co‐cultures and integration with high‐throughput analytical tools.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Platelet‐activating factor‐receptor dependent pathway mediates gemcitabine chemotherapy‐induced microvesicle particles release in pancreatic cancer cells
Authors: Thyagarajan Anita , Kadam Sayali M , Liu Langni , Kelly Lisa E , Rapp Christine M , Chen Yanfang , Sahu Ravi P 
Published: June 2021

Studies including ours have shown that pro‐oxidative stressors such as chemotherapeutic agents generate oxidized lipids with platelet‐activating factor (PAF) agonistic activity. These PAF‐agonists bind with and activate the widely‐expressed G‐protein coupled, PAF‐receptor (PAF‐R), implicated in various pathophysiological diseases including cancer. Notably, recent reports from our group have demonstrated that these PAF‐agonists are transported extracellularly via microvesicle particles (MVP). While the role of PAF‐R in mediating chemotherapy effects has been documented, its significance in chemotherapy‐induced MVP release in pancreatic cancer model has not been studied. To that end, the current studies determined the functional significance of PAF‐R in clinically relevant gemcitabine chemotherapy‐induced MVP release in human pancreatic cancer cells. Our studies using PAF‐R‐expressing (PANC‐1), and deficient (Hs766T) cells demonstrate that gemcitabine induces MVP release in a PAF‐R‐dependent manner. Blocking of PAF‐R via PAF‐R antagonist or inhibition of MVP biogenesis via acid sphingomyelinase (aSMase) enzyme inhibitor, significantly attenuated gemcitabine‐induced MVP release from PANC‐1 cells, however, exerted no effects in Hs766T cells. Importantly, lipid extracts from MVP, isolated from gemcitabine‐treated PANC‐1 cells, contained appreciable amounts of PAF‐agonists. This was measured by a well‐defined and specific approach of accessing PAF‐agonists generation via interleukin‐8 (IL‐8) secretion from PAF‐R‐expressing human epidermoid KBP cells, and not from PAF‐R‐deficient KBM cells. These results indicate that gemcitabine‐generated PAF‐agonists are transported extracellularly via MVP. Mechanistically, pretreatment with extracellular signal‐regulated kinase (ERK1/2) or p38 inhibitors, significantly abrogated gemcitabine‐induced MVP release. These findings indicate the involvement of mitogen‐activated protein kinase (MAPK) pathway in PAF‐R‐dependent gemcitabine‐induced MVP release. In sum, these studies provide the significance of PAF‐R in gemcitabine‐induced MVP release as well as the rationale of evaluating PAF‐R targeting agents with gemcitabine or gemcitabine‐based chemotherapy approaches against pancreatic cancer.Support or Funding InformationThe financial support from the NIH K22 [ES023850] and Elsa U. Pardee Foundation [670645] grants are greatly acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nutritional Ketosis and Fasting: Metabolic Inferences for Mitochondrial Function in Human Health
Authors: Hernandez Melody M. , Einstein George P. , Tulp Orien L. 
Published: June 2021

Cellular communication between healthy and sick cells during ketosis state is dynamic process. Converting our source of energy from glucose to fat is essential to morph sick cells into healthy cells. Changing our culture and environment from processed foods and toxins returning to our embryonic human; free of acquired diseases and disorders from genetic modification. The aim of this research is to free our society of the illnesses which become more and more prevalent with our daily contaminations. The research question is answered through an experiment which includes a questionnaire and lab results with case to case analysis. Achieving ketosis protects the healthy cells by regenerating our mitochondria with the appropriate fuel; which is responsible for the debilitation and apoptosis of the sick cells eliminating disease manifestations. Regenerating important signaling molecules helping regulate the expression of genes. The methodology of this research is achieving ketosis the dynamic metabolic state through different methods. One is through the fruit and vegetable diet and the other is through the ketogenic diet. Both achieve a level of ketosis in which allows the body to convert free fatty acids into ketone bodies to be used as the proper fuel for mitochondria to replenish the human body. The participants of this research are of all genders, backgrounds, and have individual health concerns and disorders with various goals for their outcome during the experiment. The results of the experiment showed overall improvement of health and wellness; some symptoms remedied while others ameliorated. Some challenges and modifications were made for children, pregnancy and severe illness.Support or Funding InformationSupport by Institutional Resources of USAT Montserrat, and the Einstein Medical Institute, NPB, FLThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Chronic Contractile Activity and Retinoic Acid on Mitochondrial Turnover in C2C12 Myotubes
Authors: Erlich Avigail Tamar , Dovijarski Nemanja , Hood David A. 
Published: June 2021

Mitochondrial turnover is necessary for the optimization and maintenance of a healthy pool of functional organelles, able to meet the metabolic needs of the muscle cell. Exercise is known to improve mitochondrial turnover by activating both mitochondrial biogenesis and mitochondrial‐specific autophagy, known as mitophagy. Moreover, research in adipocytes and liver tissue has shown the potential of the dietary supplement retinoic acid to activate mitochondrial biogenesis as well. Retinoids have also been shown to be effective in activating autophagy in cancer cells. However, the combined effect of retinoic acid isomers and exercise in facilitating mitochondrial turnover has not been described. The purpose of this project was to evaluate the effect of retinoic acid (RA) isomers combined with exercise on muscle mitochondrial turnover. Mouse C2C12 myoblasts were plated on 6‐well plates and allowed to differentiate into myotubes. Myotubes were then treated with either vehicle (DMSO), or the retinoic acid (RA) isomers 9‐cis RA or All‐trans RA (ATRA) in the absence or presence of electrical stimulation to elicit Chronic Contractile Activity (CCA; 4days, 3hrs/day). Sample collection took place 21h after the fourth day. Mitochondrial biogenesis was assessed by examining the protein content of PGC‐1α, as well as nuclear and mitochondrially‐encoded mitochondrial proteins. CCA led to a 3‐fold increase in COX subunit IV expression, as well as a 2‐fold increase in PGC‐1α. The mtDNA‐encoded COX subunit I was modestly changed by CCA, but increased 2‐fold in response to 9cis‐RA or ATRA treatment. These RA isomers also increased PGC‐1α by 3‐fold, but had no significant impact on COX IV expression. When RA isomers were combined with CCA, no additive or synergistic effects were observed. To evaluate mitochondrial degradation, we examined several lysosomal and mitophagy proteins. While autophagy proteins Beclin and p62 showed no changes with CCA or RA treatment, the LC3II to LC3I ratio was reduced 2‐fold with CCA, suggesting an enhanced clearance of autophagosomes. RA isomers had no effect on this ratio. The lysosomal fusion protein Lamp1 was increased 1.8‐fold by ATRA, but not further by CCA. Our data suggest that while RA isomers and CCA both promote mitochondrial biogenesis, RA appears to target mitochondrially‐encoded proteins, while CCA appears to have greater effects on the expression of target nuclear‐encoded proteins. CCA also may serve as a stimulus for the clearance of autophagosomes containing damaged cargo. The lack of synergy among these pathways suggests that they operate independently to stimulate mitochondrial turnover.Support or Funding InformationSupported by NSERC, CanadaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.