bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of PEA‐15 allosterically induces conformational change suited for FADD binding and negatively regulates apoptosis
Authors: Hassan Sherouk , Crespo Sergio , Wei Yufeng
Published: June 2021

Phosphoprotein Enriched in Astrocytes, 15 kD (PEA‐15) is a small non‐catalytic protein, with an N‐terminal death effector domain (DED) and an unstructured C‐terminal tail. PEA‐15 appeals to many biochemists because it regulates two important pathways. Unphosphorylated PEA‐15 interacts with extracellular regulated protein kinase (ERK) 1 and 2, preventing ERK‐dependent cell proliferation. When it is doubly phosphorylated at the two C‐terminal serine residues, PEA‐15 is recruited to the death inducing signaling complex (DISC) when it interacts with Fasassociated death domain (FADD) protein, blocking the normal recruitment and activation of procaspase‐8, which triggers the signal for apoptosis. In our lab, PEA‐15 and FADD are cloned into pET‐28b(+) vector, and are expressed in BL21(DE3) E. Coli competent cells. To mimic the phosphorylation, S104D/S116D mutant protein (PEA‐15DD) was also expressed. The proteins are purified using metal affinity (Ni2+ or Co2+) chromatography followed by ion‐exchange chromatography. The protein structures and interactions between PEA‐15DD and FADD are determined using NMR spectroscopy. Our results showed that the phosphorylation of PEA‐15 changes the conformation of the folded DED, and FADD binding does not further modulate the overall conformation. We concluded that the binding specificity of PEA‐15 is determined by the DED conformation, which is allosterically induced by the C‐terminal phosphorylation states. We further proposed that the balance of various phosphorylation states of PEA‐15 should be strictly regulated in different tissues and cell types in order to maintain the healthy conditions of the cells.

Support or Funding Information

NIH AREA R15CA179410

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Cia1:Cia2 Sub‐Complex of the Cytosolic Fe‐S Cluster Assembly Pathway
Authors: Esonwune Stephanie , Vo Amanda , Marquez Melissa , Perlstein Deborah
Published: June 2021

Iron‐sulfur (FeS) clusters are essential protein cofactors required for numerous biological functions including iron regulation, DNA synthesis and DNA repair. The Cytosolic Iron‐Sulfur Cluster Assembly (CIA) pathway is responsible for the metallocofactor assembly of extra mitochondrial FeS proteins. The CIA pathway culminates with the targeting complex, which contains the proteins Met18, Cia1 and Cia2. This complex is crucial for the last step in the CIA pathway, which is the recognition of apo‐FeS targets and insertion of their cofactors. To begin understanding the structure of this complex, we wanted to identify the residues responsible for the complex formation. We began by identifying the clusters of conserved residues required for the formation of the Cia1‐Cia2 sub‐complex since both of these proteins are essential for yeast viability, whereas the Met18 subunit is not. We alanine scanned these residues, then determined how the mutation of these residues affect Cia1‐Cia2 complex formation and the ability to support viability via genetic complementation assays. If the interactions are important in vitro, then there could be phenotypic changes in yeast cells that can alter CIA function. Our lab has previously determined that Glutamate208 within one of Cia2's conserved motifs was vital for Cia2's ability to bind to Cia1. Therefore, we scanned the surrounding conserved residues to identify additional residues important for Cia1‐Cia2 interaction. Several residues close to E208 also disrupted the Cia1‐Cia2 interaction in vitro, confirming that this conserved motif in Cia2 is the docking site for Cia1. Previously, E208A supported yeast viability, whereas, an adjacent residue, Aspartate 206 (D206), appears to be particularly important as it was the only one that failed to support viability in the complementation assay. This shows that this residue does not allow for the targeting complex to form and identify targets, negatively impacting CIA function. Now that we understand which residues are required for Cia1‐Cia2 binding, this allows for further investigation of which specific targets the complex binds to for proper CIA function.

Support or Funding Information

Boston University Undergraduate Research Opportunities Program (UROP)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Carnosol increases skeletal muscle cell glucose uptake via AMPK‐dependent GLUT 4 glucose transporter translocation
Authors: Baron David C. , Vlavcheski Filip , Vlachogiannis Ioannis , Tsiani Evangelia
Published: June 2021

Skeletal muscle is highly important in glucose homeostasis since it is quantitatively a major insulin‐target tissue. Insulin action in muscle cells activates the phosphatidylinositol‐3 kinase (PI3K)/Akt signaling pathway causing the translocation of intracellularly stored GLUT4 glucose transporters to the plasma membrane leading to increased glucose uptake. Impaired insulin action in muscle leads to insulin resistance and type 2 diabetes mellitus (T2DM).

AMP‐activated kinase (AMPK) is a cellular energy sensor and its activation increases glucose uptake by skeletal muscle cells. Finding AMPK activators is viewed as an effective approach to combat insulin resistance and T2DM. Rosemary extract (RE) has been shown to increase muscle glucose uptake and AMPK activity but the components responsible for these effects have not been identified yet. In the current study, we investigated the effect of carnosol, a polyphenol found in high concentrations in RE. L6 rat muscle cells were used to measure uptake of [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting. Carnosol stimulated glucose uptake in L6 myotubes in a dose‐ and time‐dependent manner. A response comparable to maximum insulin stimulation (196±9.2 % of control) was seen with 50μM of carnosol (2h) (182±7.8 % of control). Carnosol did not affect Akt phosphorylation while it significantly increased AMPK phosphorylation. Furthermore, the increase in glucose uptake in the presence of carnosol was significantly reduced by the AMPK inhibitor compound C (CC) while it was not affected by the PI3K inhibitor wortmannin. Carnosol increased plasma membrane GLUT4 glucose transporter levels in GLUT4myc overexpressing L6 cells and this response was abolished by the AMPK inhibitor CC. Our study is the first to show a significant increase in muscle glucose uptake by carnosol via a mechanism that involves AMPK.

Support or Funding Information

Supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
4β‐hydroxywithanolide E inhibits tumor necrosis factor α‐induced tissue factor expression and tumor‐associated coagulation in non‐small cell lung cancers
Authors: Hsieh Kan‐Yen , Lin Ya‐Han , Chang Fang‐Rong , Wu Chin‐Chung
Published: June 2021

The relation between cancer and inflammation has been widely discussed, and the onset of inflammation in tumor microenvironment promotes the progression of cancer is generally recognized. Tumor necrosis factor (TNF) α, a major inflammatory cytokine, which exists in tumor microenvironment and is constitutively produced by both stromal cells and malignant cells. The implication of TNFα in tumor growth and metastasis has been reported in several literatures. In addition, TNFα causes tissue factor (TF) upregulation in several cell types, including monocyte, endothelial cells, and cancer cells that may contribute to tumor‐associated coagulopathy and cancer metastasis. 4β‐hydroxywithanolide E (4HW) is a natural compound isolated from an edible plant, Physalis peruviana. 4HW has been reported to exhibit promising anti‐cancer and anti‐inflammatory activities. In the present study, we examined the effect of 4HW on TNFα‐induced TF expression and procoagulant activity in two non‐small cell lung cancer cell lines, A549 and H1299. At the non‐cytotoxic concentrations, 4HW potently prevented TF expression caused by TNFα in both cell lines. The procoagulant activity of TNFα‐induced TF was further studied by using plasma clotting assay. The plasma clotting time in the presence of TNFα‐treated cancer cells was obviously shorter than control group, and this phenomenon was successfully reversed by pretreating with either 4HW or anti‐TF antibody. Our results suggest that 4HW might have the potential to treat tumor‐associated thrombosis and inflammation.

Support or Funding Information

This research was supported by Ministry of Science and Technology (MOST 106‐2320‐B‐037‐003‐MYB) and Kaohsiung Medical University (KMU‐TP105H03)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Natural Products as a Source to Discover Novel Drug Targets in P. falciparum
Authors: Butler Joshua H. , Zhou Bin , Yue Jian‐Min , Cassera Maria Belen
Published: June 2021

Malaria is a deadly infectious disease that caused an estimated 216 million cases around the world in 2016 according to the World Health Organization. Plasmodium falciparum is the parasite responsible for causing the majority of malaria cases in humans. Resistance to all current frontline antimalarial therapies is a growing problem thus, there is an urgent need for new antimalarial drugs with novel targets. Natural products have a lengthy history of utility in drug discovery and remains a great source of new therapeutics. We have screened against P. falciparum in vitro cultures over 230 natural compounds isolated from various plants indigenous to the southern parts of China which have long been used in traditional Chinese medicine to treat malaria. This collection presented different degrees of antimalarial activity and toxicity toward mammalian cells. Two sesquiterpenoid dimers that exhibited potent antiplasmodial activities (<100 nM) and low toxicity to mammalian cells were selected to study their mechanism of action in more detail by inducing drug resistance in in vitro culture of P. falciparum. We were able to obtain resistant parasites against one of the selected sesquiterpenoid dimers. Clonal subcultures were subjected to next generation Illumina sequencing to identify the potential mechanism of resistance that may lead to the identification of the molecular target. In addition, a subset of similar sesquiterpenoid dimers present in this collection of natural products were screened against the resistant parasites and the analysis has outlined a starting point for structure‐activity relationship studies. We have identified a new class of promising antiplasmodial compounds due to their unique scaffold that is different from all currently known antimalarials and is likely acting through a new mechanism of action.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Montmorency tart cherry anthocyanins: dose‐dependent antioxidant activity against cholesterol oxidation
Authors: Meza Ilce Gabriela Medina , Schweiss Matthew Daniel , Barnaba Carlo
Published: June 2021

Cardiovascular disease (CVD) is a leading cause of death world‐wide, and cholesterol oxidation (CO) and cholesterol oxidation products (COPs) are widely thought to contribute to CVD by exacerbating mechanisms that produce atherosclerotic plaques. COPs not only hold atherogenic properties, but also mutagenic, carcinogenic and inflammatory properties. More recently, it has been reported that they also play an important role in the onset and progression of neurodegenerative diseases. As promoters of inflammation, cytotoxicity, and fibrogenesis, COPs drive a cycle of inflammation that increases the steady‐state level of radical and non‐radical reactive oxygen species (ROS), which in turn amplify inflammation.

Strong evidence indicates that natural products derived from plants, such as anthocyanins, poses antioxidant properties and may inhibit lipid oxidation and its metabolites. Montmorency cherries (Prunus cerasus) contain polyphenols in amounts of 3000 mg/100g dry weight, because of this they could act as a natural source of strong antioxidants. Our group has fractionated and characterized different polyphenols from Montmorency tart cherry extracts. Our preliminary studies indicate that anthocyanins have potent dose‐dependent antioxidants activity against CO suggesting that they may also be efficient at reducing CO‐induced inflammation in the CVD. We explore the antioxidant properties of Montmorency tart cherry anthocyanins against CO using in vitro systems of CVD, such as biomimetic liposomes and low‐density lipoprotein (LDL). Radical‐induced oxidation was triggered by addition of AAPH radical generator at 0.5–2 mM to a large unilamellar vesicles (LUVs) suspension under physiological conditions. Different concentrations of anthocyanins (2–100 μM) were tested over 180 minutes.

Results demonstrated that anthocyanins may have a dose‐dependent antioxidant activity, specifically targeting CO. UV‐Vis spectroscopy showed that an increase in the oxidation of liposomal cholesterol when AAPH was added to the system (Control). A dramatic increase in cholesterol oxidation was observed between 180 and 240 minutes. The kinetics associated with the formation of cholesterol hydroperoxides were delayed up 180 min, with subsequent decrease on the accumulation of 7‐ketocholesterol (7‐Keto) and 25‐hydroxycholesterol (25‐OH), which are biomarkers of CO and thus the cardiovascular inflammation. We speculate that specific compounds derived from Montmorency tart cherries have the ability of to penetrate lipid membrane and scavenge ROS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A small molecule virulence factor suppresses plant immune response
Authors: Li Bo , O'neill Erinn Marie , Mucyn Tatiana , Patteson Jon B. , Baccile Joshua , Schroeder Frank C. , Massolo Elisabetta , Dangl Jeff
Published: June 2021

Natural products play important roles in the chemical interactions between bacteria and their host. Thousands of biosynthetic gene clusters for natural products have been identified through genomic analyses, but most of them are uncharacterized (“cryptic” gene clusters). Our goal is to identify the natural products encoded by these cryptic gene clusters and investigate their functions in bacteria‐host interaction. Using transcriptomics, we identified a cryptic gene cluster that is upregulated in Pseudomonas syringe during plant infection. This gene cluster is widely distributed in plant pathogens that cause worldwide crop damage. To identify the natural product encoded by this gene cluster, we develop an approach that combines heterologous expression and metabolomics with in vitro reconstitution of enzyme activity. We identify a novel natural product, phevamine, which harbors a distinct chemical structure that partly resembles spider and wasp toxins. We discover that phevamine is a virulence factor that suppresses plant immune response. The biosynthesis of phevamine involves ATP‐Grasp type amide ligases. Our work identifies a new virulence factor that pathogens use to invade plant defense. Our work also highlights amide ligases as an important and growing class of biosynthetic enzymes for natural products.

Support or Funding Information

This work was supported by Rita Allen Foundation (B.L.), National Institutes of Health (R00 GM099904, B.L.), NSF‐IOS‐1343020 INSPIRE Track 2 (J.D.), and DOE‐BER‐0000217779 (J.D.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering A Hybrid FRET Biosensor to Study Proteolytic Activities of MT1‐MMP
Authors: Limsakul Praopim , Wang Yingxiao
Published: June 2021

The genetically‐encoded biosensors based on Förster resonance energy transfer (FRET) are extensively used for real‐time monitoring of biological processes including enzymatic activities in living cells with high spatiotemporal resolutions. However, the development of a membrane‐targeted FRET biosensor remains a challenge partly due to the poor efficiency of transportation and integration of fusion proteins at the plasma membrane, which results in high background signals. To overcome this deficiency, we introduced an alternative strategy that allows the direct assembly of FRET biosensors in situ at the cell surface by integrating an enhanced cyan fluorescence protein (ECFP; a donor) and a fluorescent dye, R‐Phycoerythrin (R‐PE; an acceptor), for FRET action. We utilized the directed evolution technology and sequence‐function analysis to engineer a monobody into an efficient R‐PE binder, PEbody, to directly capture R‐PE. Our hybrid FRET biosensor was used to monitor the enzymatic activity of the membrane type 1 metalloproteinase (MT1‐MMP) on the extracellular surface of live cancer cells. The results revealed a cytoskeleton‐dependent distribution of MT1‐MMP activities at different cell‐cell contacts, with high and low activities at loose and stable cell‐cell contacts, respectively. Therefore, integrating the directed evolution approach and rational design can be employed to systematically design novel FRET biosensors capable of monitoring the molecular dynamics at the surface and other subcellular regions in living cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phospholipids as Indicators of Castration Resistant Prostate Cancer
Authors: Ingram Lishann M. , Manosoura Maryam , Pati Sumitra , Cummings Brian S.
Published: June 2021

The association of circulating lipids with clinical outcomes of metastatic castration‐resistant prostate cancer (CRPC) is not fully understood. Interestingly, recent studies demonstrated a correlation between plasma lipids and CRPC prognosis, resulting in a proposed prognostic four‐lipid signature of (ceramide, sphingomyelin, phosphatidylcholine (PC) and phosphatidylserine (PS)). While the increase in these lipids correlated to decreased survival, the mechanisms mediating alterations in these lipids, and the correlation to resistance to treatment are not known. We addressed this gap‐in‐knowledge using in vitro models of non‐cancerous, hormone sensitive and CRPC cell lines and quantitative lipidomic analysis. An initial untargeted approach assessing both cells and media indicated that ceramides, sterols and glycerophospholipids had higher relative abundance in CRPC cells (PC‐3 and DU145), as well as hormone sensitive cells (LNCaP), as compared to a non‐cancer cell model (PNT2). Similar data was seen in media. A targeted approach focusing on lipid species shown to correlate to survival in recent publications showed significantly higher levels of 36:2 phosphatidylserine (PS) in all prostate cancer cell lines tested, and lower levels of 36:2 PC in the media of these same cells. Thus, both untargeted and targeted analysis of prostate cancer cells showed significant differences in lipidomic profiles as compared to non‐cancer prostate cells. These data suggest that the lipidomic profiles of prostate cancer cells lines mirror lipidomic profiles in the plasma of prostate cancer patients. As such, these cells may prove useful for understanding the molecular mechanism mediating lipid changes in prostate cancer patients and yield insight into the role of lipids in mediating CRPC.

Support or Funding Information

National Institute of Biomedical Imaging and Bioengineering NIBIB (EB0160100)

Department of Defense Prostate Cancer Research Program Idea Development Award (PC150431 GRANT11996600)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defects in TM6SF2 Impairs Lipidation of Nascent VLDL and Leads to Accumulation of Liver Fat
Authors: Martin Sarah A. , Smagris Eriks , Cohen Jonathan C. , Hobbs Helen H.
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is a burgeoning health problem in Western countries. Population‐based surveys indicate that one out of every three adults in the United States has excess liver fat (hepatic steatosis), the first stage of fatty liver disease. Despite the clinical importance of NAFLD, major questions remain regarding its etiology. As a step towards elucidating the pathophysiology of this disorder, we screened individuals in a population‐based study, the Dallas Heart Study (DHS) for DNA sequence variations associated with increased liver fat content. This analysis revealed that a common variant (E167K) in transmembrane 6 superfamily 2 (TM6SF2), a gene of unknown function, was associated with increased hepatic triglyceride (TG) content, reduced plasma lipid levels, and increased levels of circulating liver enzymes. TM6SF2 is expressed at highest levels in the small intestine, liver and kidney. Immunolocalization and biochemical studies localized the protein to the ER and the Golgi. To determine why individuals with the risk variant develop hepatic steatosis, we developed mice expressing no TM6SF2 (Tm6sf2−/−, KO). The Tm6sf2−/− mice have elevated levels of neutral lipids, including TG and cholesteryl esters (CE) in the liver and elevated levels of liver enzymes in the circulation, consistent with liver inflammation. At steady state the Tm6sf2−/− mice have decreased plasma levels of cholesterol and very low density lipoprotein (VLDL)‐TG. Treatment with Triton WR1339 to inhibit peripheral lipases was associated with a non‐specific and dramatic reduction in all of the VLDL associated lipid molecular species, but not apolipoprotein (Apo)‐B100. In addition, it was found that de novo synthesis of radioactive and stable labeled lipids was marginally changed in the liver, but secretion of the newly synthesized labelled lipids was dramatically decreased in the Tm6sf2−/− mouse. As expected, given these observations, VLDL was reduced in size in the KO mice. Thus, TM6SF2 plays a key role in the expansion of the nascent particle, but not in the secretion of nascent VLDL, thereby causes steatosis.

Support or Funding Information

Funding was provided by the NIH through research grants R01‐DK090066 and F32‐DK112529.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic profiling associated with deployment‐related stressors in Army personnel
Authors: Gautam Aarti , Donohue Duncan , Abu‐Amara Duna , Hoke Allison , Genfi Afia , Blessing Esther , Hammamieh Rasha , Marmar Charles , Jett Marti
Published: June 2021

Pre‐deployment identification of risk and resilience factors is crucial in developing targets to reduce or prevent posttraumatic stress symptoms in military personnel. The Fort Campbell Cohort study was designed to assess pre‐deployment biological and behavioral markers and build predictive models to identify risk and resistance for posttraumatic stress disorder (PTSD) following deployment. The aim of the current study was to use high‐resolution metabolomics profiling to identify metabolic pathways and networks associated with PTSD checklist score differences and changes across multiple pre and post‐deployment time points. In addition, we have investigated how deployment affects metabolomics profile changes within individuals independent of PTSD score changes. Untargeted metabolomics profiling of plasma has been completed using both liquid and gas chromatography with mass spectrometry. Our preliminary data indicates that deployment has complex metabolic effects that must be considered in evaluation of deployment‐associated exposures in military personnel. Our in depth‐data analysis may further elucidate pathways, novel compounds, and biomarkers associated with PTSD in its relation to military deployment. These findings may have clinical implications for understanding the pathogenesis of PTSD and provide insights to avert chronic PTSD.

Disclaimers: Research was conducted in compliance with all Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army

Support or Funding Information

Funding support from Military Operational Medicine Research Program, MOMRP is highly acknowledged.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct Transcriptional Profile Activated by SCYE1 Through JAK‐mediated STAT3 in Partially Activated Macrophages
Authors: Lee Daniel , Schwarz Margaret
Published: June 2021

Small inducible cytokine subfamily E, member 1, SCYE1 (also known as EMAP II) is a secreted factor that is locally expressed near sites of injury, labeled as a ‘find‐me’ signal, and recruits macrophages and neutrophils. Our previous studies identified that macrophages sustained protein expression of SCYE1. The transcriptional events that occur within the recruited macrophages in response to SCYE1 are unknown. The objective was to identify the transcription factor activated in partially activated thioglycollate macrophages upon exposure to SCYE1.

Partially activated murine thioglycollate‐elicited macrophages (TEPMs) were exposed to SCYE1. Following 2 hours of exposure to either vehicle or SCYE1, fractionated nuclear proteins from TEPMs were used for a luminescence based transcription factor activation screen. The acute SCYE1‐mediated transcription factor activation was assessed by immunoblotting and gene expression by quantitative PCR. Secreted cytokines were measured in supernatants of TEPMs exposed to either vehicle or SCYE1 while pre‐treated with +/− JAK1/2 inhibitor, Ruxolitinib.

In this study, we identify STAT3 (Signal Transducer and Activator of Transcription 3) as the most activated transcription factor mediated by SCYE1 in a model of partially activated, recruited thioglycollate‐elicited peritoneal macrophages. The acute and dose‐dependent phosphorylation of STAT3 (Y705) occurs between 5 and 15 minutes in response to SCYE1. Its activation is SCYE1‐specific and could be prevented by inhibition of Janus Associated Kinase (JAK) 1/2.

We profiled not only anti‐ and pro‐inflammatory genes but also that of STAT3 target genes upon exposure to SCYE1. STAT3 target genes Arg1 and Socs3, commonly activated in anti‐inflammatory macrophages, are maximal by 1 to 2 hours of exposure to SCYE1. Yet Il10 expression decreased by 1 hour suggesting that any persisting STAT3 activation was not due to Il10. There was also rapid increase of chemokine STAT3 target genes Ccl5 along with other genes activated that are typically found in pro‐inflammatory macrophages.

Unlike activated transcription, neither secreted IL‐6 nor IL‐10 was elevated by SCYE1. Consistent with a JAK‐STAT signature, both IL‐1a and G‐CSF secretion by SCYE1 was elevated and could be prevented by JAK inhibition.

Our results identify a rapid SCYE1‐mediated STAT3 activation that coincides with both pro‐ and anti‐inflammatory gene expression in partially activated macrophages.

Support or Funding Information

Funding: This publication was made possible in part by 5R01HL114977 (MAS) from NIH and the Lilly Endowment, Inc. Physician Scientist Initiative (MAS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perturbation of Homocysteine Homeostasis Leads to Cellular Epigenetic Modification And Endothelial Injury in Acute Myocardial Infarction
Authors: Chang Christopher , Yang Tzu‐Ching , Lu Shao‐Chun , Lee Ming‐Syue , Chang Po‐Yuan
Published: June 2021

Homocysteine is a highly reactive, sulfur‐containing amino acid formed as a byproduct of the metabolism of the essential amino acid methionine. Homocysteine used to be thought of as a risk factor for atherosclerosis, but emerging data have suggested that S‐adenosylhomocysteine (SAH), an intermediate metabolite of homocysteine, might be a better indicator of vascular diseases. In this study, we investigated the epigenetic role and clinical significance of SAH‐homocysteine homeostasis in acute myocardial infarction. A cell culture system was established to assess the interplay between SAH and homocysteine using human coronary artery endothelial cells (ECs). Homocysteine alone did not affect EC survival up to 500 uM; however, in the presence of adenosine/EHNA, homocysteine (25–500 uM) dose‐dependently increased intracellular SAH and promoted TUNEL‐positive apoptotic cell death. There was a concomitant downregulation of fibroblast growth factor 2 (FGF2), a critical cytokine for EC survival, through extensive DNA methylation at CpG dinucleotides in the FGF2 gene promoter. These pro‐apoptotic effects in ECs were markedly attenuated when adenosine/EHNA was removed from the culture medium. In patients with acute ST‐elevation myocardial infarction (STEMI), the plasma SAH levels quantitated by liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) were significantly higher than those in healthy controls. In contrast, the plasma homocysteine concentrations were similar between STEMI and healthy subjects. Moreover, STEMI patients who had an obvious clinical improvement also demonstrated a significant decrease in their plasma SAH levels at subsequent follow‐up. Our data conclude that the perturbation of SAH‐homocysteine homeostasis in ECs leads to cellular epigenetic modification and endothelial injury in acute myocardial infarction. Elevation of plasma SAH but not homocysteine in STEMI patients further supports the notion that SAH is a better indicator of vascular diseases than homocysteine.

Support or Funding Information

This work was supported by grants NSC101‐2320‐B‐002‐026 from the National Science Council and MOST‐106‐2314‐B‐002 ‐156 ‐MY2 (Dr. Po‐Yuan Chang) from the Ministry of Science and Technology, Taipei, Taiwan.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FUNCTIONAL CHARACTERIZATION OF THE NOVEL GENE CIA7 FROM CHLAMYDOMONAS REINHARDTII
Authors: Cantu Hector Guillermo Gonzalez , Ynalvez Ruby Agno
Published: June 2021

Heavy metal contamination presents a constant threat to biological systems. These metals interact with the catalytic‐domain of enzymes, interfering with their normal rates of activity, as well as inducing oxidative stress. Certain microorganisms have evolved molecular mechanisms to attenuate this; one of them being the expression and synthesis of low‐molecular weight, thiol‐rich proteins, termed as phytochelatins and metallothioneins. One such organism is Chlamydomonas reinhardtii. C. reinhardtii is an excellent model organism used in studies of heavy metals due to its high endogenous tolerance. In C. reinhardtii a novel gene designated as Cia7, was isolated and was hypothesized to confer tolerance to heavy metals. This was due to CIA7's conserved Cys‐residue motif. Such motif is found in a number of metal binding proteins. In this regard, this study aimed to demonstrate what role, if any, CIA7 exerts on tolerance towards Cd2+ stress in C. reinhardtii. The hypothesis was that the expression of Cia7 will contribute, at least in part, to an increased Cd‐tolerance in C. reinhardtii, as evidenced by physiological markers of health, as well as intracellular accumulation of the metal. The discovery of gene Cia7 in C. reinhardtii may lead to the elucidation of a novel mechanism of metal tolerance or homeostasis in C. reinhardtii. To functionally characterize Cia7: (1) comparative studies of chlorophyll fluorescence and cell size in WT and cia7− in the presence of Cd2+, (2) comparative studies of Cd2+ bioaccumulation in WT and cia7−, and (3) Cd2+ tolerance assays in cia7 insertional mutants were designed. The present work reports on the findings of Cd2+ induced decreased chlorophyll fluorescence and cell size in the cia7− mutant, as well as no difference in Cd‐bioaccumulation at low (5, 10μM) Cd2+ concentrations amongst the WT and cia7−. These results indicate a possible correlation amongst cia7 expression and Cd2+ tolerance. The characterization of CIA7 as a metal‐binding protein would allow for its potential usage as a metal sequestering agent in environmental applications. This would facilitate the remediation of metal‐contaminated ecosystems, which would eliminate the risk of metal poisoning in humans. Furthermore, a correlation of heavy metal exposure to cia7 expression could designate CIA7 as a biomarker for heavy metal contamination.

Support or Funding Information

College of Arts and Sciences and Office of Graduate Studies

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Notch Signaling by Src Kinase
Authors: LaFoya Bryce , Munroe Jordan A. , Albig Allan R.
Published: June 2021

Notch signaling is a form of intercellular communication which plays pivotal roles at various stages in development and disease. The Notch receptor is a single‐pass transmembrane receptor which undergoes proteolytic processing upon binding with a ligand extending from an adjacent cell's membrane. This ligation and subsequent cleavage liberates the transcriptionally active intracellular domain of Notch from the full length receptor. The emancipated Notch intracellular domain undergoes nuclear translocation where it facilitates target gene transcription. Through this series of molecular events, cells can enact changes in a neighboring cell's gene expression patterns. It is now being revealed that Notch signaling has roles which extend beyond juxtacrine signal transduction. Work in our laboratory focuses on the mechanisms by which Notch signaling integrates cell‐cell stimuli with extracellular cues. In a recent publication from our laboratory, we reported that integrin signaling is capable coordinating the Notch pathway. Here, we reveal that the regulation of Notch by integrins is carried out by Src family kinases working downstream of integrins. This is achieved through the phosphorylation of tyrosine residues, within the Notch intracellular domain, by Src. While serine/threonine phosphorylation of the Notch receptor has been well characterized and is essential to important regulatory events, the tyrosine phosphorylation of the Notch receptor is a relatively unexplored area of research. We have identified four tyrosine residues within the intracellular domain of Notch1 that are phosphorylated by Src. In order to observe the functional consequences of Src induced phosphorylation of Notch1, mutagenesis was performed at these sites. We have found that phosphorylation at these residues has implications for Notch1 intracellular domain stability and transcriptional activity. In addition to this work, we have investigated a number of polyphenolic compounds which are capable of binding integrins and regulating the Notch pathway. We report on the Notch induced ramifications of cellular stimulation with resveratrol, and related polyphenols, that alter Notch target gene transcription. Having established these results, we turn our attention towards the importance of Notch‐Src interactions and polyphenolic regulation of Notch signaling as it pertains to vascular biology.

Support or Funding Information

NIH 2R15GM102852‐02, NIH 2R15GM102852‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Astrocyte‐derived CCL20 reinforces HIF‐1‐mediated hypoxic responses in glioblastoma by stimulating the CCR6‐NF‐kB signaling pathway
Authors: JIN Peng , Park Jong‐Wan
Published: June 2021

During tumor development, stromal cells are co‐opted to the tumor milieu and provide favorable conditions for the tumor. Hypoxia stimulates cancer cells to acquire a more malignant phenotype via activation of hypoxia‐inducible factor 1 (HIF‐1). Given that cancer cells and astrocytes in glioblastomas coexist in a hypoxic microenvironment, we examined whether astrocytes affect the adaptation of glioblastoma cells to hypoxia. Immunoblotting, reporter assays, quantitative RT‐PCR, and chromatin immunoprecipitation were performed to evaluate HIF‐1 signaling in glioblastoma cells. Astrocyte‐derived chemokine C‐C motif ligand 20 (CCL20) was identified using cytokine arrays, and its role in glioblastoma development was evaluated in orthotopic xenografts. Astrocytes augmented HIF‐1a expression in glioblastoma cells under hypoxia. The expression of HIF‐1 downstream genes, cancer colony formation, and Matrigel invasion of glioblastoma cells were stimulated by conditioned medium from astrocytes pre‐exposed to hypoxia. CCL20 was secreted in a hypoxia‐dependent manner from astrocytes and busted the hypoxic induction of HIF‐1a in glioblastoma cells. Mechanistically, the CCL20/CCR6 signaling pathway upregulates HIF‐1a by stimulating nuclear factor kappa B‐driven transactivation of the HIF1A gene. Compared with the control tumors, CCR6‐deficient glioblastoma xenografts grew more slowly, with poor vascularization, and expressed lower levels of HIF‐1a and its downstream proteins. Furthermore, CCR6 expression was correlated with HIF‐1a expression in GEO and TCGA datasets from human glioblastoma tissues. These results suggest that glioblastoma cells adapt well to hypoxic stress by virtue of CCL20 derived from neighboring astrocytes.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (2016R1A2A1A05005082). Jin received a scholarship from the BK21‐plus education program and Park JW was supported by the Education and Research Encouragement Fund of Seoul National University Hospital.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
WW Domain‐containing Oxidoreductase Induces Cellular Senescence in Response to Replication Stress
Authors: Cheng Hui‐Ching , Hsu Li‐Jin
Published: June 2021

The induction of DNA damage response (DDR) to ensure accurate duplication of genetic information is crucial for the maintenance of cellular genome integrity during DNA replication. Under replication stress, cellular senescence is a DDR mechanism that prevents the proliferation of cells with DNA damage to avoid mitotic anomalies and inheritance of the damage over cell generations. A reduction in the ability of cells to trigger senescence has been suggested to be associated with cancer initiation and progression. Human WWOX gene resides within a common chromosomal fragile site FRA16D and encodes a tumor suppressor WW domain‐containing oxidoreductase. Downregulation of WWOX protein expression has been frequently found in various human cancers. Increased genome instability has been shown in the cells null for WWOX. However, the role of WWOX in DDR remains largely unclear. In this study, we demonstrated that Wwox gene knockout (Wwox−/−) mouse embryonic fibroblasts (MEFs) failed to undergo replication‐induced cellular senescence after multiple passages in culture. Strikingly, accelerated cell cycle progression and increased apoptosis were detected in late‐passage Wwox−/− MEFs. Increased γH2AX‐positive cells were observed in late‐passage Wwox−/− MEFs, indicating massive accumulation of DNA damages in these cells. The p16INK4A/Rb‐ and p53/p21cip1/waf1‐mediated signaling pathways have been suggested to control cellular senescence. We determined significantly reduced expression of p16INK4A and p21cip1/waf1 proteins in late‐passage Wwox−/− MEFs. Treatment of Wwox−/− MEFs with DNA methylation inhibitor 5‐aza‐2′‐deoxycytidine reversed p16INK4A and p21cip1/waf1 expression, and ectopic expression of p16INK4A and p21cip1/waf1 increased cellular senescence in late‐passage Wwox−/− MEFs. These results suggest that hypermethylation of p16INK4A and p21cip1/waf1 promoters may lead to the failure of senescence induction in Wwox−/− MEFs. Moreover, inhibition of ROS production by N‐acetyl‐L‐cysteine during cell replication in vitro increased protein expression of p16INK4A and p21cip1/waf1 and the induction of cellular senescence in Wwox−/− MEFs. In conclusion, our results support a crucial role for WWOX in DDR for maintaining genome integrity via the induction of cellular senescence in response to replication stress.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The surreptitious survival of the opportunistic pathogen Staphylococcus lugdunensis within macrophages as an immune evasion strategy
Authors: Heinrichs David , Flannagan Ronald , Watson David
Published: June 2021

Staphylococcus lugdunensis has acquired recognition as a relatively new and serious pathogen that is often clinically misidentified as S. aureus. To cause infection S. lugdunensis must circumvent host immunity, including the innate defenses presented by professional phagocytes such as macrophages. In this study, we demonstrate for the first time that macrophages of murine and human origin are not microbicidal towards S. lugdunensis and that phagocytosed S. lugdunensis remain viable within infected macrophages over an extended period (>24 h). Interestingly, phagocytosed S. lugdunensis, which fail to replicate, also do not intoxicate host cells, thus distinguishing the intracellular lifestyle of S. lugdunensis from that of S. aureus. Dextran pulse‐chase and LAMP‐1 immuno‐localization studies reveal that phagocytosed S. lugdunensis take up residence inside LAMP‐1‐positive phagolysosomes where the bacteria persist for at least 24 hours. Phagocytosed S. lugdunensis are modified within macrophages such that they are sensitized to in vitro stresses imposed by antimicrobial peptide treatment (e.g. polymyxin B), exposure to non‐ionic detergent (e.g. Triton‐X100), and the staphylococcal selective medium mannitol salt agar. We further demonstrate that optimal survival of S. lugdunensis requires O‐acetylated peptidoglycan since a S. lugdunensis oatA mutant, which is more sensitive to killing by lysozyme than WT, survived to a lesser extent in the phagosome. Through pharmacological disruption of lysosome function, either prior to or post phagocytosis, we demonstrate that S. lugdunensis bacteria can be coaxed into escaping the phagolysosome and replicating within both murine macrophages and primary human macrophages. Notably, however, intracellularly replicating S. lugdunensis fail to intoxicate infected macrophages as indicated by the host cells' ability to phagocytose IgG‐opsonized targets and remain refractory to vital dye staining. We propose that macrophages harboring S. lugdunensis in vivo represent a bacterial reservoir and events that disrupt macrophage function would allow these bacteria to proliferate whilst remaining protected from extracellular immune factors.

Support or Funding Information

Canadian Institutes of Health Research

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacteria, Brains, and Behavior: gut microbes and their effects on regulation of gene expression in the brain
Authors: Horstman Cara , Devries Laura , Sonnenburg Marie , Carlson Clayton
Published: June 2021

The purpose of this research is to investigate the relationship between the microbiota of the gastrointestinal (GI) system, behavioral characteristics related to Autism Spectrum Disorder (ASD), and relative gene expression of miRNAs in the brain. This research utilizes BALB/c mice (a mouse strain that demonstrates low sociability) as a prototype for ASD and C57BL/6 as a control group of mice. Each mouse strain is fed either Bifidiobacterium longum, Lactobacillus rhamnosus, both bacteria, or only the bacterial media as a negative control. These bacterial strains are both well characterized probiotics shown to change behavior and improve sociability. Behavioral characteristics throughout the 10‐week feeding period for both mouse strains, and each probiotic condition, are compared using Crawley's Sociability and Preference for Social Novelty protocol. Upon completion of the 10‐week study, the mice are sacrificed, the brains are harvested, and the RNA is extracted from the brains of the mice. PCR arrays (Qiagen) are performed to determine changes in the gene expression of brain‐specific miRNAs. Preliminary results from a 3‐week feeding study indicated several miRNAs of interest. For example, these results revealed that relative gene expression of mmi‐488‐3c in the BALB/c mice was substantially more similar to the miRNA's expression in C57BL/6 only following the Bifidobacterium probiotic treatment. The data set from the extended 10‐week probiotic treatment will provide more significant evidence that these GI bacterial probiotics cause changes in expression of important regulatory RNAs. The resultant consequences for target brain mRNAs will also be demonstrated. As this study continues, it aims to further close the gap of understanding between causal relationships between the gut microbiota, neurological changes, and social behavior of animals.

Support or Funding Information

Trinity Christian College, Vander Velde Research Fellowship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Attenuation of FFA‐induced skeletal muscle insulin resistance by rosemary extract
Authors: Vlavcheski Filip , Tsiani Evangelia
Published: June 2021

Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action in muscle leading to insulin resistance and Type 2 diabetes mellitus. Serine phosphorylation of the insulin receptor substrate (IRS) reduces insulin‐stimulated tyrosine phosphorylation leading to insulin resistance. A number of serine/threonine kinases including JNK, mTOR and p70 6SK have been implicated in serine phosphorylation of IRS‐1 and insulin resistance. Activation of the energy sensor AMP‐activated protein kinase (AMPK) increases glucose uptake and has become an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased skeletal muscle cell glucose uptake and activated AMPK. However, its effect on FFA‐induced insulin resistant muscle cells has never been elucidated. In this study, we investigated the effect of RE in palmitate‐induced insulin resistant L6 muscle cells. Glucose uptake was measured using [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting.

Exposure of L6 myotubes to the FFA palmitate (P) (0.2 mM, 16 hours) resulted in significant reduction in insulin‐stimulated glucose uptake (I: 201±1.21% and P+I: 117±15.6% of control, P<0.001 respectively) indicating insulin resistance. Importantly, exposure to RE resulted in significant restoration of insulin‐stimulated glucose uptake (179±10.5% of control, P<0.001). Treatment with palmitate increased serine phosphorylation of IRS‐1 and significantly decreased the insulin‐stimulated phosphorylation of Akt and these effects were completely abolished in the presence of RE. Treatment with palmitate increased the phosphorylation/activation of JNK, mTOR and p70 6SK whereas RE completely abolished these effects. RE increased the phosphorylation of AMPK even in the presence of palmitate. Overall, our data indicate that treatment with RE attenuates the FFA‐induced insulin resistance. RE and RE polyphenols may have potent effects against insulin resistance and deserve further study.

Support or Funding Information

Supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.