bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and characterization of a lipid peroxidase from Nitrosomonas europeae structurally related to prostaglandin H2 synthase
Authors: Selinsky Barry Steven , Cunniff Alecia M , Skaf Rebecca S , Butchy Margaret 
Published: June 2021

Prostaglandin‐endoperoxide synthase (PGHS; EC 1.14.99.1) catalyzes the two step synthesis of prostaglandin H2, an important precursor in the synthesis of prostaglandins and eicosanoids. The first step of the reaction involves the addition of two molecules of oxygen to arachidonic acid to form prostaglandin G2. In the second step, the C15 hydroperoxide in prostaglandin G2 is reduced to an alcohol, generating prostaglandin H2. PGHS has been isolated and characterized from many different eukaryotes (both vertebrates and invertebrates) and has recently been expressed from an open reading frame in the red algae Gracilaria vermiculophylla. A number of putative PGHS orthologs have been identified in bacterial genomes. In this study, a putative PGHS protein from Nitrosomosas europeae has been cloned, expressed, and characterized. Soluble Nitrosomonas protein was isolated to greater than 90% purity exploiting an N‐terminal (his)6 tag. The expressed protein exists in solution as an oligomer, likely a tetramer, and binds heme at a ratio of approximately 0.40 heme molecules per protein monomer. The expressed protein is incapable of adding oxygen to unsaturated lipid substrates, but is a capable peroxidase using both hydrogen peroxide and 15(S)‐hydroperoxy eicosapentaenoic acid as substrates. This result contrasts experiments using a similar protein previously expressed from the genome of Nostoc punctiforme, which exhibits lipoxygenase, but not peroxidase, activity. Structural modeling of the Nostoc and Nitrosomonas proteins yields superimposable structures. Mutagenesis studies of the Nostoc protein are in progress to generate peroxidase activity in the protein product.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Technique for Sickle Cell Anemia Diagnosis
Authors: Braga Camila Pereira , Grove Ryan Albert , Adamec Jiri 
Published: June 2021

Sickle cell anemia (SCA) is a global public health problem that occurs mostly in low‐ and middle‐income countries, and underserved communities. Diagnosis and early treatment are critical to prevent mortality from this disease; however, a large‐scale screening is difficult due to a need for blood collection by trained phlebotomist in designated health centers. The primary goal of this study was to test a new self‐blood collection platform to enable screening in rural and underserved communities. To address this need, a simple, miniaturized device ‐ Noviplex™ card from Novilytic was evaluated. The advantages of this device include the ability to collect 2.7 μL of biofluid as well as the fact that the collected samples are stable at ambient temperature for several weeks and can be mailed to a clinical laboratory for further testing. To obtain the whole blood samples, 1% sodium deoxycholate was added to the blood prior the application or directly to the collection disk of the device. Two mass spectrometry (MS) approaches including MALDI and Multiple Reaction Monitoring (MRM) techniques were developed and validated to 1) detect the normal peptide present in β‐globulin (HBB) from a healthy donor (VHLTPEEK, Mw= 952.50 Da); 2) detect the mutated peptide present in HBB from a SCA donor (VHLTPVEK, Mw= 922.53 Da); and 3) detect a control peptide present in HBB of both healthy and SCA donors (LLVVYPWTQR, Mw= 1,274.72 Da). For MALDI based technique, blood proteins were transferred from the dried collection disc to a PVDF membrane by a semi‐dry blot system, digested with trypsin on the membrane and matrix directly deposited on the top of the blotted sample. Presence or absence of the peptides was determined using a Bruker 7T SolariX Fourier Transform Ion Cyclotron Resonance (FTICR) MS. For MRM, the dried collection disc was transferred to a 96‐well plate, and trypsin was added to cover the collection disc completely. Following digestion, samples were directly analyzed by LC‐MRM for normal, mutated and control peptides. Both techniques were evaluated using the samples from a donor with SCA and from a healthy individual and showed good digestion efficiency and reproducible detection of the peptides of interest. In addition, both methods can be fully automatized for high‐throughput testing in clinical laboratory environment. Sample preparation times for MALDI and MRM techniques were 25 and 3 min, respectively.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of the inhibition of UCP2 on the activity and presence of Amylin in RIN‐5F cells
Authors: Manuel Gygeria , Clemons Tameka 
Published: June 2021

Amylin is a fibrillogenic protein co‐secreted with insulin from pancreatic beta cells. Amylin has been shown to function in metabolism through its influence on glucose homeostasis, which includes sending satiety signals to the brain, inhibiting glucagon secretion and delaying gastric emptying. In addition to amylin's direct role on metabolism it has also been proposed to have an impact on amyloid beta, a major component of the amyloid plaques found in Alzheimer's disease. Alzheimer's disease brains have been shown to have amyloid plaque formation and decreased expression of Uncoupling Protein 2 (UCP2), which is a protein that also has a role in metabolism. Traditionally, uncoupling proteins have been known to function as mitochondrial transporter proteins that create proton leaks across the inner mitochondrial membrane resulting in the dissipation of energy in the form of heat. Knowing that UCP2 is expressed in pancreatic beta cells and has been shown to have diminished levels in Alzheimer's disease brains the focus of this study was to examine the influence of UCP2 on the presence and expression of amylin in RIN‐5F cells (a pancreatic beta cell line). siRNA UCP2 constructs were created and immunocytochemistry experiments and ELISA assays were conducted to examine the presence of amylin and the activity level of amylin, respectively. Initial studies suggest that inhibition of UCP2 may impact the activity level and presence of amylin.Support or Funding InformationTameka Clemons at Spelman College Department of Chemistry and BiochemistryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optogenetic activation and inactivation of the neurotrophin pathway in live cells
Authors: Mondal Payel , Krishnamurthy Vishnu Vardhan , Sharum Savanna , Haack Neeka , Zhang Kai 
Published: June 2021

Signaling pathways extensively crosstalk among each other and result in different cellular phenotypes depending on the dynamic profile of protein activity. Conventional genetic and pharmacological approaches such as gene overexpression, use growth factors or inhibitors have helped us understand different aspects of signaling pathways, however these techniques provide limited means to determine contribution of a target protein for specific cellular phenotype. Therefore, to find out the molecular mechanism for a cellular outcome, there is an urgent need for a tool that can specifically activate or inactivate a protein of interest and study it's role towards a specific cell fate. Optogenetic techniques utilize light to control protein functions with high spatial and temporal resolution. Here we introduce a generalizable method for optical control of protein stability that enables fine spatiotemporal control of specific signaling pathways. This optogenetic system has been used to re‐purpose the protein degradation machinery by combining a small peptide sequence called degron, an evolved LOV (eLOV) protein and a light‐inducible nuclear export system (LEXY). Our system has been used to activate the Raf/MEK/ERK signaling cascade by light induced stabilization of a constitutively active cytosolic MEK (CA MEK). Light induced activation of the ERK pathway results in elevated phosphor‐ERK levels in human embryonic kidney cells (HEK) and rat PC12 pheochromocytoma cells as well as PC12 neurite outgrowth in the absence of growth factors. Furthermore, current non‐neuronal optogenetic methods provide fewer tools for inactivation of a specific signaling pathway compared to optogenetic activation. To bridge this gap, we have demonstrated inhibition of the ERK pathway by light induced stabilization of mitogen activated protein kinase phosphatase 3 (MKP3), an inhibitor of the ERK pathway. Thus, our system allows light‐mediated rescue of protein stability by using a degradation‐rescue strategy and offers the potential for regulation of a plethora of biological processes.Support or Funding InformationThis work was supported by the University of Illinois at Urbana‐Champaign (UIUC) (K.Z.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Tributyltin Exposures on Translation Regulation in Human Immune Cells
Authors: Ruff Amanda Elizabeth , Hamza Nafisa M , Whalen Margaret M 
Published: June 2021

Tributyltin (TBT) contaminates the environment due to its use as a biocide. It is found in human blood (ranging as high as 261 nM). TBT is able to increase the synthesis of pro‐inflammatory cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL‐1β), and interleukin 6 (IL‐6) in human immune cells. This TBT‐induced increase in pro‐inflammatory cytokines could contribute to chronic inflammation, which is a risk factor for a number of diseases including several cancers. TBT appears to utilize the ERK1/2 and/or p38 MAPK pathways to stimulate pro‐inflammatory cytokine synthesis in immune cells. MAPK pathways have the capacity to regulate translation including processes leading to the phosphorylation (activation) of eukaryotic initiation factor 4E (eIF4E), eIF4B, and the S6 ribosomal subunit. We hypothesize that TBT may influence the activation state of these translational regulators as part of its mechanism of increasing cytokine synthesis. Studies to examine the levels and phosphorylation state of eIF4E, eIF4B and S6K after varying lengths of exposure to TBT in monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs) were carried out. Initial results suggest that TBT caused increased phosphorylation (activation) of eIF4E and S6. These results may indicate that TBT is elevating the synthesis of key pro‐inflammatory cytokines in immune cells by its ability to activate translation.Support or Funding InformationNIH Grant 5U54CA163066This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ang‐II‐Induced Phosphorylation and Nuclear Export of Histone Deacetylase 5 is Mediated by an AKT‐Dependent Pathway in Vascular Smooth Muscle Cells
Authors: Truong Vanessa , Jain Ashish , Srivastava Ashok K. 
Published: June 2021

Augmented levels of angiotensin‐II (Ang‐II) are associated with the development of cardiovascular disorders such as atherosclerosis and hypertension. Exaggerated activation of Ang‐II‐induced signaling events and expression of genes linked to cell proliferation, hypertrophy and migration have been demonstrated to contribute to the development of these vascular pathologies. Ang‐II induces the phosphorylation of class II histone deacetylases (HDACs), particularly HDAC4 and HDAC5. HDACs regulate gene transcription by their ability to modify the acetylation status of the lysine residues in histone and non‐histone proteins. A heightened activation of HDACs, notably HDAC5, has been associated with vascular disorders such as atherosclerosis. We have shown earlier that Ang‐II induces the expression of early growth response factor‐1 (Egr‐1), a zinc finger transcription factor which is upregulated in atherosclerotic lesions and in animal models of vascular injury. We have also reported that the PI3Kinase/AKT pathway plays a key role in inducing the expression of Egr‐1 in vascular smooth muscle cells (VSMC). However, the role of the AKT pathway in HDAC5 phosphorylation and the contribution of HDAC5 in Egr‐1 expression in VSMC remains unexplored. Here, we show that Ang‐II induced the phosphorylation of HDAC5 in a time and dose‐dependent manner, and pharmacological blockade of the PI3K/AKT pathway by wortmannin or SC66 attenuated Ang‐II‐induced HDAC5 phosphorylation in A10 VSMC. In addition, Ang‐II treatment of VSMC resulted in the translocation of HDAC5 from the nucleus to the cytosol, and the inhibition of AKT by SC66 almost completely attenuated the nuclear export of HDAC5 induced by Ang‐II. In contrast, pharmacological blockade of the MAPK pathway by UO126 was ineffective in reducing Ang‐II‐induced HDAC5 phosphorylation. Furthermore, pharmacological inhibition of HDAC5 by MC1568 or siRNA‐induced silencing of HDAC5 suppressed Ang‐II‐induced Egr‐1 expression. In summary, our results suggest that Ang‐II induces the phosphorylation and nuclear export of HDAC5 via the PI3K/AKT‐dependent signaling pathway and that HDAC5 is an upstream modulator of Egr‐1 expression in VSMC.Support or Funding InformationSupported by Canadian Institutes of Health ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Drosophila P5B ‐Type ATPase is Required for Polyamine Transport
Authors: von Kalm Laurence , Brown David 
Published: June 2021

In eukaryotes P‐type ATPases are represented by a superfamily of membrane ion and lipid transporters divided into five subclasses. Substrates for subclasses 1–4 have been identified, however little is known about the substrate specificity of the P‐type 5A and 5B transporters. Here we show that the Drosophila P5B‐type ATPase is required for polyamine transport. The gene encoding the Drosophila P5B‐type ATPase produces multiple protein isoforms. Using a combination of genetic mutational analysis and CRISPR‐Cas9 gene editing we show that some of these isoforms are required for polyamine transport and others are not. In addition, we show that the Drosophila P5A‐type ATPase is not required for polyamine transport indicating a different substrate specificity for this transporter subfamily. In summary, we provide evidence for the first time of substrate specificity for the P5B‐type ATPase subfamily of transporters.Support or Funding InformationUniversity of Central Florida, College of Sciences Seed AwardThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tribbles Homolog 2 (TRIB2) plays a potent role in ovarian granulosa cells proliferation and function
Authors: Warma Aly , Lussier Jacques G. , Ndiaye Kalidou 
Published: June 2021

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2mRNA is rapidly induced by mitogens, has a short half‐life, and is expressed in a cell‐specific manner. We previously identified TRIB2 as a differentially expressed gene in granulosa cells of bovine preovulatory follicles. This study aimed to further investigate TRIB2mRNA and protein regulation, to identify its binding partners, and study its function in granulosa cells of bovine dominant follicles. Granulosa cells (GC) were obtained from follicles at different developmental stages: small follicles (SF: 2–4 mm), dominant follicles (DF) at day 5 of the estrous cycle (day 0 = day of the oestrous), ovulatory follicles 24 hours following injection of an ovulatory dose of hCG (OF), and corpus luteum (CL) at day 5 of the oestrous cycle. In addition to this in vivo model, an in vitro model of cultured GC was used for functional studies using the CRISPR‐Cas9 approach. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF and CL (P < 0.0001). Temporal expression of TRIB2mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 hours after hCG injection. There was a significant reduction of TRIB2 steady‐state mRNA levels in follicular walls, starting at 6 hours through 24 hours post‐hCG as compared to 0 hour (P < 0.001). Additionally, we have generated specific anti‐TRIB2 polyclonal antibodies that confirmed, in western blot analyses, TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression (P < 0.05) while inhibition of TRIB2 using CRISPR‐Cas9 resulted in significantly reduced GC proliferation (P < 0.05). These results provide strong evidence that TRIB2 is a potent regulator of GC proliferation.Support or Funding InformationResearch supported by NSERC of Canada grant #RGPIN‐2018‐04516 to KN.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of ATM Positively Regulates Rac1 Activity and Cellular Migration Through Oxidative Stress
Authors: Srougi Melissa C. , Tolbert Caitlin E. , Smith Aaron , Beck Matt V. 
Published: June 2021

Ataxia‐telangiectasia mutated (ATM) is a serine‐threonine kinase that is integral in the response to DNA double‐stranded breaks (DSBs). Cells and tissues lacking ATM are prone to tumor development and enhanced tumor cell migration and invasion. Interestingly, ATM‐deficient cells exhibit high levels of oxidative stress; however, the direct mechanism whereby ATM‐associated oxidative stress may contribute to the cancer phenotype remains largely unexplored. Rac1, a member of the Rho family of GTPases, also plays an important regulatory role in cellular growth, motility, and cancer formation. Rac1 can be activated directly by reactive oxygen species (ROS), by a mechanism distinct from canonical guanine nucleotide exchange factor‐driven activation. Here we show that loss of ATM kinase activity elevates intracellular ROS, leading to Rac1 activation. Rac1 activity drives cytoskeletal rearrangements resulting in increased cellular spreading and motility. Rac1 siRNA or treatment with the ROS scavenger N‐acetyl cysteine restores wild‐type migration. These studies demonstrate a novel mechanism whereby ATM activity and ROS generation regulates Rac1 to modulate pro‐migratory cellular behavior.Support or Funding InformationThis work was funded by High Point University's Research and Sponsored Programs (MCS), the Summer Undergraduate Research Program in the Sciences (MVB, MCS), and Undergraduate Research and Creative Works (AS, MVB, MCS).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MAP Kinases Mediate Regulation of eNOS through Phosphorylation of Different Sites
Authors: Solone Xzaviar , Wells Brady , Chrestensen Carol 
Published: June 2021

Mitogen activated protein kinases (MAPK) p38 and ERK, have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via hypothesized interactions with a pentabasic non‐canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK. In the present study, eNOS phosphorylation by MAPKs (P38, ERK, JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at pS116 contrasts with ERK, which phosphorylated pS602, and p38, which phosphorylated both sites. Literature reports that JNK1/2 activation does not alter eNOS activity while prior work in our lab shows that ERK phosphorylation negativity impacts NO output. We will present in vitro results comparing the direct impact of each kinase on eNOS activity using the oxyhemoglobin (Oxy‐Hb) assay. In HMEC‐1 cells and in vitro we observe that phosphorylation at these MAPK sites happens concurrently with activating sites (pS1179), creating antagonistically phosphorylated eNOS. These results underscore the importance of MAPK interactions with eNOS showing that each MAPK creates a unique phosphorylation pattern and NO production outcome. These findings strengthen the emerging paradigm of eNOS as a junction of multiple signaling pathways.Support or Funding InformationR15GM110634‐01A1 supported 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.
Interrogating signaling pathways activation and deactivation with homogeneous NanoBiT Cell based kinase immunoassays
Authors: Zegzouti Hicham , Hwang Brian (Byounghoon) , Engel Laurie , Goueli Said 
Published: June 2021

Cellular responses such as gene expression, enzyme activities, protein synthesis and translocation are orchestrated through activation of diverse cell signaling pathways. In these pathways, phosphorylation of specific proteins by specific kinases constitute important nodes by which the signal is transduced from an upstream activation event to downstream cellular responses. As a corollary, alteration of these kinase activities can have a detrimental effect on these cellular processes, leading to serious pathologies such as cancer, inflammation and diabetes. Therefore, monitoring these signaling events through the analysis of specific node phosphorylation states can help better understand cell behavior in health and disease states. We developed the NanoBiT cell based kinase immunoassay platform which is a suite of simple homogeneous assays to detect phosphorylation of endogenous proteins of interest in lysed cells. NanoLuc Binary Technology (NanoBiT) is a two‐subunit system based on NanoLuc luciferase that was successfully demonstrated for protein‐protein interaction (PPI) detection. We generated secondary antibody pairs linked to the two Nanoluc components, NanoBiT small and large fragments. A cell lysate containing the protein target is incubated with two primary antibodies that recognize separate epitopes on the protein. These bring NanoBiT secondary antibodies into proximity to form an active NanoLuc luciferase that makes light in proportion to the amount of target protein. When the primary antibody pair includes a phosphospecific antibody, the luminescence reflects the level of the target protein phosphorylation. Unlike current assays such as Western blot or ELISA, that are tedious, require multiple washing steps, and not easily adaptable to HTS, the NanoBiT immunoassay method presented here takes less than two hours to complete in a homogeneous “Add and Read” format. Other advantages include no cell engineering as the phosphorylation of endogenous substrate is detected in any cell type, and only a simple luminometer is required for detection. We tested this system by monitoring the activation of multiple signaling pathways including NF‐κB, mTOR/AKT and JAK/STAT, through specific nodes of phosphorylation such as pIκB, pAKT, and pSTAT3. We also tested different small or large molecule inhibitors of these pathways or their corresponding node kinase (IKK, mTOR, or JAKs) and obtained the expected pharmacology. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest, study the kinase cellular activity and regulation or identify specific kinase or pathway inhibitors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.