bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of Protein Synthesis In‐Vivo with 15N‐Glycine Labeling and MALDI‐TOF Spectrum Analysis
Authors: Wahjudi Paulin Nadi , Xiao Gary G , Lee WN Paul , Nowak Mark W , Shiva Nima
Published: June 2021

Previously, the group has demonstrated the feasibility of determining in‐vivo protein synthesis by utilizing deuterated water labeling [1] and in‐vitro protein synthesis with 15N‐glycine labeling [2]. The study is designed to give preliminary proof of concept that the 15N‐glycine labeling can also be applied for in‐vivo protein synthesis determination. Two mice were fed 20 g of 15N glycine/kg of chow for 7 days. The mice serum albumin was SDS‐PAGE purified, followed by in‐gel tryptic digestion, before MALDI‐TOF profiling. Mass spectra data for two specific peptide sequences with mass to charge (m/z) of 1479 and 1609 were analyzed for 15N enrichment and fraction of new synthesis. For m/z =1479, average enrichment of 15N in the peptide was found to be 6.06% for mouse 1 and 6.10% for mouse 2, with fraction of new synthesis found to be 97.52% and 96.54% respectively. The 15N enrichment for m/z =1609 was found to be 5.25% for mouse 1 and 6.31% for mouse 2, with fraction of new synthesis of 90.24% and 88.28% respectively. The mass spectral peaks of the heavier protein can be resolved from the natural protein by a mathematical algorithm. The capability of resolving overlapping spectra makes it possible to label proteins with lower enrichment of stable isotopes.
EFFECTS OF FATTY ACIDS ON PRODUCTION OF REACTIVE OXYGEN SPECIES (ROS) BY SKELETAL MUSCLE CELLS
Authors: Nachbar Renato Tadeu , Luchessi Augusto Ducati , Cambiaghi Tavane David , Lambertucci Rafael Herling , Hirabara Sandro Massao , Vitzel Kaio Fernando , Pinheiro Carlos Hermano da Justa , Curi Rui
Published: June 2021

Reactive Oxygen Species (ROS) are associated to muscle cell survival. These species in excess cause muscle cell damage and apoptosis. A recent study has shown that palmitic acid (PA) increases superoxide production by skeletal muscle cells in culture. Pathological conditions such as obesity and diabetes mellitus, lead to an increase in plasma levels of free fatty acids (FFA). In spite of these observations, however, the effect of other FFAs on production of ROS by skeletal muscle has not been investigated yet. In this study, the effects of oleic (OL), linoleic (LI), PA and stearic (ST) acids on ROS production by primary cultured rat skeletal muscle cells was investigated. The cells were treated with the fatty acids (25 M) during 60 minutes in the presence of the fluorescent probe dihydroethidium. Production of O2•‐ by the cells treated with ST, OL, LI and PA acids was increased by 55 ± 8%, 59 ± 6%, 62 ± 9% and 73 ± 10%, respectively. We observed that the NF‐κB activation is involved in the increased production of ROS induced by FFAs. Our results revealed that the increase in plasma FFAs in various pathological conditions, can lead to an increase of ROS production by skeletal muscle.
Expression of Human ADCK3 restores Coenzyme Q biosynthesis and Phosphorylation of Coq polypeptides in yeast abc1/coq8 mutants
Authors: Xie Letian , Hsieh Edward J. , Watanabe Shota , Allan Chris M. , Tran UyenPhuong C. , Clarke Catherine F.
Published: June 2021

Coenzyme Q (ubiquinone or Q) is a lipid electron carrier in the electron transport chain. In yeast Saccharomyces cerevisiae nine genes, designated COQ1 through COQ9, have been identified as being required for Q biosynthesis. One of these genes, ABC1/COQ8, is now thought to function as a kinase (Tauche et al., 2009). We have characterized seven independent abc1/coq8 mutants (Tzagoloff and Dieckmann, 1990); four contain mutations in kinase subdomains, and three are in uncharacterized motifs. In this study, phosphorylation of Coq3p, Coq5p, and Coq7p are dependent on Coq8p as determined by 2D‐IEF/SDS‐PAGE analyses, while Coq1p was discovered to be highly phosphorylated, but in a less Coq8p‐dependent manner. ADCK3, a human homolog of yeast ABC1/COQ8, was able to restore growth of yeast abc1/coq8 mutants on medium containing a non‐fermentable carbon source. Expression of the human ADCK3 polypeptide in three distinct coq8 yeast mutants increased steady state levels of yeast Coq4p and Coq7p, and restored phosphorylation of Coq3p, Coq5p, and Coq7p. This work is supported in part by National Institutes of Health (NIH) Grant GM45952.
Hepatic oleate synthesis restores SREBP‐1 processing and lipogenesis in SCD1‐deficient mice
Authors: Strable Maggie S , Liu Xueqing , Ntambi James M
Published: June 2021

Stearoyl‐CoA desaturase (SCD) catalyzes the de novo synthesis of monounsaturated fatty acids (MUFA) from saturated fatty acid precursors. Previous work has demonstrated that SCD1 deficiency impairs hepatic lipogenesis and protects against development of diet‐induced obesity. The objective of this study was to determine if liver‐specific MUFA synthesis restores hepatic lipogenesis and processing of the sterol regulatory element binding protein‐1 (SREBP‐1) in SCD1 global knockout mice (GKO). We produced transgenic mice expressing human SCD5 only in liver and introduced this transgene into GKO mice. Hepatic SREBP‐1 maturation is increased in the hSCD5/GKO mice while increased expression of several important lipogenic genes also occurs. Unlike GKO mice fed a high carbohydrate, very low fat diet, hSCD5/GKO mice are largely protected from the dramatic weight loss that occurs. These results suggest that the product of the reaction catalyzed by hSCD5, 18:1n‐9, is involved in the regulation of SREBP‐1 processing as well as lipogenesis. Supported by NIH.
Internalization Of Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Occurs Involving Clathrin‐Dependent Mechanisms
Authors: Somanna Naveen K , Pandey Amitabh C , Wu Edwin , Arise Kiran K , Pandey Kailash N
Published: June 2021

Atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl‐cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produce second messenger cGMP. The aim of this study was to determine whether GC‐A/NPRA is internalized involving clathrin‐coated pits. Human embryonic kidney‐293 (HEK‐293) cells were stably transfected with GC‐A/NPRA cDNA and receptor internalization studies were performed utilizing 125I‐ANP binding assay and immunoflurescence staining in NPRA‐transfected cells. Cells were treated with chlorpromazine and monodansylcadaverine, which disrupt the formation of clathrin‐coated vesicles. In the inhibitor‐treated recombinant HEK‐293 cells, 125I‐ANP/receptor complexes were internalized only by 10–15% compared with untreated control cells. A major portion of 125I‐ANP radioactivity was localized on the cell surface in both chloropromazine‐ and monodansylcadaverine‐treated cells, which accounted 85–90% compared with control cells. Immunoflourescence staining was utilized to confirm the endocytosis of ANP/NPRA complexes. The results demonstrated that the internalization of GC‐A/NPRA occurs involving clathrin‐mediated endocytotic mechanisms. The present findings provide important information towards the understanding the GC‐A/NPRA signaling mechanism, which is critical in the regulation of blood pressure and cardiovascular homeostasis.
VPAC1 Signaling Blocks Ikaros Degradation by a PKA Dependent Mechanism in Activated HuT‐78 Cells
Authors: Van der Steen Travis , Hermann Rebecca , Benton Keith , Dorsam Glenn
Published: June 2021

Over 50,000 people in the U.S. die annually from Leukemia, and over half of these people have a mutation or splicing deregulation of the tumor suppressor Ikaros (IK) protein. The phosphorylation pattern of IK controls its DNA binding activity; through the antagonistic control of casein kinase II and protein phosphatase 1. IK also contains consensus sequences for other kinases such as PKA, PI3K, and cdk1. T cell activation shifts the equilibrium to favor hyperphosphorylation of IK thereby decreasing DNA binding and cell cycle entry. Vasoactive intestinal peptide receptor 1 (VPAC1) signaling opposes the activation of the T cell receptor by blocking T cell activation through a cAMP/PKA signaling cascade. We hypothesized that VIP/VPAC1 signaling regulates the phosphorylation of IK and impedes cell cycle entry. PMA/ionomycin activation of HuT 78 cells resulted in a decrease in IK protein, but was blocked with the addition of the VPAC1 ligand VIP (10−8M). When cells were treated with either an agonist (VIP10–28) or a VIP+PKA inhibitor (H89) IK protein levels were reduced. When treated with forskolin to induce cAMP, no increase in IK was observed. These data suggest that IK is being regulated through a G protein coupled receptor by PKA in a cAMP independent mechanism. Research supported by NIH‐K01 1K01DK064828 and COBRE 2P20RR05566.
Characterization of GIV‐Gαi3, a Non‐Receptor GEF‐G protein complex involved in cell migration and metastasis
Authors: Garcia‐Marcos Mikel , Ghosh Pradipta , Ear Jason , Farquhar Marilyn Gist
Published: June 2021

G‐protein coupled receptors (GPCRs) are the canonical guanine nucleotide exchange factors (GEFs) that activate trimeric G proteins. In contrast to GPCRs, little is known about the structure or function of non‐receptor GEFs. We previously reported that GIV/Girdin is a non‐receptor GEF for Gαi‐subunits that promotes cell migration and tumor cell metastasis (PNAS, 2009, 106: 3178–83). We discovered an evolutionarily conserved GEF motif in GIV that forms a unique interface with the Gα‐subunit. Mutation of a key residue (F1685) within this motif disrupted the Gαi‐GIV interaction and made GIV GEF‐deficient. We have now extensively characterized the molecular basis for the interaction between GIV and Gαi3 and its functional consequences. We found that GIV and Gαi3 can form a stable 1:1 complex in which the rate of nucleotide exchange, but not GTP hydrolysis, by the G protein is enhanced. Based on the preferential binding of GIV to Gαi versus Gαo, we have identified W258 in the Gαi‐subunit as a critical determinant for GIV binding. Mutation of W258 to the corresponding F in Gαo rendered Gαi3 GEF‐insensitive but did not perturb interaction with other Gα binding partners, i.e., Gβγ, GPCRs, GDIs or GAPs. When either the GEF‐deficient GIV mutant F1685A or GIV‐insensitive Gαi3 mutant W258F were expressed in HeLa cells they failed to undergo cell migration and to promote Akt signaling. These findings provide valuable information on the structural and biochemical basis for the biological functions of GIV as a Gαi activator which may be useful in the design of small molecules to disrupt the Gαi‐GIV interface for therapeutic purposes.

Support: NIH DKI7780 and CA100768 (M.G.F). Susan G. Komen KG080079 (M.G‐M). NIH/T32 DK07202 and RSA from American Gastroenterology Association (P.G). McNair Student Research Scholarship (JE).
Estrogen Receptor‐alpha Expression in Striatal Tissue for Future Use in Studying the Effects of Bisphenol A in the Mammalian Brain
Authors: Dickinson Katherine Jane
Published: June 2021

Bisphenol A (BPA) is a chemical that leaches out of some food and beverage containers made of polycarbonate plastic and into our food. BPA has structural similarities to estrogen, a hormone commonly associated with the reproductive system that also has non‐reproductive functions. For instance, estrogen affects the striatum, the brain region that controls the motor system, and the hippocampus, the brain region that controls learning and memory. Estrogen affects these brain regions by binding to estrogen receptor‐alpha (ERα). In the studies presented here, ERα protein was detected using immunoassays of protein extracts from these brain regions from adult female rats and 6–8 days old male and female rats. It was determined that ERα is present in both the striatum and the hippocampus. However, the amount of ERα expressed depends on the type of tissue. Overall, female striatum expressed more ERα than male striatum. Surprisingly, preliminary experiments indicate that ERα expression was greater in striatum than in hippocampus. Because ERα was shown to be present in striatal and hippocampal tissue, the next step is to determine if ERα is present in cultured neuronal cell lines from the striatum and hippocampus. If so, the neurons can be exposed to BPA, and the effects of BPA on molecular functions in these brain regions can be observed. This work was supported by funds from the Florence M. Rooney endowment at CSB/SJU, a CSB/SJU Professional Development grant (DGS), and the CSB/SJU Undergraduate Research Program.
The importance of STAT6 regulated genes activated by Type I Interferon
Authors: Yu‐An Hsu , Lei Wan
Published: June 2021

Interferons (IFN) are natural defense factors in vertebrates. This family of cytokines regulates antiviral, anti‐tumor, immuno‐modulatory activities, and regulation of cell differentiation. The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)‐STAT (1, 2, 3, 5 isotypes) signaling pathway. Although several studies have indicated that IFN‐α and IFN‐β can activate complexes containing STAT6, the biological role of this activation is still unknown. It is known that Daudi cells and B cells transformed with Epstein‐Barr virus (EBV) resistant to the anti‐proliferative effect of IFN‐α display a decrease in type I IFN‐mediated activation of STAT6. Therefore, it is suggested that complexes containing STAT6 may play a more general role in gene activation necessary for the anti‐proliferative effects of type I IFN. To determine the IFN‐activated‐STAT6 regulated genes, we performed a Chip‐chip experiment using anti‐STAT6 antibody and subsequently determined the mRNA expression levels of those identified genes. A total of 151 genes were identified by Chip‐chip. The qPCR was performed on Daudi cells treated with IFN‐α or IFN‐β at 1, 3, 6, 12, 24, and 48 hours. Our results revealed that the STAT6 may be a positive or negative regulator on gene expression levels and the expression levels of those genes are time dependent. A more detail molecular studies will be performed to determine the biological functions of those genes.
Role of p44/42 activation in TBT‐ induced losses of granzyme B and perforin in human natural killer cells
Authors: Abraha Abraham , Whalen Margaret
Published: June 2021

Natural killer (NK) cells are key players in innate immunity and are responsible for early defense against tumors and viruses. It is well known that both perforin and granzyme B are released from NK cells upon target cell contact. The environmental pollutant, tributyltin chloride (TBT) has been reported to decrease human NK cell lytic function with accompanying activation of p44/42 and decreased levels of granzyme B and perforin. The current studies are designed to examine the role of the mitogen activated protein kinase (MAPK), p44/42, activation in TBT‐induced losses of granzyme B and perforin. p44/42 activation was blocked by the MEK inhibitor, PD98059. NK cells were exposed to PD98059 for 1 h followed by 1 h exposure to 200 or 100 nM TBT. Following the incubation period, NK cells were washed twice with media and incubated for 24 h period in compound‐free media. Western blot analysis using an antibody to granzyme B revealed that PD98059 was able to block 37% of the 200 nM TBT‐induced loss of granzyme B. Moreover, western blot analysis using an antibody to perforin revealed that PD98059 was able to block 42 % of the 200 nM TBT‐induced loss of perforin. Thus our data show a role for TBT‐induced activation of p44/42 in the decreases in granzyme B and perforin in NK cells and that the TBT‐induced p44/42 activation is not solely responsible for the losses of granzyme B and perforin protein levels.
Tributyltin causes activation of the MAP3K, c‐Raf, but not Ras in human natural killer cells
Authors: Celada Lindsay , Whalen Margaret
Published: June 2021

The toxic chemical tributyltin (TBT) has been found in human blood. TBT is employed in various industrial applications and due to these uses contaminates the environment. Human natural killer (NK) cells are a subset of lymphocytes capable of destroying tumor cells, virally infected cells, and antibody‐coated cells. It has been shown that TBT is able to diminish the lytic function of NK cells. Previous studies have demonstrated that exposure to TBT leads to the activation of Mitogen‐Activated‐Protein‐Kinase (MAPK) and MAPK‐Kinase (MAP2K) in NK cells. TBT also results in the activation of the MAPK Kinase‐Kinase (MAP3K), c‐Raf, at an exposure time of ten minutes in NK cells. Activation/phosphorylation of c‐Raf (Ser338) was seen with exposure to 300, 200, 100, and 50 nM TBT (2 fold at 300–100 nM and 1.45 fold at 50 nM). Increased phosphorylation of Ser259 was also seen with 300–200 nM TBT (2 and 1.7 fold, respectively). Ras is a GTPase which is known to lead to the activation of the MAP3Ks. In its inactive state, Ras is bound to GDP; but when activated, it binds GTP. The focus of this study was to see if there was a TBT‐induced activation of Ras that leads to activation of Raf in NK cells. It was found that exposure to TBT in these cells did not result in the activation of Ras; indicating that there is an alternative mechanism for activation of Raf by TBT.

Supported by NIH grant S06 GM008092‐34
PPB is a Novel Serine/Threonine Phosphatase of Akt and is Involved in Myogenesis
Authors: Duan Xueyan , Tao Shen , Gao Xinxin , Zhang Jin , Lin Xia , Feng Xin‐Hua
Published: June 2021

PI3 kinase/Akt signaling pathway plays a central role in many biological functions. There are two phosphorylation sites on Akt (Thr308 and Ser473), which are phosphorylated by PI3‐kinase/PDK1 and mTOR‐rictor kinase complex, respectively. Recently, PH domain and leucine rich repeat protein phosphatase 1(PHLPP1) was identified to dephosphorylate pSer473. The identity of phosphatase on Thr308 is still elusive.

We screened for Akt phosphatase on Ser473 and Thr308 in mammalian cells. Of 45 phosphatases, we identified PPB (Protein Phosphatase for Akt/PKB) as a potent phosphatase that dephosphorylates both Thr308 and Ser473. This was supported by both in vivo and in vitro phosphatase assays. Fluorescence resonance energy transfer (FRET) analysis using AktAR reporter indicates that PPB reduces Akt activity in live cells. However, PPB dead mutant doesn't dephosphorylate Akt. All these data suggest that PPB dephosphorylates Akt and reduces its kinase activity.

PI3K/Akt pathway plays an important role during myogenesis. To study the effect of PPB on myogenesis, we generated C2C12 stable cells that over‐express PPB. When C2C12 control and stable cells were differentiated for up to 3 days, the expression of two important myogenetic markers, myosin heavy chain (MHC) and myogenin was abolished, However, this can be rescued with gain‐of‐function Akt, suggesting the inhibition of myogenesis in C2C12 cells by PPB was through its dephosphorylation of Akt.
PHLPP Phosphatase Regulation of EGFR Expression
Authors: Niederst Matthew J , Cava Gloria Reyes , Kunkel Maya , Newton Alexandra
Published: June 2021

The PH domain leucine‐rich repeat protein phosphatase (PHLPP) opposes growth factor‐dependent signaling by specifically dephosphorylating Akt and protein kinase C on a key regulatory site, the hydrophobic phosphorylation motif. Here we show that PHLPP also suppresses the amplitude of the Ras/ERK pathway by a distinct mechanism which controls the cellular levels of EGF receptor (EGFR). Depletion of PHLPP1 or PHLPP2 in normal or cancer cell lines results in dramatically enhanced EGF‐mediated activation of Ras, MEK and ERK. The increased amplitude of ERK pathway signaling correlates with a robust increase in signaling‐competent EGFR protein levels. Live‐cell imaging of EGFR kinase activity using a genetically‐encoded FRET reporter reveals that depletion of PHLPP isoforms results in a dramatic increase in EGF‐stimulated EGFR kinase activity. QPCR analysis reveals that EGF receptor mRNA levels are an order of magnitude higher in MEFs lacking PHLPP1 compared to wild‐type MEFs. Thus, PHLPP isoforms control the levels of signaling –competent EGFR by decreasing mRNA levels of the receptor. The finding that PHLPP isoforms control EGF receptor levels establish these phosphatases as key regulators of two critical oncogenic pathways, both the PI3K pathway via effects on Akt and, more globally, growth factor signaling by effects on levels of signaling‐competent receptor.
Characterization of a putative AS regulatory phosphorylation site implicated in vascular endothelial nitric oxide production
Authors: Thompson Ricci , Corbin Karen , Pendleton Laura , Eichler Duane C.
Published: June 2021

Argininosuccinate synthase (AS) catalyzes the rate‐limiting step of the nitric oxide (NO) cycle in vascular endothelial cells (EC); however, little is known about its regulation. Recently, we reported that AS is regulated by phosphorylation. Using in silico, as well as proteomic analysis, we have identified a putative phosphorylation site at serine 328 which is located adjacent to a caveolin binding motif located in the central region of AS tertiary structure. In this report, we examined the role of the S328 site relative to supporting NO production in ECs by mutation analysis, mutating S328 to either an alanine (S328A) or to the phospho‐mimetic aspartate (S328D). These constructs were overexpressed in ECs, and NO production was measured after stimulation with either the calcium ionophore plus ortho‐vanadate, or vascular endothelial growth factor (VEGF). In both cases, the S328D AS mutant produced significantly higher NO than wild type AS (n=3, p<0.05), while the S328A mutant had no effect over background. Notably, the S328A mutant also did not support cell viability when the BAECs were serum starved before VEGF incubation. These data suggest that the S328D mutant promotes stimulated NO production and maintains cell viability, and supports the proposal that S328 may be a biologically significant site for AS regulation.
The nerve growth factor receptor TrkA exists as a pre‐formed homodimer before ligand binding
Authors: Shen Jianying , Maruyama Ichiro
Published: June 2021

The nerve growth factor (NGF) family of neurotrophins plays a central role in the development, growth, and maintenance of the nervous system. These neurotrophins transduce their effects by interacting with two types of cell surface receptors, the Trk family of tyrosine kinase receptors, and p75NTR. Two classes of NGF‐binding sites, high and low affinities, are present on the surface of neurons. When TrkA and p75NTR receptors are co‐expressed, the association rate is dramatically increased, creating a new site consistent with the NGF high affinity binding site. These results suggest that the receptors interact each other prior to ligand binding.

In the present study, we have analyzed the structures of the TrkA receptor for NGF by bimolecular fluorescence complementation and luciferase fragment complementation assays as well as by chemical crosslinking of the receptor expressed on the cell surface. These analyses demonstrated that before ligand binding, the majority of receptors exist as a homodimer in living cells. Using Brefeldin A, a lactone antibiotic that disassembles the Golgi apparatus and block anterograde transport of the receptors from ER to Golgi, furthermore, it has been found that TrkA homodimers are initially formed in ER before reaching to the cell surface. This work provides new insights into understanding of transmembrane signaling by TrkA receptors.
Role of calcium in the structure and function of protocadherin of the liver, kidney and colon
Authors: Behrendt Marc , Krahn Michael Peter , Rizk Sandra , Alfalah Marwan , Naim Hassan Y.
Published: June 2021

Protocadherin LKC (PLKC) shows characteristics of a potential tumor‐suppressor by inducing contact inhibition of cell proliferation after downregulation of beta‐catenin signaling. Here, we expressed an YFP‐tagged version of PLKC in Chinese Hamster Ovary (CHO) cells to directly assess its potential function in non‐polarized cells and implication in changing cell morphology. The results indicate that PLKC exhibits a significant calcium‐dependent adhesion activity which is not mediated by the actin cytoskeleton. Interestingly, this activity is nearly as strong as that of E‐cadherin and stronger than that of N‐cadherin. PLKC is localized at sites of cell‐cell interaction concomitant with morphological alteration of the transfected CHO cells. Nevertheless, no significant change could be assessed in the trans‐epithelial resistance in the PLKC‐expressing cells versus mock transfected controls indicating that PLKC per se is not directly implicated in triggering cell polarity. The mode of interaction of PLKC molecules in the cell and at the sites of cell‐cell interaction revealed a calcium‐dependent dimerisation of PLKC in the Golgi membranes and at the cell surface, while calcium is not required for dimerisation in the ER. The results shed light on a dual role for calcium in the structure and function of PLKC.
Binding of STIM1, but not Orai1 to TRPC4 activates Store‐operated Calcium entry in Mouse Lung Endothelial Cells
Authors: Sundivakkam Premanand , Freichel Marc , Levitan Irena , Vogel Stephen M , Flockerzi Veit , Malik Asrar B , Tiruppathi Chinnaswamy
Published: June 2021

We have previously shown that the TRPC isoform TRPC4 is essential for Ca2+ store release‐activated Ca2+ entry (SOCE) in mouse lung endothelial cells. Here we show that upon stored Ca2+ release, STIM1 binding to TRPC4 activates Ca2+ entry in mouse lung endothelial cells (MLECs). We observed thrombin‐induced Ca2+ entry as well as IP3‐induced inward cationic current in wild‐type (WT) MLECs. Interestingly, thrombin‐induced Ca2+ entry and IP3‐induced inward current were abolished in TRPC4 knock‐out (TRPC4−/−) MLECs. Knockdown of TRPC4 expression using siRNA prevented thrombin‐induced Ca2+ entry, but knockdown of TRPC1 partially inhibited Ca2+ entry in WT MLECs. We observed the expression of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1 and the CRAC channel protein Orai1 in WT and TRPC4−/− mice LECs. Knockdown of STIM1 using siRNA abolished thrombin‐induced Ca2+ entry in WT MLECs, whereas knockdown of Orai1 failed to prevent thrombin‐induced Ca2+ entry in WT MLECs. Moreover, co‐immunoprecipitaion experiments revealed the interaction between TRPC4 and STIM1 upon ER stored‐Ca2+ release. These findings suggest that TRPC4 interacts with STIM1 to form a functional store‐operated Ca2+ entry channel complex in MLECs. Thus, the interaction between TRPC4 and STIM1 may play a critical role in the regulation of lung vascular inflammatory responses induced by the pro‐coagulant protease thrombin.
Analysis of cyclic AMP‐coupled signature molecules in human micro vascular smooth muscle cells shows link to stress‐associated proteins
Authors: Jeyaraj Selvi C , Unger Nicholas T , El‐Dahdah N. Paul , Sessler Richard J , Zirwas Matthew , Pleister Adam , Green‐Church Kari B , Chotani Maqsood A
Published: June 2021

Vascular stress or local injury may trigger an inflammatory response leading to increased production and release of prostaglandin E2 (PGE2) and prostacyclin (PGI2), activation of Gs‐coupled receptors, and increased intracellular concentrations of cyclic AMP (cAMP). This study elucidated the targets of cAMP signaling in the vessel wall, specifically in human vascular smooth muscle cells explanted from skin punch biopsy arterioles of healthy volunteers (microVSM). Intracellular concentrations of cAMP were increased by treating quiescent cells with forskolin (10 μM, a direct activator of adenylyl cyclase). cAMP activated both A‐kinase and Rap1 GTPase signaling (within 15 min), and increased reactive oxygen species (26±3 %,≤1h). Differential‐in‐Gel electrophoresis, utilized to identify global changes in protein profiles, showed detectable changes in known proteins associated with the cytoskeleton, gene expression, protein ‐synthesis, ‐folding, ‐targeting, ‐secretion, and the extracellular matrix. Notably, the cellular oxidative stress‐associated protein peroxiredoxin‐II and the p53 regulated growth and differentiation gene N‐myc downstream‐regulated gene 1(NDRG 1) were increased (≥2‐fold). These studies point to a crucial role of cAMP in eliciting a protective cellular response to maintain vessel wall integrity in response to stress or injury.
Stimulation of GTP Cyclohydrolase I by Phosphorylation Upon T Cell Activation
Authors: Chen Wei , Li Li , Brod Torben , Saeed Omar , Dikalov Sergey , Harrison David G.
Published: June 2021

We have found that phosphorylation of GTP cyclohydrolase‐1 (GTPCH‐1), the rate‐limiting enzyme for BH4 biosynthesis, increases its activity and BH4 levels in endothelial cells. To determine if this occurs in non‐endothelial cells, we stimulated T cells in vitro (anti‐CD3) and in vivo (OVA immunization in OT2 mice). Naïve T cells had nearly undetectable levels of BH4 and minimal expression of endothelial and inducible NOS. Anti‐CD3 stimulation robustly induced T cell eNOS and iNOS and increased biopterin to 5.4 pMol/mg protein. Biopterin induction was similar in CD4 and CD8 cells. Western blots showed that this was associated with GTPCH‐1 phosphorylation at S72. Pharmacological inhibition of casein kinase II prevented GTPCH‐1 phosphorylation and blunted the increase in T cell BH4. Inhibition of GTPCH‐1 with diaminohdroxypyimidine (DAHP, 500 μm) prevented T cell BH44 accumulation and increased T cell superoxide produciton, which was dependent on uncoupling of both eNOS and iNOS. GTPCH‐1 inhibition also promoted TH2 polarization in memory CD4 cells. OVA immunization in vivo confirmed a marked increase in T cell BH4. Thus, T cell activation is associated with GTPCH‐1 phosphorylation, which increases GTPCH‐1 activity and biopterin production. In inflammatory settings where BH4 oxidation is favored, this could modulate T cell function. Supported by NIH R01 HL39006 and a VA Merit Grant.
Conformational effects induced by YC‐1 and carbon monoxide binding to soluble guanylate cyclase
Authors: Lopez‐Pena Ignacio , Kristianto Jasmin , Gerber Nancy Counts , Esquerra Raymond
Published: June 2021

Soluble guanylate cyclase binds the gaseous ligands NO and CO. Active sGC starts a signaling cascade by converting GTP to cGMP, resulting in vasodilation and neurotransmission. NO activates sGC at least 200‐fold, while CO‐sGC only marginally increases activity. However, in the presence of the allosteric effector YC‐1, the CO complex is activated close to that of the NO complex. Our objective is to understand the molecular mechanism of how YC‐1 binding induces sGC activation by CO. We used MCD to probe the electronic environment of the heme sensing site, and FRET to probe conformational changes as a function of YC‐1 binding. Our findings indicate that the heme is not affected by YC‐1 binding, and that the conformational effects of CO/YC‐1 resemble those caused by NO. These results suggest that a region outside of the heme environment can bind small molecules and induce conformational changes required for sensitization to CO. This work was supported by the following grants: NIH SCORE GM52588(RE) and NIH MCMHD MD000544 (RE), and fellowships NIH MBRS MARC 5T34 GM08574 (IL) and NIH RISE R25‐GM59298 (JK)
Diacylglycerol production at the Golgi: regulation by Ca2+
Authors: Kunkel Maya , Newton Alexandra
Published: June 2021

Protein kinase C and protein kinase D are potently activated by agonist‐evoked increases in diacylglycerol. Using live cell‐imaging probes for kinase activity, we have previously shown that both kinases are robustly active at the Golgi following stimulation of Gq‐coupled receptor signaling pathways, displaying activation signatures at the Golgi that are distinct from those at the plasma membrane. Here we address the mechanism by which signals received at the plasma membrane cause these two protein kinases to be activated at the Golgi. Using FRET‐based reporters to image diacylglycerol production, we show that Ca2+ is necessary and sufficient to transduce signals from the plasma membrane to the Golgi. First, chelation of intracellular Ca2+ prevents UTP‐dependent increases in diacylglycerol at the Golgi, but not plasma membrane. Second, raising intracellular Ca2+ by treating cells with thapsigargin induces diacylglycerol production at the Golgi. Thus, agonist‐evoked increases in intracellular Ca2+ cause increases in Golgi diacylglycerol, allowing this intracellular membrane to serve as a platform for signaling by protein kinases C and D.

This work was supported by NIH GM43154 and NIH DK54441.
Role of base pairing partner on DNA‐protein crosslinking from guanine oxidation
Authors: Perez Zitadel Anne , Madison Amanda , Stemp Eric D. A.
Published: June 2021

Oxidative DNA damage contributes to aging, cancer, and other degenerative diseases. Guanine is particularly vulnerable to oxidation, creating radicals which form irreversible crosslinks to proteins. Here, we investigate how the reactivity of the guanine radical in the oligonucleotide 5′‐ATATGATATGGATATGATAT‐3′ depends on its base pairing partner(s). We synthesized DNA duplexes with cytosine derivatives or mismatches across from guanine. Guanine radicals were created photochemically and the DNA was analyzed by gel shift experiments. In the study of mismatches, the duplexes contained mismatches across from the 5′‐G of the GG doublet. Under both nondenaturing and denaturing conditions, crosslinking decreased in the order of G:G > G:A > G:T = G:C. The enhanced reactivity for the G:G and G:A mismatches is attributed to a lowered oxidation potential near the GG doublet. Cytosine derivatives crosslinked in the order of 5‐bromocytosine > cytosine > 5‐methylcytosine. These results are consistent with the presence of more of the more reactive radical cation with the 5‐bromocytosine, since it is a weaker base than cytosine or 5‐methylcytosine; melting temperature experiments indicated minimal differences between duplexes with the different cytosines. These results show that the reactivity of guanine in oxidative DNA‐protein crosslinking reactions depends on its base pairing partner.
Role of Cooperative ATP hydrolysis in RecA protein filaments
Authors: Reyes Ricardo M. , Gruenig Marielle C. , Cox Michael M.
Published: June 2021

The RecA protein forms filaments on single‐stranded DNA in the first step leading to RecA‐promoted DNA strand exchange. While bound to DNA, RecA promotes ATP hydrolysis with a kcat of 30 min−1. A mutant form of RecA, called RecA K250R, hydrolyzes ATP at 1/6 the rate of the wild type protein. When the mutant and wild type proteins are mixed on ssDNA, they form mixed filaments but do not affect each other. The mixed filament hydrolyzes ATP at a rate commensurate with the fraction of subunits present as the mutant. However, when mixed filaments initiate DNA strand exchange, many changes ensue. These effects of the mutant protein will be discussed.
Effects of the antioxidants in pomegranate juice on DNA‐protein crosslinking via guanine oxidation
Authors: Rodgers Kasey , Marzvanyan Liana , Madison Amanda , Stemp Eric
Published: June 2021

DNA‐protein cross‐linking is a direct result of oxidative damage. Here we investigated effects of antioxidants from pomegranate juice on DNA‐protein crosslinking via guanine oxidation. Cross‐linking was detected by gel shift assay. After flash quench treatment, pUC19 exhibited a drop in mobility and highly cross‐linked DNA remained in the wells. With addition of POM brand pomegranate juice to samples, the plasmid runs as a single band even after oxidation via the flash‐quench technique. Significant inhibition of cross‐linking is evident at a 1:500 dilution of the juice, a concentration that has physiological relevance. Individual compounds found in the juice (gallic acid, quercetin, catechin, cyanin, ellagic acid, and p‐coumaric acid ) were investigated. These compounds were active down to concentrations of 1μM. Transient absorption spectroscopy results showed a decay of guanine radical concomitant with the formation of phenoxy radical, suggesting that the phenolic components repair guanine radical by reduction.
Induction of DNA damage repair defect by the exogenous expression of BRCA2 BRC repeat in Trypanosoma brucei
Authors: Hall Mack , Misra Smita , Chaudhuri Minu , Chaudhuri Gautam
Published: June 2021

The DNA recombination repair protein BRCA2 is conserved and is present in many eukaryotes including the protozoan parasite Trypanosoma brucei. One of the major functions of BRCA2 is to haul the DNA recombinase RAD51 from the cytosol to the nucleus. Binding of BRCA2 to RAD51 is mediated through the consensus BRC repeats of BRCA2 and this binding is critical for homologous recombination. Yeast 2‐hybrid analysis studies revealed that a single BRC repeat from TbBRCA is enough to bind to different isoforms of TbRAD51. We tested whether over‐expression of a single BRC repeat unit of TbBRCA2 will increase the parasites sensitivity to DNA damage. In order to evaluate the importance of BRCA2/RAD51 interactions on the DNA recombination/repair processes in T. brucei, we have amplified a single BRC repeat from its ORF. We cloned this amplicon into pLew79MHTAP vector, and stably transfected the procyclic form of the parasite. Transfected cells were selected with phleomycin. Expression of the BRC repeat was evaluated by RT‐PCR and Western blot analyses. To evaluate the effects of the over‐expression of a single BRC repeat on DNA damage sensitivity, procyclic forms of T. brucei over‐expressing a single BRC repeat were exposed to increasing concentrations of methyl‐methane sulfonate. Over‐expression of a single BRC repeat significantly increased the sensitivity of the parasite cells to this DNA damaging reagent.
Human DNA glycosylase NEIL1 but not NEIL2 preferentially collaborates with proteins of DNA replication to carry out LP‐BER in replicating cells
Authors: Hegde Muralidhar L , Hegde Pavana M , Theriot Corey A , Tomkinson Alan A , Li Gu‐Min , Hazra Tapas K , Mitra Sankar
Published: June 2021

We have previously shown that NEIL1, activated in S‐phase, functionally interacts with the PCNA, flap endonuclease 1 (FEN1), replication protein A (RPA), suggesting its preferential involvement in repairing replicating genome. NEIL1 along with NEIL2, has higher activity with damaged base in fork or bubble DNA that mimics replication/transcription intermediates. Here we show that both NEIL‐FLAG immunoprecipitates (IPs) from human cells contained replicative DNA polymeraseδ (Polδ), replication factor‐C (RFC) and DNA ligaseI (LigI), along with PCNA and FEN1. However, the NEIL1‐IP contained ~3–5 fold higher level of these proteins than the NEIL2 IP. Although, both the NEILs binarily interact with these proteins, only NEIL1 activates Polδ and RFC stimulates NEIL1 but not NEIL2. NEIL1 IP eluate could efficiently repair a base lesion via long‐patch repair (LP‐BER) pathway. We reconstituted NEIL1‐initiated LP‐BER using purified proteins and showed the dependence on PCNA and its loading by RF‐C. Repair initiated by C‐terminally truncated NEIL1 lacking the interaction domain was significantly less efficient compared to that initiated with full‐length NEIL1. Further, NEIL1 has significantly higher ability to repair 5‐OHU using replication proteins. These results showed that NEIL1 preferentially collaborates with proteins of DNA replication to carry out a distinct sub pathway of BER during DNA replication. (USPHS PO1 CA 92584 and APDA fellowship to MLH).
NMR study of the effect of Zn on conformational activation of rat DNA polymerase β
Authors: KIRBY THOMAS W , BEARD WILLIAM A , WILSON SAMUEL H , LONDON ROBERT E
Published: June 2021

The DNA repair enzyme DNA Polymerase β (Polβ) fills gapped DNA using a two‐metal ion (usually Mg2+) mechanism. Crystal structures show that the transition from an “open” binary complex (Polβ·gapped DNA) to a “closed” ternary complex (Polβ·gapped DNA·dNTP) activates formation of the active site geometry involved in Mg2+ coordination. Other divalent metals can also function in place of magnesium, and some can cause a decrease in fidelity. We have investigated the effect of Zn by using [methyl‐13C] methionine‐labeled Polβ in NMR experiments to study the conformational changes undergone by Polβ in solution during its catalytic cycle The methionine probes are located in the lyase domain (M18), and in different parts of the polymerase domain, i.e. the catalytic subdomain (M155, M158, M191, and M236); and the nascent base‐pair binding subdomain (M282). The transitions that correspond to the “open” to “closed” structures (with Mg2+ present) can be monitored by the chemical shifts of the methionine methyl groups. In particular, the large chemical shift change and broadening of the M282 resonance detects the rearrangement of helix N (in the nascent base‐pair binding subdomain) induced by the formation of the activated ternary complex. In this work we show that these same responses can be achieved for a binary complex in the presence of Zn2+ ions. (Supported by the Intramural Research Program of the National Institutes of Health)
Role of a strictly conserved residue in N‐glycosylic bond cleavage by human Thymine DNA Glycosylase
Authors: Maiti Atanu , Drohat Alexander C.
Published: June 2021

Thymine DNA glycosylase (TDG) promotes genomic integrity by removing thymine from mutagenic G·T mispairs arising from deamination of 5‐methylcytosine and follow‐on base excision repair enzymes restore a G·C pair. Previous studies suggested that Asn140, a strictly conserved active site residue is important, for base excision, yet its catalytic role had not been investigated rigorously. To further our understanding of the catalytic mechanism of TDG, here we determined the contribution of Asn140 to the substrate binding and chemical steps of the reaction. Isothermal titration calorimetry (ITC) experiments show that TDG‐N140A variant binds substrate analogs with the same tight affinity as wild‐type TDG, indicating Asn140 does not contribute to substrate binding. Single turnover kinetics experiments show that TDG‐N140A exhibits no detectable base excision activity for a G·T substrate, and its excision rate is vastly diminished (by ~104.4fold) for G·U, G·FU, and G·BrU substrates. Our findings indicate that Asn140 is essential for the chemical step, but does not contribute substantially to substrate binding. Thus N140A variant provides a useful platform for investigating the role of other residues in forming the reactive enzyme‐substrate complex. This work was supported by an NIH grant.
ANALYSIS OF GENETIC VARIATION BETWEEN WILD AND MUTANT STRAINS OF HYPER‐PECTINOLYTIC Aspergillus niger USING RAPD‐ PCR
Authors: OKAFOR UZOMA ANTHONY , Okochi Veronica Ihuaku , Ebuehi O Albert O , Okpuzor Joy , Adekunle Adedotun A
Published: June 2021

Improving the value of agrowastes generated via fungi degradation has become a priority in most large biomass generating countries for economic and ecological reasons.

Wild strain of Aspergillus niger previously isolated from agrowastes in Lagos Nigeria, was subjected to UV‐mutagenesis; through the use of a selective culture medium containing pectin and 2 deoxy D glucose (2dg) to isolate hyper pectinolytic mutant strains. The polymerase chain reaction (PCR) that includes random amplified polymorphic DNA (RAPD) was employed to investigate the influence of UV‐mutagenesis in inducing mutation in the wild strain.

Genetic relationship among isolates was determined by pair‐wise comparisons of the shared number of DNA bands on an agarose gel. The results showed that there was considerable genetic variation among isolates. The wild and the mutant strains of the fungal isolates shared an average of only 20% and 42 % of the bands in their RAPD profiles using OPA 2 and OPA 4 respectively, while they shared an average of 60 and 56% of the bands using OPC 3 and 8 respectively. The results indicated that there is genetic variability between the wild and mutant strains; signifying the possibility of the use of UV mutagenesis for generation of hyper‐pectinolytic fungi for effective biodegradation of agrowastes and enzyme production.
Transcription arrest at G quadruplex DNA‐forming sequences
Authors: Tornaletti Silvia , Beckett Joshua S. , Broxson Christopher
Published: June 2021

G quadruplex DNA is a four stranded non‐B DNA structure that can transiently form in G repeats by stacking of G quartets. G4 DNA formation has been associated with promoter regions and a functional role of this structure in gene regulation has been proposed. G4‐forming sequences also correspond with genomic regions particularly susceptible to genomic instability. Transcription has a central role in formation of these structures and in the instability associated to these sites. However, little is known about how G4 DNA is processed when encountered by an elongating RNA polymerase. Here we describe the effect of a G4 DNA‐forming sequence contained in the human c‐myb proto‐oncogene on T7 RNA polymerase (T7RNAP) transcription. This G‐rich sequence consists of three repeats of sequence (GGA)4 that fold into G4 DNA under physiological conditions. We found that this G4 forming‐sequence is a strong block to T7RNAP progression. Surprisingly, arrest is only observed when G4 DNA is located in the transcribed strand of template DNA. Consistent with formation of G4 DNA in the transcription template, T7RNAP arrest is strictly dependent on the presence of KCl, a cation required for formation and stability of G quartets. Our findings have broad implications for mechanisms of transcription regulation and mutagenesis associated with GGA repeats in vivo. (This work is supported by a Bankhead‐Coley New Investigator Grant 08BN‐07).
MicroRNA analysis of the role of RecA2 in plant mitochondrial DNA replication in A. thaliana
Authors: Wright Ashley Nicolle , Cupp John
Published: June 2021

The RecA proteins have been shown to be important enzymes in the recombination processes of plant mitochondria, but current studies have yet to identify the specific functions and the level of involvement of the individual proteins in mitochondrial DNA maintenance. The lack of viable RecA2 mutants has significantly halted the progress of characterizing the specific role of the RecA2 protein, which is dual targeted to mitochondria and chloroplasts. We have created viable RecA2 mutants by inducible gene silencing, from which we will be able to finally observe the physiological effects of silencing RecA2 expression. Induced microRNA expression at specific times of induction is being used to characterize the effects of blocked expression of RecA2 on further plant development. Expression is being characterized by western and slot blot analysis of protein and RNA levels of both the wild type and mutant plants. We will show evidence for whether the induced knockouts of the RecA2 protein result in reduced growth, which will provide new information about the protein's involvement in the maintenance of mitochondrial DNA.
Fluorophore optimization for FRET based ribozyme studies
Authors: Zaborowicz Matthew , Rohlman Christopher
Published: June 2021

The focus of this research is fluorophore optimization for FRET analysis of group I introns. Group I introns are ribonucleic acids. These introns have the ability to splice themselves from the larger RNA to which they are bound and, once modified into ribozymes, can act as reaction catalysts. FRET is used to study the kinetics and folding of these Group I introns. Current protocols for the synthesis of fluorescently labeled substrates require a large excess of the fluorophore relative to the RNA substrate used in the ribozyme study. To develop an optimized reaction, a Tetrahymena ribozyme substrate was designed with both a fluorescein label and the ability to be modified with the fluorophore tetramethyl rhodamine. A study was conducted to determine the optimum conditions necessary to achieve a 90% yield and a functional FRET substrate. Results from this study are generally applicable to synthesis of FRET probes for RNA folding and catalysis. This research was supported by the Albion College Foundation for Undergraduate Research, Scholarly and Creative Activity.