bluuebunny/crossref_metadata_2025 huggingface dataset:
TNF‐alpha inhibits alpha‐ENaC2 expression through oxidative stress pathway in lung epithelial cells
Authors: Xu Haishan , Chu Shijian
Published: June 2021

The α‐subunit of the epithelial sodium channel ENaC plays an important role in lung fluid absorption. In human lungs, α‐ENaC is expressed as several splice variants. The α2‐ENaC is one of the major variants and possesses channel function. Previous studies show that α1‐ENaC expression is suppressed by TNF‐α but it was unclear whether α2‐ENaC variants in human cells are similarly affected. In this study, we aim to investigate TNF‐α inhibits α2‐ENaC expression via increased reactive oxygen species. We found that TNF‐α reduces α2‐ENaC expression and antioxidants attenuate this inhibition. Our results demonstrate that 1. TNF‐α inhibits ENaC1 and ENaC2 mRNA expression and promoter activity; 2. H2O2 inhibits ENaC1 and ENaC2 mRNA expression and promoter activity; 3. Antioxidant inhibits TNF‐α suppressed ENaC2 mRNA expression but not ENaC1; 4. TNF‐α receptor‐binding domain but not lectin‐like domain mediates the inhibition of α2‐ENaC expression. These data suggest that TNF‐α suppresses α2‐ENaC expression in human lung epithelial cells through the inhibition of its core promoter, and this inhibition is dependent on TNF‐α mediated oxidative stress. This work was supported by a grant from the Department of Veterans Affairs.
Genetic analysis of mammalian Cdk7 kinase submodule of TFIIH: uncoupling of RNAPII CTD Serine 5 phosphorylation and transcription
Authors: Mäkelä Tomi P , Helenius Katja , Yang Ying , Tselykh Timofei , Pessa Heli K , Frilander Mikko J
Published: June 2021

The Cdk7 submodule has been suggested to function as a general activator of RNA Polymerase II (RNAPII) transcription by phosphorylating Serine 5 of the C‐terminal domain (CTD) and of cell cycle through T‐loop phosphorylation of Cdks. Genetic analyses in mammalian cells have suggested that Cdk7 is not essential in post‐mitotic cells, and indeed our recent results show that downregulation of Cdk7 is associated with adipogenesis, and suggest that Cdk7 acts as a roadblock to adipogenesis through inhibitory phosphorylation of PPARγ on Serine 112. We analyzed cell cycle and transcriptional changes following loss‐of‐function of Cdk7 kinase in mouse fibroblasts (MEFs) and human cancer cells. Our results show that in both exponentially growing MEFs and U2OS osteosarcoma cells loss of Cdk7 kinase, either through conditional knockout of Mat1 or Cdk7 knockdown, led to a G1 cell cycle arrest associated with decreased Cdk2 phosphorylation followed by cell death. These results are consistent with a role for Cdk7 in activating cell cycle Cdks. The adipogenic effects we previously noted following Mat1 loss in MEFs suggested that Cdk7 would not regulate general mRNA transcription. Comparison of expression profiles of Mat1‐/‐ and control MEFs using Affymetrix expression arrays demonstrated changes in less than 1% of mRNAs, indicating that Cdk7 activity is not crucial for transcription of most genes. However, analysis of RNAPII CTD phosphorylation demonstrated a dramatic drop in Ser‐5 phosphorylation and a concomitant decrease in Ser‐2 phosphorylation. The unexpected results demonstrate that the level of RNAPII CTD Ser‐5 phosphorylation is not correlated with general transcription. In addition, no significant defects were noted in RNA splicing or polyadenylation.
Effects on translation initiation and malignant transformation of insertion of Mouse Mammary Tumor Virus (MMTV) into the eukaryotic initiation factor 3e (eIF3e) gene
Authors: Chiluiza David , Callahan Robert , Rhoads Robert E
Published: June 2021

Previously we showed that eIF3e forms at least part of the binding site for eIF4G in the 48S initiation complex (LeFebvre, JBC. 281, 22917, 2006). The eIF3e gene is a common integration site for MMTV, which causes expression of truncated forms of the protein (eIF3e‐sh) and induces mammary epithelial tumors in mice (Marchetti, J. Virol. 69, 1932, 1995). We hypothesize that disruptions in cap‐dependent translation contribute to tumor formation. In the present study, we analyzed the effects of eIF3e‐sh in both cell‐free translation systems and stably transfected mouse mammary epithelial cells. Both eIF3e and eIF3e‐sh inhibited the cell‐free translation of capped luciferase mRNA. Cells expressing eIF3e‐sh continued to divide after reaching confluence, were more resistant to serum‐starved apoptosis, and were smaller than control cells. Expression of eIF3e‐sh caused a reduction of overall translation rate but, paradoxically, a shortened doubling time. The rate of protein synthesis was highly dependent on cell density, and the inhibitory effect of eIF3e‐sh on translation was not observed at high density. It is well known that a decrease in cap‐dependent translation favors an increase in IRES‐driven translation. We are testing cap‐dependent versus IRES‐driven translation to determine whether this could explain the differences in both protein synthesis and cell phenotype (Supported by NIH Grant No. R01 GM20818).
Dinitrobenzene Isomers Structure‐ Dependent Stimulation of Neuronal Nitric Oxide Synthase Catalyzed Electron Flux in the Absence of Calmodulin
Authors: Joshi Chintamani N , Miller R Timothy
Published: June 2021

Neuronal nitric oxide synthase (nNOS) requires calmodulin (CaM) ‐ binding for efficient electron transfer converting L‐arginine to L‐citrulline and nitric oxide. The present studies focus on electron transfer to and through CaM‐free nNOS. Turnover rates (min−1) of nNOS‐mediated NADPH oxidation (NADPHox) and acetylated cytochrome‐c reduction (AcCyt‐cred) were used to estimate total electron flux and O2. production. Dinitrobenzenes (DNBs) stimulated the rate of NADPHox by CaM‐free nNOS in an isomer‐ and concentration‐ dependent manner. Rates for NADPHox by CaM‐free nNOS at the limit of solubility of the DNBs (160 ?M) were 189 ± 5 min−1, 94 ± 3 min−1 and 38 ± 2 min−1 for 1,4‐, 1,2‐ and 1,3‐DNB, respectively, compared to the solvent control (7 ± 1 min−1). Highly stimulated rates of AcCyt‐cred were observed in presence of all the three DNBs. The nNOS reductase construct without the CaM binding domain (aa 745‐1729) behaved in a manner similar to holo‐nNOS in the presence of DNBs, in the NADPHox and AcCyt‐cred assays. These results indicate that CaM‐free nNOS generates ROS at environmentally‐relevant levels of DNBs (ASTDR). Considering the absolute amount of constitutive NOS expressed in organisms; chronic exposure to low levels of DNBs may lead to continuous oxidative stress.

Supported by ES011982 and RR008124 to RTM and UTEP respectively.
Intein‐mediated protein splicing of human Eg5 kinesin
Authors: Korona Dagmara , Korona Boguslawa , Wojcik Edward
Published: June 2021

HsEg5 is a homotetrameric BimC/kinesin‐5 family member that plays a vital role in the mitotic spindle. This crucial role has identified HsEg5 as a promising target for cancer chemotheraphy.

The aim of a project is to clarify the role of the sequence regions in kinetic mechanism of HsEg5 and to determine the various conformational states in which this protein occurs while performing its biological function. Intein‐mediated protein ligation (IPL) was used as a tool for segmental labeling of proteins of interest. Based on various criteria including the presence of cysteine required for peptide bound formation (native or artificially introduced), the breaking points for IPL were designed, so that the separately expressed fragments of protein could be joined together. The rebuilt protein has been obtained in a very high yield compared to values reported in the literature. The resulting IPL product reconstructed from fragments exhibits catalytic activity of native enzyme, however, due to the amino acid exchange (substitution for cysteine), the activity differs from that of wild type enzyme. The segmentally labeled protein is then subjected to analysis by Fourier‐Transform Infrared Spectroscopy (FTIR).
CaMKII Phosphorylates Phospholamban via an Ordered Sequential Kinetic Mechanism
Authors: Huynh Khai Quang , Pagratis Nikos
Published: June 2021

Many of the cellular responses to Ca++ signal are modulated by a family of multifunctional Ca++/calmodulin dependent protein kinases (CaMKs): CaMKI, CaMKII and CaMKIV. CaMKII has an autoregulatory domain that restricts enzyme activity in the absence of Ca++/CaM. Ca++/CaM binding alone produce maximal activity of CaMKII, whereas CaMKI and CaMKIV have an activation loop that requires phosphorylation by an upstream CaMK kinase for maximal activity. In addition to its role in memory and other neuronal functions, CaMKII appears to mediate many of the pathologic changes that occur during cardiac hypertrophy and heart failure. In order to further understand the role of CaMKII, we investigated the kinetic mechanism of CaMKII (IIα, IIβ, IIδ, IIγ) in comparison with those of CaMKI and CaMKIV by analyzing their steady state kinetics using phospholamban as a phosphoacceptor. The results indicated that (a) CaMK family's reaction mechanisms were of the sequential type in which all substrates must bind to enzyme before any product is released; (b) While CaMKIIs catalyzed Ordered Bi Bi reaction where phospholamban was the first substrate to bind and ADP was the last product to be released, CaMKI and CaMKIV exhibited random sequential mechanism where either phospholamban and ATP can bind to the free enzyme and (c) Constant α (ratio of apparent dissociation constants for binding peptide in the presence and absence of the second ligand) of all isozymes for ATP and peptide was higher than 1 indicating that the binding of phospholamban to CaMK decreased its affinities toward ATP. Our results suggested a unique kinetic mechanism for CaMKIIs in which peptide substrate binding may be an initiating factor for the catalysis and may play a role in the regulation of the enzyme activity in cells.
Structural Insights into the Mechanism of Tartrate Dehydrogenase: A versatile enzyme catalyzing multiple reactions
Authors: Malik Radhika , Viola Ronald E
Published: June 2021

Tartrate Dehydrogenase (TDH) is a member of the family of R‐hydroxy acid dehydrogenases, occupying a unique branch. TDH catalyzes several interesting transformations which vary depending on the nature and the stereochemistry of the respective substrates. Structural and functional information on this unusual enzyme will provide a better understanding of the evolution of enzymes within this species, as well as guide its engineering for chiral synthon production. Here we report the first structure of TDH solved to 2.0 Åresolution by SAD phasing using selenomethionine labeled protein. The enzyme is a dimer with residues from both monomers forming each active site cleft. The complex with NADH, Mg2+ and the intermediate analog oxalate represents the closed state of the enzyme. Based on the structures we have identified putative substrate and metal binding residues. Their proposed roles in the catalytic mechanism have been confirmed by site directed mutagenesis and kinetic analysis. In particular, the role of D225 as the base responsible for initial proton abstraction from the C2 hydroxyl of the substrate has now been established. A product complex provides additional insights which complement our mechanistic picture of this unusual enzyme. These results will now be used to guide the reengineering of this enzyme.
Characterization and Hybridization of Disease‐Associated Human Adenylosuccinate Lyase Mutants
Authors: Antonopoulos Christina H. , Ariyananda Lushanti De Zoysa , Lee Peychii , Currier Jenna M. , Colman Roberta F.
Published: June 2021

Adenylosuccinate lyase (ASL), a catalyst of key reactions in purine biosynthesis, is a homotetramer in which different regions of three subunits contribute to each of four active sites. Human ASL deficiency is an inherited metabolic disease associated with autism and mental retardation. We have constructed, expressed, purified and characterized two disease‐associated ASL mutants (L311V and R396H) that are located in different regions of the enzyme. L311 is in the central helical region away from the active site, whereas R396 is in close proximity to the entrance to the active site. The Vmax for L311V and R396H are 8.6 and ~2.0 μmol/min/mg, respectively, as compared to 11.9 for wild type; however, all three enzymes have comparable affinities for adenylosuccinate. L311V has ~25 % less tetramer than wild type while R396H has similar amounts of tetramer as wild type. At 37 °C the specific activities of these enzymes decrease with time and reach a limiting specific activity without significantly changing the amount of tetramer. Most ASL deficient patients are compound heterozygotes (e.g. L311V/R396H). In vitro generation of hybrid tetramers from L311V and R396H, only one of which has a His6‐tag, followed by their purification on a Ni‐NTA column and characterization, is in progress in this laboratory and yields insights into the function of ASL in compound heterozygous patients. (Supp. by Autism Speaks and HHMI.)
X‐ray structure of NS1A effector domain from the influenza A/Udorn/72 virus
Authors: Xia Shuangluo , Monzingo Arthur F , Robertus Jon D
Published: June 2021

The nonstructural protein NS1A from influenza virus is a multifunctional virulence factor and a potent inhibitor of host immunity. It has two functional domains, an N‐terminal 73 amino acids RNA‐binding domain, and a C‐terminal effector domain. Here we present the crystallographic structures of the wild type and W817A mutant of the effector domain of the influenza A/Udorn/72 virus. Structure comparison with the effector domain from mouse‐adapted influenza A/PR8/34(PR8) virus strain reveals a similar monomer conformation, but a different dimer interface. The W187A mutation abolishes the dimer interaction completely, and the CPSF30 binding pocket is exposed to the solvent in the mutant structure. It indicates that the dimer interface observed in the structure of PR8 effector domain is likely to be a crystallographic packing effect. A model of full length NS1A is made based on the structures of N‐terminal and C‐terminal domain. It provides new insight to this important and unusual protein.
In vitro silencing of a putative phospholipid translocase results in an increase in glucose uptake in 3T3 L1 adipocytes
Authors: Hurst Sarah Elizabeth , Peretich Amanda , Minkin Steven , Dunlap John , Biggerstaff John
Published: June 2021

Atp10c is a putative phospholipid translocase which encodes for a type IV P‐type ATPase. Atp10c gene, located on mouse chromosome 7, appears to be a strong candidate for diet‐induced obesity and diabetes mellitus type II. Our in vitro studies using a 3T3‐L1 murine adipocyte cell line revealed that when Atp10c expression was silenced or knocked down using siRNA, glucose uptake and fat accumulation increased, while insulin resistance decreased. Furthermore, the silencing effect of Atp10c caused an increase in Glut 1 and CEBP‐α gene expression and a decrease in Glut 4 and PPAR‐γ gene expression. To further investigate the role of Atp10c in insulin resistance and glucose uptake, immunolocalization of Atp10c by fluorescence microscopy was employed. Data from these experiments suggest that Atp10c not only co‐localizes with caveolin‐1 and GLUT4 at the nuclear membrane, but also can be visualized as a punctate pattern in and around the nucleus. This finding suggests that Atp10c additionally may localize to the trans‐Golgi network or endosome, strengthening its biological role in protein trafficking in the exocytic and endocytic pathways.
Male‐female differences in post translational modifications of mitochondrial proteins
Authors: Lagranha Claudia Jacques , Steenbergen Charles , Murphy Elizabeth
Published: June 2021

Premenopausal women have reduced cardiovascular disease compared to men. The mechanisms underlying such gender differences are unclear. Using a rat Langendorff model, we found that females (F) had smaller infarcts than males (M) after 30 minutes of ischemia. We hypothesized that this protection is mediated by altered protein levels or post‐translational modifications (PTM), and we used a proteomic approach to investigate these differences. Isolated mitochondria from M and F rats were analyzed by 2D fluorescence difference gel electrophoresis (2D‐DIGE) followed by mass spectrometry (MS). 2D‐DIGE analysis of M and F revealed differences in 20 proteins. PDH‐E1α and aldehyde dehydrogenase (ALDH) were both located at multiple pI values, suggesting PTM. Furthermore, for PDH E1α, F had higher levels at high pI values and lower levels at low pI values while the reverse was true for ALDH; these data suggest M‐F differences in PTM. Consistent with M‐F differences in PTM, using ProQ to identify phosphoprotein, we found increased PDH‐E1α phosphorylation in M compared to F. Quantitative MS identified 742 proteins, but no significant M‐F difference was found in any of these proteins. These data suggest that PTM plays a bigger role in M‐F differences than changes in expression of mitochondrial proteins.
Murine Genotype Alters Palatal Tissue Response to Nicotine
Authors: Serrano Maria Juliana , Yu Wenli , Svoboda Kathy
Published: June 2021

Our previous work demonstrated that nicotine inhibited palatal fusion in vitro in a dose dependent manner. We asked if the mouse genotype would be a factor in nicotine teratology.

METHODS

Palatal shelves from mouse strains (CD1, ND4, C56 and 129S) E13.5 were cultured in serum free media (control), and nicotine (0.6 mM and 6 mM). Palates were harvested at 24 hours, 48 hrs and 72 hrs and fixed in 4%PFA for paraffin embedding and H&E sections.

RESULTS

CD1 and ND4 strains were consistent in the palatal fusion pattern within litters. The shelves in all the embryos are not fused at a specific age (E 13.5).C56 and 129Shad variable palate fusion. Some of the embryos had fused palates at embryonic day 13.5, while other embryos had undeveloped palatal shelves. The tissue cultured palates from CD1 and ND4 mice treated with 0.6mM nicotine fused, while palate treated with 6 mM nicotine retained epithelial seam. Palates from C56 mice had an epithelial seam using low and high doses of nicotine. 129S palate shelves 6mM nicotine have two layers of MEE in the midline (non fusion).

CONCLUSIONS

The genetic background of mice influenced nicotine responses in palatal tissues.

Grant Funding Source

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Dynamics of Cholesterol Exchange in the Oxysterol Binding Protein Family
Authors: Canagarajah Bertram J , Hummer Gerhard , Hurley James H
Published: June 2021

The oxysterol‐binding protein‐related protein (ORP) family is essential to sterol transfer and sterol‐dependent signal transduction in eukaryotes. The crystal structure of one ORP family member, yeast Osh4, is known in apo and sterol‐bound states. In the bound state, a 29 residue N‐terminal lid region covers the opening of the cholesterol‐binding tunnel, preventing cholesterol exchange. Equilibrium and steered molecular dynamics (MD) simulations of Osh4 were carried out to characterize the mechanism of cholesterol exchange. While most of the structural core was stable during the simulations, the lid was partly opened in the apo equilibrium MD simulation. Helix α7, which undergoes the largest conformational change in the crystallized bound and apo states, is conformationally coupled to the opening of the lid. The movement of α7 helps create a docking site for donor or acceptor membranes in the open state. In the steered MD simulations of cholesterol dissociation, we observed complete opening of the lid covering the cholesterol‐binding tunnel. Cholesterol was found to exit the binding pocket in a step‐wise process involving (i) the breaking of water‐mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.
Chemical and Biochemical Models of Parrondo's Paradox
Authors: Osipovitch Daniel C. , Gordon Thomas , Barratt Carl , Schwartz Pauline M.
Published: June 2021

Parrondo's paradox is a mathematical concept describing two losing games played in an alternate fashion to give a winning outcome. Our objective was to devise computational models of chemical and biochemical systems that were analogous to the paradox. The model systems were created using Kintecus, a chemical kinetics simulation program based on the Arrhenius equation. Model I is a multi‐step system with a temperature sensitive catalyst. The predicted concentration of the target product after 15000 sec at 300K is 6.5x10−3 M and at 480K is 1.6x10−2M. Paradoxically, if the model is run with an oscillating temperature profile between 300K and 480K (23 cycles), then it predicts that considerably more product, 1.25x10−1M is made; this is more product than predicted at any fixed temperature between 300K and 480K. There are few examples of chemical reactions that are actually conducted under oscillating temperature conditions. Model II describes the thermal cycling conditions of PCR under a two‐temperature protocol (2T‐PCR). The computational model predicted that little double‐stranded DNA was synthesized at either 330K (57C) or 370K (97C) but that with thermal cycling (26 cycles in 23000sec), dsDNA is produced almost exponentially until substrates/primers are consumed. This model will allow prediction and optimization of 2T‐PCR reactions. These models suggest that thermal cycling should be explored further in chemical and biochemical systems. This research was supported by the University of New Haven.
Characterization of Fatty Acid Transport across Human Brain Microvessel Endothelial Cells (HBMECs)
Authors: Mitchell Ryan W. , Miller Donald W. , Hatch Grant M.
Published: June 2021

Endothelial cells lining the blood capillaries of the Blood Brain Barrier (BBB) are tightly packed thus regulating the transport of substances from the blood into the brain, including fatty acids. Fatty acids are essential for both the developing and adult mammalian brain. Since most fatty acids in the brain enter from the blood, we examined the mechanism of their transport across Human Brain Microvessel Endothelial Cells (HBMECs). HBMECs were plated onto transwell plate inserts and then incubated for up to 4 h with [1‐14C]oleate in the apical medium. Radioactivity in the basolateral medium was temporally examined. There was a near linear increase in [1‐14C]oleate incorporation into the basolateral media in the presence of albumin indicating a protein acceptor is required for oleate transport. The presence of phloretin, a non‐specific fatty acid uptake inhibitor, significantly decreased [1‐14C]oleate transport into the basolateral medium. In addition, siRNA knockdown of fatty acid transport protein‐1 (FATP‐1) or fatty acid translocase (FAT/CD36) significantly decreased [1‐14C]oleate incorporation into the basolateral media. In summary, transport of oleate across HBMECs is, in part, a transcellular process. Oleate transport across HBMECs is mediated by both diffusion and carrier mediated processes. (Supported by the Manitoba Health Research Council and the Canadian Institutes of Health Research)
Distinct pools of OXPAT suggest roles in lipid trafficking
Authors: Tansey John T , Bartholomew Sadie R , Hlavin Erica M , Summerfield Taryn L , Ackerman William E
Published: June 2021

Muscle and liver tissues surfeited with neutral lipid have been implicated in the development of insulin resistance and type II diabetes. PAT proteins coat lipid storage droplets (LSDs) and regulate lipid storage. The most recently discovered PAT family member, OXPAT (also known as LSDP5 or MLDP), localizes to two distinct intracellular pools: the LSDs and a cytosolic pool. Characterization of the cytosolic pool reveals that OXPAT resides on a discreet structure. Sucrose gradient ultracentrifugation and non‐denaturing gradient gel electrophoresis indicate that the particles have a density of 1.10‐1.17 g/ml and a diameter of approximately 15 nm (575 kDa). Immunoprecipitation of these particles using an OXPAT specific antibody fails to precipitate ADRP. In contrast, both ADRP and OXPAT coexist on the more buoyant LSD. CHO cells ectopically expressing OXPAT have increased neutral lipid stores (1.5 fold more triacylglycerols and 3.8 fold more total cholesterol). Additionally, there is a change in the morphology of these LSDs (the size of the largest droplet in each cell increased from 1.5 to 3.1 microns when OXPAT was expressed). OXPAT shifts from the cytosol to the LSD upon treatment of cells with oleic acid to increase neutral lipid deposition. Collectively these data suggest a new role for OXPAT in neutral lipid trafficking and storage. This work was supported in part by a K award (HD049628) to WEA.
Long‐chain acyl‐CoA synthetase 3 (ACSL3) mediates transcriptional control of hepatic lipogenesis
Authors: Bu So Young , Mashek Mara T , Mashek Douglas G
Published: June 2021

Long‐chain acyl‐CoA synthetases (ACSL) and fatty acid transport proteins (FATP) activate fatty acids (FAs) to acyl‐CoAs and each isoform of ACSL and FATP has different tissue distribution patterns, intracellular locations and substrate preferences. Since FAs and acyl‐CoAs affect the regulation of various transcription factors that govern hepatic energy metabolism, we hypothesize that distinct pools of intracellular FAs and acyl‐CoAs governed by ACSL or FATP isoforms differentially regulate hepatic gene expression. To test this hypothesis, we knocked down each ACSL and FATP isoform in rat primary hepatocyte cultures using siRNA and analyzed reporter gene activity of transcription factors (PPAR‐gamma, SREBP and LXR‐alpha). Of the different ACSL and FATP isoforms, knockdown of ACSL3 significantly decreased PPAR‐gamma reporter gene activity. ACSL3 knockdown also down regulated the expression of lipogenic genes. These findings were further supported by metabolic labeling studies showing that ACSL3 knockdown resulted in decreased [C14]acetate incorporation into lipids. In summary, these results indicate that activation and channeling of FAs by ACSL3 mediates lipogenic pathways in the liver. The mechanism by which ACSL3 regulates these transcriptional factors governing lipogenesis needs to be further investigated.
ADAM13 is Required for Xenopus Head Development by Regulating Class B Ephrin and Wnt Signaling
Authors: Wei Shuo , Bridges Lance C. , Williams Phoebe , Xu Guofeng , Whittaker Charles A. , Shah Anoop , O'Neill Shannon , White Judith , DeSimone Douglas
Published: June 2021

Protein ectodomain shedding by disintegrin metalloproteinases (ADAMs) has emerged as a key regulatory event in cell signaling. We previously showed that an ADAM metalloproteinase, ADAM13, is expressed in cranial neural crest (CNC) cells in Xenopus laevis, and that overexpression of a protease dead mutant of ADAM13 prevents CNC migration. To further understand the roles of this gene in vertebrate development, we carried out loss‐of‐function studies in X. tropicalis, a closely related frog species with a diploid genome. Knockdown of ADAM13 by antisense morpholinos (MOs) resulted in abnormal head development, including reduced CNC induction and perturbed eye field patterning. The metalloproteinase activity of ADAM13 was required for these functions in vivo. Using cultured cells, we identified membrane‐bound ephrins (Efns) B1 and B2 as substrates for ADAM13. Knockdown of ADAM13 protected endogenous EfnB1/B2 in developing embryos, indicating that these two ephrins are in vivo substrates for ADAM13. The defects caused by ADAM13 MO could be rescued by blocking forward EfnB signaling, restoring canonical Wnt signaling, or by ectopic expression of the transcription factor Slug. Our data suggest a novel EfnB‐Wnt‐Slug signaling cascade through which ADAM13 controls CNC induction and eye field patterning.
Effects of Nonstructural Proteins (NS) of Respiratory Syncytial Virus (RSV) on Host Interferon Response
Authors: Swedan Samer Fuad , Barik Sailen
Published: June 2021

RSV is a Pneumovirus of the Paramyxoviridae family. To replicate efficiently, RSV interacts with the host cell to inhibit cellular innate immunity. RSV has two nonstructural proteins, NS1 and NS2, which have no homology with any other gene in biology. Both are dispensable for RSV replication in cell culture, but needed for robust virus infection in animals. Both NS proteins have interferon (IFN) antagonist activity. The project's goal is to define the functions of the NS1 and NS2 proteins of RSV in viral replication and pathogenesis. More specifically, the amino acid residues of the NS proteins essential for IFN antagonism will be identified and host proteins that interact with the NS proteins will be determined. The central hypothesis is that NS1 and NS2 antagonize IFN production by interacting with components of IFN induction and response cascades. To test this hypothesis, techniques such as site‐directed mutagenesis of NS genes, luciferase reporter assays, and IFN functional assays will be employed. This will provide an integrated view of the structural determinants of NS proteins on IFN antagonism and the effects of altering these determinants on RSV biology. Initial results indicate that NS1 and NS2 prevent the induction of IFN‐β and implicate the ten c‐terminal residues of NS2 in the degradation of STAT2.

Research sponsored by Alabama EPSCoR and NIH Grant AI059267
Autophagy in skeletal muscle: The effects of age and diet
Authors: Wohlgemuth Stephanie Eva , Nguyen Alex , Lees Hazel Anne , Marzetti Emanuele , Leeuwenburgh Christiaan
Published: June 2021

Effects of age and life‐long calorie restriction on regulation of autophagy were studied in skeletal muscle of male Fischer344xBrownNorway rats. Autophagy is the primary pathway for degradation of long‐lived proteins and organelles. We investigated the expression of autophagy regulatory proteins in plantaris (fast‐twitch) and soleus (slow‐twitch) muscles of 8, 27 and 38‐month‐old rats, fed ad libitum or 40% calorie restricted (CR). Muscle mass decreased with age. The decrease was less pronounced in soleus than plantaris, and attenuated by CR in both muscles. The expression of the autophagy regulatory proteins Beclin‐1 and Atg7 increased with age in both muscles. In contrast, the LC‐3 II/I ratio increased in the soleus, but decreased in the plantaris. CR stimulated autophagy in the soleus but had no marked effect in the plantaris. In conclusion, the response of autophagy to age and CR appears to be muscle type specific, with implications for autophagy's diverse roles in aging. This study was supported by the University of Florida Institute on Aging and Claude D. Pepper Older Americans Independence Center (1 P30 AG028740) and by grants to CL (NIA R01‐AG17994 and AG21042).
Charactrization of anti‐aging effect in Halophyte extracts, Suaeda asparagoides
Authors: Hong Heeok , Ahn Jeung‐Youb
Published: June 2021

The aim of this research was to develop anti‐skin aging product using anti‐oxidant effective materials in halophyte plant, Suaeda asparagoides (SA Ex).

We tried out several experimental works in in vitro and in vivo (animal study and human clinical study). To find the anti‐oxidant effects in this study we used DPPH, photo‐hemolysis, luminol‐dependent chemiluminescence assay and radical scavenging assay. Anti‐oxidant effect shown 87.8% free radical scavenging activity in ethylacetate fraction of SA Ex. Biochemical enzyme inhibition assay was done by collagenase, hyaluronidase, and elastase. Among the Collagenase, hyaluronidase and elastase inhibition activity, collagenase inhibition activity was shown strong effect definitely. MTT assay was employed for cell cytotoxicity effect. We also investigated formula stability and stabilized formula was applied to in vivo test which was the skin sensitization test on hairless mouse (HR‐1).

In conclusion, first, we have got outcome material registration on International Cosmetic Ingredient Dictionary (ICID). Second, this research report is first of all about isolation of components in Suaeda asparagides. Third, we made guarantee that is data on material safety and stability by in vitro and in vivo.

These anti‐oxidative fraction and materials could be re‐evaluated the biological effects on research area. The data show us potential anti‐aging biological ability and the materials were expected as a functional ingredient of industrial products in the future.
Heteroarotinoids elevate tissue transglutaminase in human erythroleukemia cells
Authors: Wilds Brandon W , Miller Matthew R , Dawson Ashley N , Birckbichler Paul J
Published: June 2021

Tissue transglutaminase (tTg) is postulated to play a role in apoptosis and/or differentiation in cultured cells. All trans‐retinoic acid (RA) is a naturally occurring molecule that binds to retinoic acid receptors responsible for apoptosis and differentiation. RA induces tTg activity in human erythroleukemia (HEL) cells, but in general, results in high toxicity. Heteroarotinoids (Hets) are less toxic synthetic structural analogues of RA and are defined as systems with one aromatic ring and one heteroatom (N, S, or O). We propose that Hets have a role in differentiation and/or apoptosis of HEL cells. To test this, HEL cells were cultured for 48 h with RA (tTg induction), DMSO (negative control) and Hets 1, 2, and 4 (experimental compounds). Hets 1, 2 and 4 showed significant growth inhibitions, lowered cell viability and induced apoptosis relative to DMSO and RA. tTg was quantitated in HEL cells using an inhibition ELISA assay. When an anti‐tTg monoclonal antibody was added to a sample containing tTg, an antigen‐antibody complex was formed. When this sample was added to a 96‐well microtiter plate containing attached tTg, less color was produced relative to an antibody sample that was not premixed with tTg. Preliminary results in Het 2 and Het 4‐treated cells showed elevated levels of tTg. The results appear promising for our hypothesis of a role for Hets in differentiation and/or apoptosis of HEL cells.
SUMO Mediated Response Pathways to Genotoxic Stress
Authors: Ro Kevin , Wohlschlegel James
Published: June 2021

The different pathways undertaken by cells in response to genotoxic stress are essential to their survival. In this study, we investigated how the SUMO conjugation pathway may play an important role in responding to various environmental stresses. SUMO is a member of the ubiquitin family of small protein modifiers and its covalent attachment to cellular targets is a critical regulatory step for a diverse range of cellular processes that includes transcriptional regulation, cell cycle progression, and DNA damage response. Specifically, we analyzed how the cell's viability and SUMO conjugation patterns changed when exposed to both acute and long term treatments of ethanol, hydrogen peroxide, hydroxyurea, nocodazole, and benomyl. To study how different stresses affected SUMO conjugation pathway, and subsequently cell viability, yeast strains deficient in key SUMO conjugation enzymes were knocked‐out and subjected to the stresses. Analysis using Western blots and growth assays reveal that several of the stresses used increased SUMOylation and affects cell growth. Proteomic analysis using a linear ion trap‐orbitrap hybrid mass spectrometer was also done on the ethanol subset and 119 SUMO substrates were found to be present in greater abundance in ethanol treated cells versus untreated cells. We are currently further characterizing the functional role of this set of SUMO targets in the cellular stress response.
Compound C inhibition of adipocytes differentiation: The role of an increase in p21 in suppressing the clonal expansion of preadipocytes
Authors: Lee Woo Hyung , Nam Minwoo , Kim Sang Geon
Published: June 2021

AMP‐activated protein kinase (AMPK) activation inhibits adipocytes differentiation. This study investigated the effect of compound C (CC), a widely used AMPK inhibitor, on differentiation of 3T3‐L1 preadipocytes. CC treatment blocked hormone‐induced preadipocyte differentiation due to inhibition of mitotic clonal expansion, which was accompanied by the failure of degradation of p21, a cyclin‐dependent kinase inhibitor. CC increased the level of p21 protein, but not its mRNA, in preadipocytes incubated in a hormone‐free medium. Cycloheximide decreased the basal p21 level, which was inhibited by CC treatment, supporting the stabilization of p21 by CC. Treatment of AICAR or metformin, AMPK activators, failed to induce p21 or inhibit the ability of CC to increase p21 level. In conclusion, CC inhibits proliferation of preadipocytes as a consequence of an increase in p21 content, which might result from p21 stabilization, and the increase in p21 level by CC might not be associated with AMPK inhibition.
Angiopoietin like 2, an orphelin ligand, is a potent vasodilator
Authors: Farhat Nada , Thorin‐Trescases Nathalie , Mamarbachi Maya , Drouin Annick , Perrault Louis , Allen Bruce , Thorin Eric
Published: June 2021

Angiopoietin‐like 2 (Angptl2) is a member of the angiopoietin family whose function and receptor remain unknown. Our objectives were to identify the Angptl2 receptor and study its physiological role in the vasculature. In vitro, exogenous addition of Angptl2 (1 nM) induced a dilation (73.6% + 8.7 of maximum dilation, n=8) of isolated preconstricted mesenteric arteries from normal C57BL6 mice. An Angptl2‐GST fusion protein was purified from the media of stably transfected HEK 293 cells by affinity chromatography on glutathione Sepharose. To purify the Angptl2 receptor, membranes were prepared from human arterial endothelial cells, solubilized, and incubated with Angptl2‐GST (25 mg), or GST, precoupled to glutathione Sepharose beads. After extensive washing, bound proteins were eluted using Laemmli sample buffer and resolved on SDS‐PAGE. Bands were visualized using Sypro Ruby, excised, and sequenced by mass spectrometry (LC‐MSMS). The identified proteins included the EGF receptor (EGFR). Furthermore, shRNA knock down of Angptl2 in HUVECs was associated with reduced phosphorylation of p38, JNK and ERK MAPKs. In conclusion, Angptl2 is a potent vasodilator whose signaling may involve transactivation of the EGFR and activation of the 3 major MAPK cascades. The Angptl2 receptor remains to be identified.
Magnesium ions inhibit Apaf‐1 apoptosome assembly by competition with cytochrome c (Cc)
Authors: Smith Jeffrey B. , Smith Lucinda , Garcia Patrick L.
Published: June 2021

The disassembly of mammalian cells by apoptosis generally depends on oligomerization of the Apaf‐1:caspase‐9 apoptosome in the cytoplasm, which is evoked by the release of Cc from mitochondria. A soluble fraction (S100) was prepared from the 911 line of immortalized human embryonic retinoblasts or from 911 cells transiently transfected with human Apaf‐1 with carboxy‐terminal V5 epitope and hexahistidine tags. Apoptosome activation was assayed by monitoring the rate of caspase‐3 like activity with a fluorogenic tetrapeptide. Apoptosome activation was absolutely dependent on the addition of Cc and markedly stimulated by the addition of a nucleotide (MgATP or MgdATP). The Km of Cc was 0.6 μM bith a ransfected with human Apaf‐1 with a V5 epitope tag and hexahistidine. The Ki of free Mg2+ was 0.3 mM, which is half the 0.6 mM basal free Mg2+ level of 911 and HeLa cells (Abstract 6098). While MgATP was required for maximal apoptosome activation, excess MgATP did not inhibit prodced activation and had apoptosome activation and had no inhibitory effect Cc‐dependent apoptosome activity. Transfection of 911 cells with Apaf‐1V5His increased apoptosome activation by Cc by approximately 10 fold relative to that of cells transfected with empty vector, which indicates that Apaf‐1in S100 is rate limiting. Mg2+ inhibited apoptosome activity caused by transfected Apaf‐1V5His similarly to that of endogenous Apaf‐1 in S100 from nontransfected 911 cells. Mg2+ inhibition of Cc‐dependent apoptosome activity was highly cooperative with a Hill coefficient of 4 to 7, which indicates that Mg2+ can switch off apoptosome assembly. Apoptosome assembly was observed by sedimentation velocity in glycerol gradients and western analysis of Apaf‐1V5His and procaspase‐9 and the large subunit. Mg2+ prevented apoptosome assembly and autoprocessing of procaspase‐9 to subunits. These results show that Mg2+ potently inhibits apoptosome assembly, presumably by blocking the binding of Cc to monomeric Apaf‐1. Supported by grant GM60383 from NIH.
Investigating the role of a metacaspase in the mushroom fungus Schizophyllum commune
Authors: Goldberg Alyssa R. , Horton J. Stephen
Published: June 2021

The present study was designed to help elucidate the role of a metacaspase in the life cycle of the mushroom‐producing fungus Schizophyllum commune. Results from a wide variety of previous studies indicate that apoptosis is essential to the development and maintenance of multicellular organisms. Metacaspases (found in fungi, plants and protists) are hypothesized to have similar activity to caspases, which are key components in apoptosis. The role of one metacaspase in S. commune will be assessed by disabling one of the four genes that code for metacaspase proteins in this organism. A "knockout" DNA construct of the gene has recently been constructed in our laboratory. This construct will be homologously integrated into the genome via DNA‐mediated transformation, producing a "null mutant". The null mutant will be examined to determine any possible effects on mushroom production and reproduction in this species. We hypothesize that without a completely functioning apoptotic mechanism in the mutant strains to remove aged and sterile cells, normally reproductive fruiting bodies may develop into sterile structures or not form completely. The results of these experiments should help us gain an understanding of the mechanisms regulating mushroom development in this model organism. Supported by a Union College IEF Grant to A.R.G.
Dissecting the function of the HigB addiction module toxin
Authors: Hurley Jennifer Marie , Woychik Nancy Ann
Published: June 2021

Toxin‐antitoxin (TA) modules were initially discovered on bacterial plasmids that facilitate plasmid maintenance via post segregational killing. More recently, chromosomally encoded TA operons found in free‐living prokaryotes have been demonstrated to aid in the cellular response to stress. TA modules encode a stable toxin that inhibits cellular growth, and a labile cognate antitoxin, that inhibits the action of the toxin through the formation of a toxin/antitoxin complex. In addition, these modules have been implicated in pathogenesis and antibiotic tolerance. TA systems also represent a new class of targets for antibiotic treatment of pathogens resistant to existing therapeutics.

The goal of this project is to identify the precise function and intracellular target of the HigB toxin, which is a member of the RelE family of toxins. When expressed, RelE or HigB inhibit protein translation through mRNA cleavage that is mediated by the association of the toxins with the Escherichia coli 50S ribosomal subunit. However, the specificity of mRNA cleavage and the nature of the ribosome‐dependent toxin activity appear to be distinct between the two toxins. We are currently investigating additional mechanistic details of HigB toxicity. Research supported by the Virus‐Host Interactions in Eukaryotic Cells T32 Training Grant from the NIH‐NIAID awarded to Dr. Sidney Pestka.
Effect of SB203580 on apoptosis related gene expression pattern of SEB in human PBMCs
Authors: Mason Tricia Nicole , Hopkins Aaron Matthew , Mendis Chanaka
Published: June 2021

Here we attempt to utilize a well characterized set of apoptotic related genes that are key to the pathogenesis of Staphylococcal Enterotoxin B (SEB) induced lethal shock in human peripheral blood mononuclear cells (PBMC) to evaluate the effectiveness of a potential inhibitor of apoptosis related events. We utilized SB203580 (p38 specific inhibitor) to alter the expression pattern of SEB induced apoptosis related genes in a time dependent manner. After designing primers using stringent conditions, alterations to the known gene expression patterns of heparanase precursor (HEP), Caspase 1, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10, Ubiquitin specific protease, and SOD were analyzed using reverse‐transcription (RT‐PCR). Our results showed the effectiveness of SB203580 in inhibiting apoptosis related gene activity in a time dependent manner. We believe that our investigation may also reveal crucial information to better understand the complexity of apoptosis related signaling events that take place in SEB induced human PBMC module. The work also provides important information in inhibiting pathway inter‐connectors such as p38 to abolish unwanted cellular behavior.
Attenuation of pheromone responsiveness in yeast in the absence of the glucose sensor Gpr1.
Authors: Wright Stephen E , Willhite Grant , Muncy Zac , Schlafer Danielle
Published: June 2021

G protein coupled receptors are known to associate with other GPCRs as either homo or heterodimers. Haploid strains of the yeast saccharomyces cereviseae express only two types of GPCRs which mediate either pheromone or glucose sensing via very different signal transduction pathways. We have observed a reduction in the activity of the pheromone pathway in strains lacking either the glucose receptor (Gpr1p) or its associated G protein (Gpa2p). Cells lacking either of these components had reduced levels of pheromone‐induced projection formation or growth inhibition. This suggests that the two receptors may interact during signal transduction.
Tetraoxyethylene isooctylphenyl ether(Militarin‐5) induces apoptosis in A549 cells
Authors: Kim Tae‐Woong , Lim Mi‐Hee , Lim Dong‐Uk , Yoon Deok‐Hyo , Cho Jae‐Youl , Park Haeil , Sung Jae‐Mo , Hwang Ki‐Chul
Published: June 2021

Tetraoxyethylene isooctylphenyl ether(Militarin‐5) is one of the derivatives of Militarin, which is found from Cordyceps militaris. Cordyceps militaris is a parasitic fungus and has long been used as a Chinese medicine to treat illnesses, promote longevity, increase athletic power, and relieve exhaustion and anticancer. In this study, Militarin‐5 was synthesized, and, especially, we focused on the mechanisms of cell death exhibited by Militarin‐5 against human lung adenocarcinoma epithelial cell line, A549. Militarin‐5 inhibited growth and induced apoptosis in A549 cells in a dose‐dependent manner as determined by MTT assay, DNA fragmentation and flow cytometry histograms by propidium iodine staining. In Western blot analysis, apoptosis in A549 cells was associated with the activation of caspase‐3, ‐9 and proteolytic cleavage of PARP, decrease of Bcl‐2/Bax protein ratio, and release of cytochrome C from mitochondria into cytosol. We also found that Militarin‐5 inhibited the expression of COX‐2 in A549 cells. These results show that apoptosis of Militarin‐5 toward A549 cells is mediated by mitochondria‐dependent death‐signaling pathway. Thus, it indicated that Militarin‐5 may be a potential candidate in future research and development of bioactive ingredients as anticancer agents. Supported by grant from KOTEF, Korea