bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular dynamics studies of polymerase X/DNA complexes in the presence of OxoG on the templating strand
Authors: Benitez Benedetta Alessandra Sampoli , Arora Karunesh , Bogdanovic Jasmina , Barbati Zachary , Schlick Tamar
Published: June 2021

The African Swine Fever Virus (ASFV) is an encapsulated deoxyvirus known to induce a lethal infection in domestic pigs. The double‐stranded DNA viral genome encodes a total of 151 proteins, including a minimal DNA repair system, with a repair polymerase, pol X, which catalyses a single‐nucleotide filling in gapped DNA. Pol X, is the smallest naturally occurring DNA‐directed DNA polymerase described so far. It belongs to the X family of polymerases and shares sequence and structure similarity with human DNA polymerase β (pol β). However, where pol β is a moderate fidelity enzyme, pol X is much more error tolerant. In this study, we are exploring several pol X/DNA complexes in which the templating base pair opposite to the incoming nucleotide contains the damaged base 7,8‐Dihydro‐8‐oxoguanine (oxoG). OxoG is one of the major known products of oxidation of DNA by reactive oxygen species (ROS). Since the African swine fever virus's components are in an active state of respiration, it is conceivable that the virus may encounter this common ROS‐induced DNA lesion. Kinetics data have shown that pol X accommodates A opposite to oxoG, with efficiency even higher than the non‐damaged G:C base pair. Thus, the goal of this molecular dynamics study is to explore combinations of oxoG:A, oxoG:G and oxoG:C systems to elucidate, at an atomistic level, the kinetics data. Research was supported by the Rose M. Badgeley Charitable Trust.
Effect of quadruplex DNA formation on repair of endogenous DNA damage
Authors: Beckett Joshua , Broxson Christopher , Bloom Linda , Tornaletti Silvia
Published: June 2021

Quadruplex (G4) DNA is a non canonical four stranded DNA structure that can form in G repeats by stacking of planar arrays of four hydrogen bonded guanines called G quartets. G4‐forming sequences often correspond to genomic regions characterized by genomic instability. We hypothesize that because of its unusual structural properties, G4 DNA may impair the recognition and repair of DNA damage, leading to mutagenesis. To test this hypothesis we have measured the efficiency of repair of abasic (AP) sites, the most common lesion spontaneously generated during cellular metabolism, when they are formed in the G4 forming sequence from the c‐myc gene. We find that removal of a single AP site by human AP endonuclease on the G4 containing substrate was dramatically reduced when the AP site was located in the loop of the G4 structure compared to the unstructured control. This effect was due to decreased enzymatic activity and not to inefficient binding of AP endonuclease to the lesion. Our findings suggest that inefficient repair of lesions formed in G4 DNA may contribute to the mutagenesis associated with these structures.
Either Intravenous or Inhaled Anesthetic for Elective Otorhinological Surgery Does Not Induce Oxidative Stress
Authors: Braz Mariana Gobbo , Braz Leandro Gobbo , Fecchio Denise , dos Anjos Ferreira Ana Lucia , Braz Jose Reinaldo Cerqueira , Tang Guangwen , Yeum Kyung-Jin , Salvadori Daisy Maria Favero
Published: June 2021

Oxidative status of patients undergoing surgery with either intravenous or inhaled anesthetics was studied. Thirty adult patients classified as physical status I, who were scheduled for otorhinological surgery, were randomized to receive propofol (n=15) or isoflurane (n=15) anesthesia. Blood samples were drawn before (T1) and 2 h after the beginning of anesthesia (T2) and on the following day of surgery (T3). Plasma pro‐inflammatory IL‐6 cytokine and apoptosis evaluated in T helper cells were detected by flow cytometry; oxidative DNA damage in lymphocyte by the comet assay; and antioxidant performance by fluorometry. IL‐6 increased in T3 for both groups. While propofol decreased oxidized purines in T2 compared to T1 (p=0.01), isoflurane did not induce oxidative DNA damage. Both anesthetics tend to increase antioxidant performance in T2 (p=0.06 for propofol and p=0.1 for isoflurane). Propofol decreased the frequency of apoptotic cells in T2 and T3 compared to T1. The current study indicates that both anesthetics did not induce oxidative stress in patients undergoing elective surgery and that propofol might exert antioxidant protection.

Supported by FAPESP, Brazil #2006/59625‐6; 2010/05611‐0
Anthocyanins of Black Soybean (cv. Cheongja 3) Induce Autophagy via AMPK Activation in U2OS cells
Authors: Choe Yun-Jeong , Ha Tae Joung , Ko Kyoung-Won , Lee Sun-Young , Kim Ho-Shik
Published: June 2021

Anthocyanins (ATC) were reported to be able to induce autophagy, but the mechanism of which remains still to be clarified. In U2OS cells, ATC extracted from black soybean (cv. Cheongja 3) induced hallmarks of autophagy such as LC3's punctuate pattern formation and the conversion of LC3‐I to LC3‐II. The induction of autophay was accompanied with phosphorylation of multiple protein kinases including Erk1/2, p38MAPK, JNK, AKT, and AMP‐dependent protein kinase (AMPK). While anthocyanins‐induced autophagy was inhibited by none of each inhibitor of ERK1/2, p38 MAPK, JNK, and AKT, suppression of AMPK by compound C as well as knockdown of AMPK using AMPK siRNA reduced anthocyaninsi‐nduced autophagy. Activation of AMPK by AICAR treatment also induced autophagy. Notably, reduction of autophagy via inhibition of AMPK enhanced anthocyanins‐induced apoptosis. Therefore, it can be suggested that anthocyanins induce autophagy by activation of AMPK, and autophagy may inhibit anthocyanins‐induced apoptosis.

This research has been supported by the Biogreen 21 Program (Code#PJ007186), Rural Development Administration Korea.
Structural and functional studies of disulfide bonded hemolysin A
Authors: Apolinario Michael Angelo , Weaver Todd Michael
Published: June 2021

The research implemented hemolysin A as a model to investigate the protein‐structure paradigm within beta‐helix structure‐function. Hemolysin A belongs to the Two‐Partner Secretion pathways; functioning in an energy independent manner where the A‐component is secreted in B‐dependent fashion. In the secreted form, HpmA functions as a robust hemolysin. Past results shown that truncated form of HpmA harbors a beta‐helix structure and facilitates dose‐dependent, uni‐directional and biphasic template‐plate assisted hemolysis. In order to better understand the HpmA structure‐function relationship we have created a set of site‐directed mutants, where cysteine residues have replaced serine 99 and asparagine 100. The double mutant harboring cysteines, S99CN100C, has been expressed, purified and characterized via hemolytic activity measurements and size exclusion chromatography coupled to multi‐angle light scattering. The results indicate that engineered cysteine residues inhibited template‐assisted hemolytic activity and established a disulfide bonded macromolecular assembly. These results may allow a more general model of beta‐helix directed protein aggregation to be developed for virulence factor activation and neurodegenerative propagation. NSF (RUI) awards MCB0744754 and MCB1050435 supported the research.
Role of p97 AAA ATPase in the autophagic‐lysosomal degradation of hepatic CYP2B1
Authors: Acharya Poulomi , Correia Maria Almira
Published: June 2021

CYP2B1 is a phenobarbital‐inducible hepatic cytochrome P450 that is turned over via autophagic‐lysosomal degradation (ALD). We have previously shown that the ubiquitin‐dependent proteasomal degradation of hepatic CYPs 3A requires endoplasmic reticulum (ER)‐membrane extraction by the p97 AAA (ATPase involved in a variety of different cellular activities) ATPase. We now report that lentiviral shRNA‐mediated p97 knockdown (91%) in rat hepatocytes cultured in a collagen‐matrigel sandwich resulted in a 5‐fold increased CYP2B content. Following p97 knockdown a 10‐ and 14‐fold stabilization of the parent and high molecular mass K63‐ubiquitinated CYP2B species respectively, is further observed upon treatment with the ALD‐inhibitors (I) 3‐methyladenine (a suppressor of autophagosome formation) and NH4Cl (an established lysosomal inhibitor). S35‐pulse‐chase followed by immunoprecipitation 24 h later verified that CYP2B degradation was indeed impaired upon p97 knockdown. Significant accumulation of CYP2B1 in the microsomal but not cytosolic fraction on ALD‐I treatment indicated a near complete impairment of its ER to cytosol extraction under these conditions. We document for the first time that p97 is also essential for CYP2B ALD. Our findings thus support the growing number of reports implicating p97 in autophagy.
Functional Roles for The Proteasome in Retro‐translocation of Polytopic Membrane Proteins for ERAD
Authors: Smith Nathan James , Adle David , Lee Jaekwon
Published: June 2021

The endoplasmic reticulum‐associated degradation (ERAD) system, known for its role in quality control of secretory proteins, is responsible for regulating Pca1, a cadmium‐extruding P‐type ATPase that plays a major role for cadmium detoxification in yeast Saccharomyces cerevisiae. Pca1 contains an N‐terminal, cadmium responsive degron, which targets it for degradation in the absence of cadmium to the proteasome. The mechanism by which the integral membrane protein Pca1 is removed from the ER for degradation by the cytoplasmic proteasome is unknown. Here we define the roles for the molecular factors involved in ERAD in dislodging Pca1 from the ER membrane. Pca1 is stabilized in cells lacking Doa10 E3 ligase and Ccd48 AAA‐type ATPase activity in ERAD. Unexpectedly, inhibition of proteasome activities results in accumulation of full‐length Pca1 in the ER membrane with no significant detection in the cytosol, indicating a role for the proteasome in efficient removal of Pca1 from the membrane. Experiments also display a physical interaction between the proteasome and Pca1 in an ubiqutinylation dependent manner. Analysis of another membrane bound ERAD substrate, STE6*, displays a similar phenomenon. Support provided by NIH 5RO1ES016337
Role of ADP‐ribosylation factor domain protein 1 (ARD1/TRIM23) on lysosome biogenesis and function
Authors: Kang Gi Soo , Cates Krystal , Pacheco-Rodriguez Gustavo , Meza-Carmen Victor , Daniels Mathew , Connelly Patricia , Moss Joel , Vaughan Martha
Published: June 2021

ADP‐ribosylation factor‐domain protein 1 (ARD1/TRIM23) is a 64‐kDa multi‐functional protein containing an N‐terminal E3 ubiquitin ligase sequence, a C‐terminal GTPase (ADP‐ribosylation factor, ARF) domain, and an internal GTPase‐activating region. ARD1 has been implicated in the regulation of innate immunity, as well as the internalization and degradation of growth factor receptors, but biological functions associated with its intracellular localization in lysosomes and Golgi, remain unknown. Therefore, we compared the lysosomal proteome of livers from ARD1‐null (KO) and control mice. Lysosome‐associated membrane proteins (LAMPs) 1, 2, and 3 were significantly more abundant in the livers of KO mice than those of wild‐type (WT) mice. Levels of LAMP2 in mouse embryonic fibroblasts (MEFs) from ARD1‐KO mice were also higher than those from WT mice. Electron microscopic analysis of mouse liver revealed a larger number of primary and secondary lysosomes in KO than WT mice. In addition, the mean sizes of the primary and secondary lysosomes appeared smaller in KO liver samples than in WT. These findings suggest that ARD1 may have a role in lysosome function and warrant further investigation of ARD1 participation in lysosome biogenesis and/or autophagy.
Tripartite motif protein 23 (TRIM23) regulates degradation of epidermal growth factor receptor (EGFR)
Authors: LE KANG , Pacheco-Rodriguez Gustavo , Moss Joel , Vaughan Martha
Published: June 2021

Tripartite motif protein 23 (TRIM23), also known as ADP‐ribosylation factor‐domain protein (ARD) 1, is a multi‐domain 64‐kDa protein that contains ADP‐ribosylation factor, GTPase‐activating protein, and E3 ubiquitin ligase sequences. That EGFR levels were higher in ARD1−/− than in wild type MEFs was known, but the mechanism responsible remained unknown. To explore that question, we used co‐immunoprecipitation (co‐IP) to evaluate possible interactions of ARD1 and EGFR. Endogenous ARD1 and EGFR in HeLa cells were each immunoprecipitated by antibodies against the other, as was cytohesin‐1, the only known activator of the ARD1 ARF domain. Overexpression of ARD1, intended to enhance the interaction between ARD1 and EGFR, decreased the total cell EGFR. We then found that EGFR levels were increased after ARD1 depletion in HeLa cells. It appears that the interaction of ARD1 and EGFR can be important in regulation of EGFR degradation, and thereby the EGFR content of HeLa cells as well as MEFs.
Separation of proinsulin conversion intermediates
Authors: Mackin Robert Brian
Published: June 2021

The processing of proinsulin (PI) to its final product, insulin, involves three different proteases (proprotein convertase 1 (PC1), PC2 and carboxypeptidase E) and a number of possible cleavage intermediates. The two major intermediates, des‐(31,32)‐PI and des‐(64,65)‐PI are of interest as potential biomarkers for predicting diabetes and as substrates for examining the specificity of prohormone processing. Unfortunately, these intermediates are not commercially available and they are extremely difficult to purify because they differ from the precursor by the absence of two basic amino acids, and from each other only in the identity of the two missing residues.

We have combined trypsin/carboxypeptidase B digestion to generate the intermediates, with anion‐exchange chromatography to separate the reaction products. We were able to obtain three peaks of PI‐related material. Each of these peaks was characterized by performing reduction and alkylation of the peptides, followed by reversed‐phase high performance liquid chromatography. Peak 1 contains only the substrate, PI; peak 2 contains only the final products, insulin and C peptide; and peak 3 contains only the two cleavage intermediates, des‐(31,32)‐PI and des‐(64,65)‐PI. While we are continuing our efforts to separate the intermediates from each another, we are currently using the combined intermediates to examine the specificity of PC1 and PC2.
Phosphoinositide 3‐kinase (PI3K) activation and actin dynamics within force sensing anchors are required to stabilize VLA‐4 integrin‐mediated leukocyte adhesion
Authors: Rullo Jacob , Becker Henry , Hyduk Sharon , Cybulsky Myron
Published: June 2021

During inflammation, leukocytes modulate α4β1 (VLA‐4) integrin avidity in order to rapidly form stable nascent adhesive contacts to VCAM‐1‐expressing endothelial cells and resist detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization under conditions of fluid forces remains poorly understood. We report that mechanical forces imparted by flow induce formation of upstream tension‐bearing anchors attached to the VCAM‐1‐coated surface and Rac, but not Cdc42, small GTPase‐dependent polymerization of F‐actin within these structures. These structures are critical for adhesion stabilization, since disruption of actin polymerization dramatically inhibits VLA‐4‐dependent resistance to detachment, but does not affect VLA‐4 expression, affinity modulation, clustering or constitutive linkage to F‐actin. A key mediator of force‐induced Rac activation and actin polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension‐bearing anchors and blockade of PI3K or deficiency of PI3Kgamma isoform reproduced the adhesion defect produced by inhibition of actin polymerization. Our results implicate a novel mechanism in the leukocyte adhesion cascade necessary for stabilization of nascent adhesive contacts.

Research supported by a CGS CIHR Doctoral Award.
Endothelial cell IQGAP1 is an important regulator of the transendothelial migration of leukocytes
Authors: Sullivan David P. , Muller William A.
Published: June 2021

The movement of leukocytes across the endothelium (transendothelial migration, TEM) into tissue is a complex process that is critical for both beneficial and deleterious inflammation. Our lab recently identified a novel endothelial cell compartment, (the Lateral Border Recycling Compartment, LBRC) that delivers critical membrane proteins to surround the migrating leukocyte and is required for TEM. Despite its importance in inflammation, the protein composition of the LBRC is largely unknown. Using biochemistry and subcellular fractionation we recently identified IQGAP1 as a protein associated with the LBRC. Our data indicate that IQGAP1 is required for TEM and is likely involved with regulating LBRC function during TEM. siRNA mediated knockdown of IQGAP1 expression in endothelial cells significantly impairs their ability to support TEM without altering the structure or size of the LBRC. Furthermore, IQGAP1 appears to be recruited to the site of transmigration along with the LBRC. These data suggest that IQGAP1 is involved in the regulation of LBRC trafficking during TEM. This research was supported by F32 AI084454 to DPS and R01 HL046849, R37 HL064774, and R21 HL102519 to WAM.
The effect of Peroxiredoxin 6 (Prdx6) mutation at its active site on its interaction with piGST
Authors: Zhou Suiping , Lien Yu-Chin , Shuvaeva Tea , DeBolt Kristine , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Prdx6, a 1‐Cys member of the selenium‐independent peroxiredoxin superfamily, has both peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of recombinant Prdx6 mediated by πGST is required for the peroxidatic catalytic cycle (PNAS 101: 3780, 2004). In a previous abstract, we reported that transfection with a plasmid construct expressing πGST into MCF7, a cell line that lacks endogenous πGST, significantly increases the lipid peroxidase activity in the cells. A dramatic increase in the interaction between Prdx6 and πGST in cells upon their treatment with tert‐butyl hydroperoxide was shown by the Duolink Proximity Ligation Assay (DPLA). To examine the role of the peroxidase or PLA2 active site amino acids of Prdx6 on interactions with πGST, plasmids expressing mutated Prdx6 (C47S or S32A) were transfected into Prdx6 null mouse pulmonary microvascular endothelial cells. Interaction between the 2 proteins as shown by DPLA was abolished by mutation at the active site for peroxidase activity (C47), but mutation of the active site for PLA2 activity (S32) had no effect. These data are consistent with the hypothesis that oxidation of the catalytic cysteine in Prdx6 is required for its interaction with πGST.
Effect of an added spacer on the ‘double lockdown’ inhibition of kinesin‐1 by tail domains
Authors: Hackney David Daniel , Yeo Young Woon
Published: June 2021

Kinesin‐1 is a motor protein that produces processive movement along microtubules using the energy of ATP hydrolysis. Under physiological conditions, kinesin‐1 is autoinhibited by folding at a central hinge to allow the tail domains to bind to the head (motor) domains. Even though both the tails and heads are dimeric, inhibition is produced by binding of only one tail domain to a dimer of heads. The recent determination of the structure of a head‐tail complex by X‐ray crystallography (Kaan, et al., Science 333, 832 (2011)) indicated that a single tail domain formed a bridge between two head domains, effectively cross‐linking them. This suggested a ‘double lockdown’ mechanism in which ADP release is inhibited because the coupled undocking of the neck linker cannot occur while maintaining both the cross link between heads at the coiled‐coil neck and the new cross link provided by the tail domain. Insertion of flexible spacers at the start of the coiled‐coil domain should allow greater relative movement of the two head domains and make it more difficult for tails to tie them down with an additional cross link. We have tested a series of single amino acid substitutions and inserts and deletions and find only small differences in the affinity of tails. Long spacers of 6 and 12 amino acids, however, decrease affinity of heads for tails by 13 and 34‐fold respectively.

Supported by NIH grant 1R01NS058848.
Mutational evidence for a substrate activation site in phosphoenolpyruvate carboxylase
Authors: Halaby Steve , Grover Scott D
Published: June 2021

The interaction of phosphoenolpyruvate carboxylase (PEPc) with its substrate PEP is unusual because PEP serves as both substrate (as Mg‐PEP complex) and allosteric activator (as free, uncomplexed PEP). Based on results from mutational analysis and the location of these residues within the same pocket on the surface of maize phosphoenolpyruvate carboxylase, we propose that the residues R753, N755, N942, and I956 contribute to the substrate activation site. Although they are not located in the enzyme's active site, modification of any of these residues via site‐directed mutagenesis significantly reduces the enzyme's affinity for the substrate PEP. Using the program Computed Atlas of Surface Topography of proteins, it was determined that pocket 217 houses these four residues in addition to R232 which, based on previous mutational analysis, is also believed to be involved in substrate activation. Mutations R753E and N755A displayed a 9‐fold increase and a 3‐fold increase, respectively, in the Km. R753E displayed a 3 fold reduction in the Vmax, while N755A only showed a one third reduction. N942D showed a two‐fold increase in Km and roughly a 1/3 reduction in Vmax. Preliminary studies of I956F showed a 52‐fold loss of affinity for substrate. These results suggest that these five residues within pocket 217 are critical to the activation of PEPc by its substrate. This work was supported by the NIH MBRS‐RISE grant GM61331.
Potassium channels as operators of alveolar epithelial repair
Authors: Girault alban , Privé Anik , Trinh Nguyen Thu Ngan , Bardou Olivier , Maillé Emilie , Brochiero Emmanuelle
Published: June 2021

A common feature of inflammatory lung diseases such as acute lung injury and acute respiratory distress syndrome (ARDS) is extensive damage and remodeling of alveolar epithelium. A better understanding of determinants of alveolar regeneration is thus necessary to develop strategies able to restore alveolar integrity. Recent studies have shown that K+ channels (KCh) are key components of cell proliferation and migration processes, necessary for tissues repair. We therefore postulated that KCh play a role in respiratory epithelial repair.

Using a wound‐healing assay, we first showed that glibenclamide and clofilium, inhibitors of KATP and KvLQT1 channels, decrease repair rates of A549 and primary alveolar ATII cell monolayers and their inhibitory effects are additive. The role of KvLQT1 and KATP was then confirmed by a molecular approach using specific siRNAs. On the contrary, KvLQT1 and KATP activators significantly stimulate the wound repair. We also evaluated the influence of KCh on cell growth and we showed that clofilium dose‐dependently inhibits alveolar cell growth and induces an accumulation of cells arrested in G0/G1 phase.

In conclusion, we demonstrated that KCh, particularly KvLQT1 and KATP, are involved in epithelial repair processes and should be identify as novel targets to promote alveolar repair necessary to ARDS resolution.

Project funded by Canadian Institute of Health Research.
The inhibitory effect of an anti‐DPP IV monoclonal antibody 6A3 on the DPP IV/polymeric fibronectin adhesion is most likely due to a conformational change
Authors: Cheng Hung-Chi , Hung Ting-Ting , Liu Ju-Fang , Wu Jun-Yi
Published: June 2021

The epitope of an anti‐rat endothelial Dipeptidyl peptidase IV (DPP IV) monoclonal antibody (mAb) 6A3, capable of inhibiting polymeric fibronectin (polyFN)‐mediated lung‐metastatic cancer cell adhesion to DPP IV, has been mapped to D329KTTLVWN of rDPP IV in close proximity to L311QWLRRI, the putative FN‐binding motif. There has, however, never been direct evidence in confirming this putative motif. For that purpose, the pulldown assay for various DPP IV fragments with polyFN‐conjugated gelatin beads was performed. Surprisingly, fragments harboring L311QWLRRI did not show any significant binding activity, whereas N‐terminal fragments excluding L311QWLRRI strongly bound to polyFN. Since the 6A3 epitope‐equivalent domain is structurally located within the adenosine deaminase (ADA)‐binding region of hDPP IV, binding of 6A3 may mimic that of ADA. Upon superimposition of the rDPP IV structure with that of either hDPP IV or ADA‐bound hDPP IV, the overall structural similarities were extremely high, except one apparent difference within an approximately 10 amino acid‐region between naked DPP IVs and ADA‐bound hDPP IV near their N‐terminals. We therefore re‐hypothesize that 6A3 causes a conformational change of this newly proposed 10 amino acid FN‐binding motif of DPP IV in inhibiting DPP IV/polyFN binding. This study was supported by NSC 99‐2320‐B‐006 ‐028 ‐MY3 and NSC 99‐2627‐B‐006‐023.
Cigarette smoke‐stimulated epithelial‐mesenchymal transition through Src activation
Authors: Forman Henry Jay , Zhang Hongqiao , Liu Honglei , Borok Zea , Davies Kelvin J.A. , Ursini Fulvio
Published: June 2021

Epithelial‐mesenchymal transition (EMT) is implicated in the pathogenesis of lung fibrosis and cancer metastasis, both associated with cigarette smoke (CS). CS, the major cause of lung cancer CS has been reported to promote EMT. Nonetheless, the mechanism whereby CS induces EMT remains largely unknown. We investigated the induction of EMT by CS and explored the underlying mechanisms in the human non‐small cell lung carcinoma (H358) cell line. We demonstrate that CS exposure decreases E‐cadherin and increases N‐cadherin and vimentin, markers of EMT. Pretreatment with N‐acetyl cysteine (NAC), a potent antioxidant and precursor of glutathione, abrogated changes in these EMT markers. Furthermore, CS activated Src kinase (shown as increased phosphorylation of Src at Tyr418) and the Src kinase inhibitor, PP2, inhibited CS‐stimulated EMT changes, suggesting that Src is critical in CS‐stimulated EMT induction. NAC addition to the medium abrogated CS‐stimulated Src activation. Catalase had no effect on Src activation at 5 min of CS exposure, but abrogated it at 5 min. Taken together, these data suggest that CS initiates EMT through Src, which is activated by CS through oxidation or alkylation.
Molecular Analysis of Site‐directed Mutants of Phosphoethanolamine Methyltransferase from the Malaria Parasite Plasmodium falciparum
Authors: Alpert Tara , Lee Soon Goo , Jez Joseph
Published: June 2021

Plasmodium falciparum, which is responsible for malaria in humans, requires rapid division and efficient phospholipid synthesis to survive. Unlike mammals, Plasmodium require phosphoethanolamine methyltransferases (PfPMT) to synthesize phosphocholine for membrane biogenesis. Disruption of the PfPMT gene causes severe growth, reproduction, and viability defects. Thus, this enzyme is a potential anti‐malarial target. To examine the role of residues in the phosphobase binding site and a potentially catalytic histidine in PfPMT, site‐directed mutagenesis, functional assays, and protein crystallography were used. Point mutants targeting the phosphobase binding site and the catalytic histidine were expressed in E. coli and purified by affinity chromatography. Assays of the phosphobase site mutants indicate that electrostatic interactions provided by Arg179 and Lys247 are required for activity. Tyr27, Tyr160, Tyr175, and Tyr181 are also functionally important. Mutation of His132 resulted in proteins with no detectable activity; however, these proteins still bound substrates with affinity comparable to wild‐type. To further investigate the structure of this mutant, the H132A protein was crystallized and a 1.19 Å resolution data set collected. The resulting structure supports a critical role for the histidine in the reaction mechanism. Research funded by the Howard Hughes Medical Institute.
Citric acid cycle and nitrogen assimilation enzyme activities in glutamate dehydrogenase mutants of S. cerevisiae
Authors: Sieg Alex , Trotter Pamela J.
Published: June 2021

In Saccharomyces cerevisiae reductive amination of the citric acid cycle intermediate α‐ketoglutarate to glutamate is the main pathway for nitrogen assimilation. This is catalyzed by the NADP‐dependent glutamate dehydrogenases (Gdh1p & 3p), and the reverse reaction by an NAD‐dependent enzyme (Gdh2p). It has been proposed that, in GDH‐mutant strains, either the proline utilization (PUT) or the glutamine synthetase‐glutamate synthase (GS/GOGAT) pathway is the alternative nitrogen assimilation pathway to the exclusion of the other. Using a GDH‐null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway‐specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). Growth of the null mutant was only slightly impaired on all nitrogen sources tested, confirming other pathways for nitrogen assimilation exist. Enzyme assays point to GS/GOGAT as the alternate pathway when ammonia, glutamine or proline are the sole nitrogen sources. In contrast, GOGAT activity was decreased on urea and Put2p activity was slightly increased. These results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH‐dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serve as the sole compensatory pathway. (Supported by NIH grant R15‐GM069372 to P.J.T.)
Evidence of a dual role for adiponectin in liver regeneration following partial hepatectomy
Authors: Correnti Jason , Hoek Jan
Published: June 2021

Adiponectin is an adipokine that has been shown in multiple contexts to be hepatoprotective. In order to investigate adiponectin's role in liver regeneration following partial hepatectomy (PHx) we compared cell cycle kinetics in Adiponectin KO and WT mice. Adiponectin KO mice exhibit ~6h delay in the onset of the cell cycle markers PCNA and cyclin A as well as BrdU incorporation after PHx relative to WT controls. Despite the later onset of cell cycle progression, adiponectin KO mice upregulate PCNA and cyclin A in an accelerated manner relative to WT animals, and by 54 h after PHx there is no detectable difference in remnant liver weight, indicating a compensatory regulatory mechanism in the absence of adiponectin. Pretreating WT animals with the anti‐diabetic drug rosiglitazone raised serum adiponectin levels significantly and this correlated with suppression of cyclin A expression 36 h after PHx. Rosiglitazone treatment had no effect on cyclin A expression in adiponectin KO mice, suggesting that inhibition of regeneration by rosiglitazone requires adiponectin. These findings suggest that adiponectin enhances the onset of liver regeneration but may play an inhibitory role during S‐phase.
Role of unique miRNAs in development of obesity and type 2 diabetes
Authors: Yadav Hariom , Rane Sushil G
Published: June 2021

Obesity is a result of increased energy intake and/or decreased energy expenditure. This energy imbalance varies in different individuals; certain people gain weight more rapidly than others when subjected to similar high calorie diet. However, the etiology and mechanistic insight into these variations are poorly understood. Studying this phenotype in humans is complicated due to variation in diet, physical activity, etc. Therefore, we developed diet induced obese (DIO) and diet resistance (DR) mice to investigate the phenomenon of variable weight gain in response to a high fat diet. C57BL/6J mice were fed high fat diet and bred for multiple generations to derive pure DIO and DR colonies. The DIO and DR mice had similar body weight at birth, but responded differently to HFD (i.e. DIO gained more weight than DR), despite ingesting similar caloric intake. We next inquired the mechanistic reasons for this phenotypic difference. Interestingly, we observed that the body temperature was significantly increased in DR mice, compared to the DIO mice. Consistent with this, we observed increased BAT, muscle and mitochondria specific genes and decreased WAT specific genes in the white adipose tissue (WAT) of DR mice. Considering these mice are genetically similar, we hypothesized that epigenetic modification, such as miRNAs, may play a role in the phenotype diversity. miRNAs are small oligonucleotides that regulate gene expression at the transcriptional and translation levels. We thus profiled expression levels of miRNAs in the WAT of DIO and DR mice. Interestingly, we observed that 17 miRNAs were upregulated, 8 miRNAs were down regulated, while few miRNAs were switched on or off depending on whether the WAT was derived from DIO or DR mice. These data suggest that miRNAs are differentially expressed in the context of adiposity. Further work is in progress to understand the molecular targets of these miRNAs and to definitively ascertain whether change in miRNA expression is an early and causative event in the pathogenesis of obesity
Gender specific expression of myo‐inositol‐3‐phosphate synthase in Drosophila melanogaster
Authors: Batugedara Gayani D. , Eldon Elizabeth D. , Klig Lisa S.
Published: June 2021

myo‐Inositol, an essential metabolite, plays an important role in development, osmoregulation, and diseases such as Alzheimer's, autism, Down syndrome, bipolar disorder and spinal cord defects. myo‐Inositol‐3‐phosphate synthase (MIPS) converts glucose‐6‐phosphate to myo‐inositol‐3‐phosphate. In Drosophila melanogaster, MIPS has a computationally determined MW of 62kDa and is 59% identical (72% similar) to the human MIPS protein. Initial experiments revealed the molecular structure of the native MIPS protein in D. melanogaster to be a tetramer. MIPS RNA has been shown to be highly expressed in the fly head, hindgut, ovary and testis. Western blot analyses using crude lysates of male and female wild‐type whole flies, heads, gonads and carcasses revealed multiple isoforms of the D. melanogaster MIPS protein. Moreover, gender specific differential expression of the approximately 63kDa, 62kDa and 40kDa MIPS isoforms is apparent. Multiple isoforms of this protein have been demonstrated in other organisms, however this is the first demonstration of gender specific expression of the MIPS isoforms. These isoforms may be generated by alternate splicing of the MIPS transcript. In silico experiments demonstrate the existence of cDNAs that could encode some of the MIPS isoforms. These studies contribute to understanding the role of inositol synthesis in development and neurological disorders.
When the close relationship between function and substrate metabolism in ex vivo working mouse hearts goes beyond differences in genetic background
Authors: Vaillant Fanny , Lauzier Benjamin , Poirier Isabelle , Gélinas Roselle , Frayne Isabelle Robillard , Thorin Eric , Rosiers Christine Des
Published: June 2021

Transgenic mice are valuable study model for cardiovascular research. The choice of appropriate control is, however, crucial given the impact of mouse genetic background on various parameters, including cardiac function. In this study, we tested for a link between the cardiac functional and metabolic phenotype of 5 different mouse strains (Bl6J, Bl6CR, SJL, 129SV and ATX). We used our model of working hearts perfused ex vivo with physiological concentrations of 13C‐labeled carbohydrates (CHO) and fatty acids (FA) to assess flux through energy producing pathways. Results reveal marked differences in most functional and metabolic flux parameters measured amongst all control strains. Most striking was the 129SV heart, which showed a better ex vivo function and a shift from FA to glucose oxidation. Beyond all these variations, we found significant correlations between specific metabolic flux and functional parameters, suggesting the role of i) glycolysis as a determinant of cardiac work (cardiac power: r=0.54; dP/dtmax; contractility: r=0.43), ii) CHO oxidation of heart rate (r=0.51) and stroke volume (r=−0.72), and iii) FA oxidation of MVO2 (r=0.70) and coronary flow (r=0.64). Our results highlight a close link between specific functional and metabolic flux parameters in mouse hearts, which goes beyond differences in genetic background and ought to be considered for data interpretation. Supported by the CIHR.
The underlying mechanisms by which estrogen regulates energy metabolism and body composition
Authors: Ezzat-Zadeh Zahra , Dodge Bonnie G , Elam Marcus , Feresin Rafaela , Browne Jenna , Kim Jeong-Su , Arjmandi Bahram H
Published: June 2021

Ovariectomized rats gain excess body weight (BW) even when they are pair‐fed to sham animals. This higher BW, contrary to common belief, does not result in higher bone mineral density (BMD). The purpose of the present study is to evaluate the potential mechanisms underlying these paradoxical observations. Eighteen 10‐mo old female Sprague‐Dawley rats (n=6/group) were divided into either a sham‐operated group (sham) or two ovariectomized groups receiving 17β‐estradiol at the dose of 10μ/kg BW (ovx+E2) or solvent vehicle (ovx) twice per week. At the present time (100 days post‐surgery), animals have been scanned using DXA for total BMD, fat mass (FM) and lean mass. Currently, the mean BW of the ovx group without E2 is significantly higher than the animals in the sham and ovx+E2 groups (sham: 396.80 ± 13.51 vs. ovx: 437.83 ± 13.25 g). This significant increase in BW is related to a significant increase in mean total FM (sham: 211.0 ± 11.57 vs. ovx: 248.66 ± 11.20 g). DXA scans indicated a trend in BMD loss in the ovx control group (P=0.9). Animals will be sacrificed once the loss of mean total BMD in ovx rats reaches a significant level (P<0.05). This study intends to investigate the mechanisms by which ovarian hormone deficiency alters energy metabolism by assessing anabolic and catabolic factors as well as oxidative and inflammatory loads in selected tissues such as muscle and bone.
Avocado oil improves dyslipidemia and increase mitochondrial resistance to oxidative stress in streptozotocin (STZ)‐induced diabetic rats
Authors: Ortiz-Avila Omar , Sámano-García Carlos Alberto , Calderón-Cortés Elizabeth , Clemente-Guerrero Mónica , Noriega-Cisneros Ruth , Saavedra-Molina Alfredo , Cortés-Rojo Christian
Published: June 2021

Increased mitochondrial oxidative damage is a feature of diabetes. It has been demonstrated that the enrichment of mitochondrial membranes with low unsaturated‐fatty acids may confer resistance against deleterious effects of ROS on respiratory chain complexes (RCC). Avocado oil is rich in oleic acid, which has beneficial properties in lipid metabolism. Since diabetes is also characterized by dyslipidemia, the aim of this work was to explore if avocado oil may improve dyslipidemia and increase resistance of RCC to ROS. The daily administration of avocado oil to STZ rats at a dose equivalent to 3.5g oleic acid normalized cholesterol and triglycerides levels after 90 days. The activity of complexes III and IV from liver mitochondria of STZ rats was inhibited in 92 and 72% by 25 μM Fe2+ and avocado oil increase their resistance by 67 and 27%, respectively. Fe2+‐induced lipoperoxidation was lowered in STZ rats that consumed avocado oil. These results suggest that avocado oil consumption is beneficial at systemic and subcellular levels probably due to its high oleic acid content. The authors appreciate the financial support from CONACYT (130638 to CCR), PROMEP (PTC‐266 to CCR) and CIC‐UMSNH (2.16 to ASM)
ARH3 catalyzes degradation of mitochondrial matrixaccumulated Poly (ADP‐ribose)
Authors: Mashimo Masato , Niere Marc , Agledal Line , Dölle Christian , Kasamatsu Atsushi , Kato Jiro , Moss Joel , Ziegler Mathias
Published: June 2021

Poly (ADP‐ribose) (PAR) metabolism, which is mainly mediated by members of the Poly (ADP‐ribose) polymerase (PARP) and Poly (ADP‐ribose) glycohydrolase (PARG) families, is involved in several cellular pathways including DNA stability, apoptosis, and mitosis. PARG, initially identified as a PAR degrading‐enzyme, exists as multiple isoforms with different subcellular localization, resulting from alternative splicing of the single gene. ADP‐ribosylhydrolase 3 (ARH3) also has PAR‐degrading activity. The enzymes participating in a putative PAR metabolism in mitochondria are still unknown.

An alternatively spliced, small human PARG isoform (PARG55) and ARH3 are located in mitochondria owing to a mitochondrial targeting sequence at their N‐terminus. Using a modified PARP1 construct to produce PAR selectively in mitochondria, ARH3‐deficient cells were shown to have more mitochondrial PAR accumulation than wild‐type cells. Overexpression of ARH3 decreased mitochondrial PAR content, while overexpression of PARG55 had no effect. PARG55 was shown to lack exon5, which is essential for PAR‐degrading activity. These data suggest that ARH3 rather than PARG55 is the primary enzyme involved in catalyzing the degradation of PAR in mitochondria.
Lysyl Oxidase Propeptide Promotes Adipogenesis through Inhibition of FGF‐2 Signaling
Authors: Griner John , Zhu Meijun , Rodgers Carl , Du Min
Published: June 2021

Lysyl oxidase (LOX), an extracellular enzyme, catalyzes the oxidative deamination of lysine residues in collagen and elastin. LOX is synthesized as an inactive 50kd prepro‐protein and secreted where it is cleaved into an active 32kd enzyme and an 18kd propeptide (PP). Fibroblast growth factor‐2 (FGF‐2) leads to the expansion of fibrogenic progenitor cells, and it is reported that PP inhibits FGF‐2 signaling. We hypothesized that PP regulates adipogenesis via inhibition of FGF‐2 signaling. 3T3‐L1 cells were treated with FGF‐2 and adipogenesis was evaluated by expression of PPARγ and Oil‐Red O staining. PP was cloned into pFLAG expression vector and expressed in DH5α cells. 3T3‐L1 cells were allowed to differentiate with PP and adipogenic medium. Recombinant PP inhibited FGF‐2 signaling by up‐regulation of the adipogenic marker PPARγ, whereas the phosphorylation of ERK1/2 and AKT, two key pathways activated by FGF‐2, was reduced. Separately, we cloned PP into pCMV mammalian expression vector, and made a point mutation resulting in an Arg152 to Gln substitution, which is expected to diminish its inhibitory effect on FGF‐2 signaling. Similarly, transfection with WT PP increased expression of PPARγ and C/EBPα, whereas the PP point mutation diminished the adipogenic promoting potential of LOX‐PP. For the first time, our data show that PP enhances adipogenesis, an effect mediated by inhibition of FGF‐2 signaling.
Flipping out! Overexpression and Localization of a Novel Flippase, ATP10C
Authors: Hurst Sarah Elizabeth , Paulusma Coen , Dunlap John , Biggerstaff John , Dhar Madhu
Published: June 2021

Atp10c, located on mouse chromosome 7, is a putative phospholipid translocase or flippase which encodes for a type IV P‐type ATPase. Based on numerous studies in yeast and in mice, we hypothesize that ATP10C due to its flippase nature plays a role in diet‐induced obesity and diabetes; mice heterozygous for Atp10c display this phenotype. For our investigation, we decided to look at Atp10c/ATP10C in order to gleam information about its biological role and/or function. In order to do this, an ATP10C‐GFP plasmid construct was commercially obtained and transfected into HEK293T cells. Immunoblots with these cells revealed band at the expected molecular weight, about 165 kD. When the fusion protein was transfected into UPS‐1 cells, we discovered that unlike other type IV P‐type ATPases, ATP10C does not require chaperone proteins, CDC50A or 50B, for function, nor does it act on phosphatidylserine in the lipid bilayer. Next, immunolocalization with known protein markers by fluorescence microscopy was employed and we were able to show that the ATP10C‐GFP fusion protein localized to the plasma membrane and appeared as a punctate pattern in the nucleus of HEK293T cells, thus strengthening its role in protein trafficking and cell signaling pathways. Funding for this research was provided by the American Diabetes Association.
Progression of Obesity‐Related Heart Dysfunction in Two Rat Models
Authors: Martins Ricardo Fonseca , Gorr Matthew William , Youtz Dane Juel , Teich Steven , Wold Loren Eugene
Published: June 2021

The prevalence of obesity in the US has increased substantially over the past decades, with upwards of 33.8% of the population obese and 68% overweight. In childhood, 11.7% – 14.6% of preschoolers in developed countries and 16.4% of adolescents in the US are considered obese. Several animal models have been utilized to study the impact of obesity on the heart. In rats, the most prevalent models are the Zucker Fatty rats (ZF), a genetically modified rat with a leptin receptor deficiency, and normal Sprague Dawley rats (SD) fed a high fat diet. In the present study, we examined echocardiographic indices for each model in order to evaluate heart function. Values for IVRT/HR were significantly higher in ZF rats compared to Zucker Lean rats (0.058±0.010 ms/bpm vs, 0.045±0.009 ms/bpm, p<0.05) at 19 weeks of age, however the values for SD rats were not significantly different after eating a high fat diet for 10 weeks. E/A ratio was not significantly different in ZF rats compared to lean controls, however was significantly decreased in SD rats fed a high fat diet compared to rats fed a normal diet (0.98 vs. 1.11; p<0.05). Diastolic dysfunction was evident in both models, although the values for the ZF rats indicated less relative diastolic impairment than the SD rats fed a high fat diet. Continued work will examine the progression and mechanistic differences in these models, and their relevance to human obesity.
AIMing at Metabolic Syndrome: Towards development of novel therapies for modern metabolic diseases via macrophage‐derived AIM
Authors: Miyazaki Toru , Kurokawa Jun , Arai Satoko
Published: June 2021

Metabolic syndrome is a cascade of metabolic diseases starting with obesity and progressing to atherosclerosis and is often fatal because of serious cardiovascular problems such as heart/brain infarction and hemorrhage. Accumulating evidence has revealed a critical involvement of inflammatory responses triggered by lesional macrophages in its pathogenesis. Importantly, we found that macrophages are associated with the progression of these diseases not only in the induction of inflammation but also in the production of apoptosis inhibitor of macrophage (AIM), which we initially identified as a soluble factor expressed by macrophages. At atherosclerotic plaques, AIM is highly expressed by foam macrophages and inhibits apoptosis of these cells. This results in the accumulation of macrophages, causing inflammatory responses within the lesion, and ultimately disease progression. In adipose tissue, macrophage‐derived AIM is incorporated into adipocytes through CD36‐mediated endocytosis, thereby reducing the activity of cytosolic fatty acid synthase. This unique response stimulates lipolysis, resulting in a decrease in adipocyte size, which is physiologically relevant to the prevention of obesity. The lipolytic response also stimulates inflammation of adipocytes in association with the induction of metabolic disorders subsequent to obesity. Thus, AIM is involved in the progression of metabolic syndrome in both advancing and inhibitory fashions. Regulation of AIM could therefore be therapeutically applicable for metabolic syndrome.