bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the Antimicrobial Activities from Secondary Metabolites in Plant Families (Boraginaceae, Fabaceae, Lamiaceae, and Lauraceae)
Authors: Compean Kassandra , Ynalvez Ruby
Published: June 2021

For several decades, there has been an increased interest in the antimicrobial activities of different extracts obtained from traditional medicinal plants. There are more than 20,000 species of plants used in traditional medicines. Drugs can be derived from natural products, which are usually secondary metabolites and their derivatives. The increased prevalence of antibiotic‐resistant bacteria emerging from the extensive use of antibiotics may render the current antimicrobial agents insufficient to control at least some bacterial infections. Therefore, the search for new antimicrobial agents is an important line of research.

The objective of this study was to determine the antimicrobial activities of plant extracts from Sassafras albidum, Ehretia anacua, Melissa officinalis, Eysenhardtia texana, and Melissa odorata. The ethanol and aqueous extracts were prepared for each of the five plant species and tested against Staphylococcus aureus and Pseudomonas aeruginosa. To test the antimicrobial activity, the disk diffusion method was performed using streptomycin as a positive control. Zone of inhibitions were measured using a vernier caliper. Results showed an antimicrobial potential for the Ehretia anacua (family: Boraginaceae) ethanol extract against Staphylococcus aureus; therefore, it was tested for more antibacterial and antifungal activities, using acetone, ethanol, and diethyl ether extracts. Ehretia anacua was also subjected to qualitative and quantitative phytochemical analysis to determine the exact constituents exhibiting these biological activities.
Generation and Characterization of a Stable Inducible O‐GlcNAcase Cell Line
Authors: Maduka Austin , Groves Jennifer , Zachara Natasha
Published: June 2021

O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification that is added and removed from nuclear, cytoplasmic, and mitochondrial proteins by the O‐GlcNAc transferase (OGT) and the O‐GlcNAcase (OGA), respectively. Suggesting that O‐GlcNAc plays a role in cell survival, elevated O‐GlcNAcylation protects cells from many stressors including myocardial ischemia reperfusion injury. To target O‐GlcNAc as a potential cardioprotective therapeutic, we need a better understanding of the mechanisms by which O‐GlcNAc modulates protein function and how OGT and OGA are regulated during times of injury. Recent studies in our laboratory aim to understand the regulation of OGA by its oxidative stress‐dependent binding partners. As existing antibodies do not effectively purify OGA, the goal of this project is to generate and characterize U2OS (human osteosarcoma) and H9C2 (rat myoblast) stable cell lines overexpressing wild type or catalytically dead His6‐tagged OGA. As constitutive overexpression of OGA is toxic, protein expression will be controlled using a tetracycline‐on system (Invitrogen, pT‐Rex). First, we will determine the expression, localization, and activity of His6‐OGA in these cell lines. Ultimately, we will perform a large‐scale isolation of His6‐OGA from oxidatively stressed cells to validate OGA's interactome. These cells lines will be vital for performing physiological studies that will provide molecular insight into the regulation of OGA and the role of O‐GlcNAc in cell survival and cardioprotection.
Redox Control of Protein Arginine Methyltransferase 1 (PRMT1) Activity
Authors: Hevel Joan , Morales Yalemi , Nitzel Damon , Price Owen , Gui Shanying
Published: June 2021

Elevated levels of asymmetric dimethylarginine (ADMA) correlate with risk factors for cardiovascular disease. ADMA is generated by the catabolism of proteins methylated on arginine residues by protein arginine methyltransferases (PRMTs), and is degraded by dimethylarginine dimethylaminohydrolase (DDAH). Reports have shown that DDAH activity is down regulated and PRMT1 protein expression is upregulated under oxidative stress conditions, leading many to conclude that ADMA accumulation occurs via increased synthesis by PRMTs and decreased degradation. However, we now report that the methyltransferase activity of PRMT1, the major PRMT isoform in humans, is impaired under oxidative conditions. Oxidized PRMT1 displays decreased activity, which can be rescued by reduction. Mutagenesis studies demonstrate that the oxidation event involves two cysteine residues on PRMT1. We demonstrate a hydrogen peroxide concentration‐dependent inhibition of PRMT1 activity that is readily reversed under physiological H2O2 concentrations. We additionally show that PRMT1 purified from oxidatively challenged human cells displays impaired activity compared to PRMT1 purified from healthy human cells. Our results challenge the unilateral view that increased PRMT1 expression necessarily results in increased ADMA synthesis, but rather demonstrate that enzymatic activity can be regulated in a redox‐sensitive manner. Our findings suggest that modulation of protein arginine methylation could play a large role in the oxidative stress response.
Defining the Mechanism of O‐GlcNAc Signaling in Stress‐Induced Autophagy and Cardioprotection
Authors: Fahie Kamau , McCartin Michael , Wolfgang Michael , Zachara Natasha
Published: June 2021

Ischemic heart disease causes global morbidity and mortality. Therapies for treating myocardial infarction and ischemia reperfusion injury (IR/I) have remained elusive. Current research demonstrates that the dynamic modification of intracellular proteins by O‐linked b‐N‐acetylglucosamine (O‐GlcNAc) confers cardioprotection in models of IR/I. However the molecular mechanism(s) of O‐GlcNAc mediated cardioprotection are unknown. In this study we investigated the hypothesis that O‐GlcNAc regulates the cardioprotective process of autophagy. Utilizing destabilizing domain technology, we generated inducible constructs that regulate the protein levels of the enzymes that catalyze the addition and removal of O‐GlcNAc, O‐GlcNAc transferase (OGT) and O‐GlcNAcase, to facilitate conditional control of cellular O‐GlcNAcylation. We show that enhancing global O‐GlcNAcylation increases autophagic flux, whereas reducing O‐GlcNAc levels suppresses peroxide‐induced autophagy. In addition, AMP‐activated protein kinase (AMPK) signaling, which promotes autophagy, is enhanced when O‐GlcNAc levels are raised pharmacologically. As such, we assessed the O‐GlcNAcylation state of pro‐autophagic regulators. AMPK is O‐GlcNAc modified or associates with O‐GlcNAc modified proteins in a stress dependent manner. Together, our data suggests that O‐GlcNAc can regulate numerous points in the autophagic pathway and may promote cardioprotection through the upregulation of autophagy. AHA Award 13POST17100083
Visualization of Bcl‐2 Family Proteins (BIK and BAK) Interactions Using Fluorescence Microscopy
Authors: Kumarjiguda Deepa , Normand Caitlyn , Roecklein‐Canfield Jennifer
Published: June 2021

Kaposi's sarcoma is a skin cancer that results in lesions all over the body. Human Herpesvirus 8 (HHV8) is an oncovirus that causes Kaposi's sarcoma. There has long been a link between virus‐encoded proteins that disrupt host cell signaling pathways and cancer in the human host. Programmed cell death, or apoptosis, is an important cellular pathway that halts unreasonable cell growth and disruption of this pathway can lead to tumor development. The human Bcl‐2 family of proteins is involved in both pro and anti apoptotic signals. KSHV expresses a Bcl‐2 homolog, vBcl‐2, which has been shown to inhibit apoptosis in the host cell by several mechanisms. Previous studies and our work have shown a physical interaction between a human pro‐apoptotic protein, Bik, and the KSHV vBcl2 protein, suggesting this interaction is relevant in the anti‐apoptotic function of the virus. Understanding the functional role of this interaction will help elucidate the complex mechanism used by the virus to escape the normal cell death process. We have analyzed this interaction by look at the binding interface in several mutant BIK proteins and their localization in the cell. These interactions are visualized by using fluorescence microscopy. Determining the location of these host proteins during viral infection and expression of vBcl2 is critical to understanding the role the vBcl2‐hBIK interactions plays in apoptosis.
HCV‐Host Cell Interactions: Role of Ebp‐1 in Hepatitis C virus Replication
Authors: Mishra Priya , Dixit Updesh , Pandey Ashutosh , Pandey Virendra
Published: June 2021

Persistent hepatitis C virus ( HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish chronic HCV infection are poorly understood. Also, the role of specific cellular factors in chronic HCV infection is not well established. We have recently identified a cellular factor ErbB3 binding protein‐1 (Ebp1) that specifically interacts with HCV RNA genome and is inhibitory to HCV replication. Ebp1 is a double stranded RNA binding protein that is a member of a family of proliferation‐regulating proteins. Ebp1 has two isoforms, p42 and p48, which are synthesized from two differently spliced mRNAs from the same gene. Our preliminary results show that overexpression of Ebp1 (the longer as well as the shorter isoform) has a strong negative impact on HCV replication. We also found that Ebp1 binds to the HCV 5′NTR. We established that Ebp1 is mainly localized in the cytosol, a site of HCV replication. Further experiments showed that Ebp1 interacts not only with HCV viral proteins NS5A and NS5B, but also with interferon‐inducible double stranded RNA activated protein kinase PKR, which is critical for cellular antiviral and anti proliferative responses induced by interferon. Taken together our results suggest the critical role of Ebp1 in negatively regulating Hepatitis C virus infection. We propose to further identify the role of PKR in Ebp1 mediated signaling in Hepatitis C virus infection. Elucidation of the role of Ebp1 in activating PKR or vice versa will provide valuable insight into the molecular mechanisms of these interactions and their implication on HCV‐replication and associated pathogenesis.
Pathogenetic Pathways of Aberrant O‐glycosylation of IgA1 in an Autoimmune Disease, IgA Nephropathy
Authors: Novak Jan , Takahashi Kazuo , Suzuki Hitoshi , Yamada Koshi , Reily Colin , Stewart Tyler , Ueda Hiroyuki , Hall Stacy , Moldoveanu Zina , Raska Milan , Julian Bruce , Renfrow Mathew
Published: June 2021

IgA nephropathy (IgAN), a frequent cause of end‐stage renal disease, is an autoimmune disease wherein immune complexes consisting of IgA1 with galactose‐deficient O‐glycans (Gd‐IgA1; autoantigen) and anti‐glycan autoantibodies deposit in the glomeruli and induce renal injury. Serum IgA1 has 3‐6 clustered core 1 O‐glycans, some of which may be deficient in galactose. Patients with IgAN usually have elevated serum levels of Gd‐IgA1. The mechanisms involved in production of Gd‐IgA1 are not fully understood.

Using IgA1‐producing cell lines, we have analyzed the heterogeneity of IgA1 O‐glycosylation and the corresponding biosynthetic pathways. IgA1 secreted by cells from IgAN patients vs. healthy controls had more O‐glycans and more galactose‐deficient sites. These changes were associated with differential expression/activity of key glycosyltransferases, elevated for an initiating enzyme GalNAc‐transferase 14 and for GalNAc‐specific sialyltransferase (ST6GalNAc‐II) and, conversely, decreased for the galactosyltransferase (C1GalT1) and C1GalT1‐associated chaperone Cosmc. These findings were confirmed by siRNA knock‐down.Expression of these enzymes can be affected by cytokines that further enhance the enzyme imbalance, resulting in increased Gd‐IgA1 production.

In summary, Gd‐IgA1, the key autoantigen in IgAN, is produced by IgA1‐secreting cells due to dysregulation of multiple key enzymes. These findings thus provide insight into possible targets for future disease‐specific therapy.

Supported by NIH grants DK078244, DK082753, GM098539 and by a gift from IGA Nephropathy Foundation of America.
Macrophage Liver Kinase B1 Interacts with IKKβ and Inhibits Inflammatory Signaling in Response to LPS
Authors: Liu Zhaoyu , Zou Ming‐hui
Published: June 2021

Liver Kinase B1 (LKB1), a serine/threonine ki nase, is a tumor suppressor and metabolic regulator. Recent data suggest that LKB1 is essential in regulating homeostasis of hematopoietic cells and immune responses. However, its role in macrophages and innate immune system remains unclear. Here we report that macrophage LKB1 inhibits pro‐inflammatory signaling in response to lipopolysaccharide (LPS). LPS‐induced pro‐inflammatory cytokines and pro‐inflammatory enzymes were monitored in bone marrow‐derived macrophages (BMDMs) isolated from myeloid cell‐specific LKB1 knock out mice and their wild type littermate control mice. LPS induced higher levels of pro‐inflammatory cytokines and pro‐inflammatory enzymes in BMDMs from LKB1 KO than those from wild type mice. Consistently, LPS‐induced higher levels of NF‐κB activation in LKB1 deficient macrophages than the wild type. Further, LPS stimulation significantly increased LKB1 phosphorylation at serine 428, which promoted its binding to IKKβ resulting in the inhibition of NF‐κB. Finally, LPS injection caused higher levels of cytokine release and more severe tissue injury in the lung tissues of LKB1 KO mice than those of control mice. We conclude that LKB1 inhibits LPS‐induced NF‐κB activation in macrophages.
Selective Up‐Regulation and Activation of Matrix Metalloproteinase 11 (Mmp11), Non‐Autonomous Cell Death and Pathological Dysfunctions upon Loss of Ran‐binding Protein 2 (Ranbp2).
Authors: Ferreira Paulo , Patil Hemangi , Cho Kyoungin
Published: June 2021

Ranbp2 harbors functional and pathogenic idiosyncratic activities by controlling cell‐type selective, stress‐elicited or disease‐prone substrates. To probe Ranbp2 roles in neurodegeneration, we generated mice with loss of Ranbp2 expression in cone photoreceptor neurons and retinal pigment epithelium (RPE). We found that loss of Ranbp2 promotes the degeneration of the cone photoreceptors and RPE. Further, we found that degeneration of cone photoreceptors promoted the non‐autonomous death of nearby healthy rod photoreceptors. In regards to the RPE, we found that RPE degeneration led to the derangement of the cellular organization of RPE and extrusion of atrophic RPE cells to the subretinal space. Strikingly, these manifestations were followed by the breakdown of the blood‐retinal barrier that was characterized by extravasations of the nearby choriocapillaris bead. The cone and RPE degenerations were accompanied by erosion or disorganization of intracellular structures, subcellular localization of some partners of Ranbp2 and early and selective up‐regulation and activation of Mmp11, but not of any other type of matrix metalloproteinase tested. Finally, we found that functional loss of subset of Ran GTPase‐binding domains (RBDs) of Ranbp2 recapitulated the up‐regulation of Mmp11 and pathologies caused by Ranbp2 loss. Collectively, the findings support that activation of Mmp11 by Ranbp2 is under control of a subset of its RBDs and that Ranbp2‐dependent Mmp11 activation promotes the non‐autonomous death and dysfunction of healthy tissues or cells neighboring damaged cells with Ranbp2 deregulation.
Creating a 3D matricellular environment to promote islet expansion for diabetes therapy – the role of SPARC family proteins
Authors: Viloria Katrina , Ryall Claire , Dandagama Amanda , Asher Sharan , Lhaf Fadel , King Aileen , Jones Peter , Kocher Hemant , Jones Lucy , Hill Natasha
Published: June 2021

Loss of the extracellular matrix following enzyme harvest of islets for transplantation renders them susceptible to a new environment, and is hypothesized to contribute to decreased graft function and survival. The matrix provides structural support for islets as well as mediating growth factor and cytokine interactions. SPARC, or secreted protein acidic and rich in cysteine, is a collagen‐binding matricellular protein that plays a role in matrix assembly and regulates cellular responses to the extracellular environment. Our data shows that SPARC is expressed by stromal cells within islets, and that SPARC expression in these cells is regulated by metabolic factors. Furthermore, SPARC inhibits growth factor signalling in both beta cells and primary islet tissue. Our data suggests that understanding the effect of matricellular proteins will be necessary to create a matrix environment that supports islet expansion. We have developed a model in which matricellular protiens are incorporated into a 3D collagen matrix to test the effect of matricellular proteins on primary islet growth and survival.
Endothelial Deficiency of the Extracellular Matrix Protein CCN1 Alters Developmental and Pathological Angiogenesis
Authors: Chaqour Brahim , Krupska Izabela , Yan Lulu , Lau Lester , Grant Maria , Chintala Hemabindu
Published: June 2021

CCN1 is a secreted cysteine‐rich and integrin‐binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre/Lox system is associated with EC hyperplasia and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate‐early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF‐R2) activation and downstream signaling pathways required for tubular network formation. CCN1 increased the expression of and association between Src homology 2 domain–containing protein tyrosine phosphatase‐1 (SHP‐1) and VEGF‐R2 which leads to rapid dephosphorylation of VEGF‐R2 tyrosine preventing EC hyperproliferation.Concordantly, ectopic expression of CCN1 prevents the exuberant ischemia‐induced retinal neovascularization in an oxygen‐induced retinopathy model by altering the balance of activation of VEGF‐R2 and SHP‐1. These data highlight novel functions of CCN1 as a naturally optimized molecule fine controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.

Supported by NEI‐NIH grant EY022091‐01
miR‐625# and miR‐206 participate to the cellular response to matrix rigidity
Authors: Rio Marc , Dina Christian , Busson Pierre‐François , Bélaadi Néjma , Loirand Gervaise , Guilluy Christophe
Published: June 2021

The extracellular matrix (ECM) affects many aspects of cell growth and behavior. Not only do cells respond to the composition of the ECM, but they also respond to its physical properties. In a seminal paper, Pelham and Wang observed that cells develop large integrin‐based adhesions on rigid surface, whereas on soft substrate adhesions remain immature and small. Since this cellular response to ECM stiffness is central in various aspects of biology, from physiology to disease, many efforts have been directed recently to understand how cells “sense” and respond to the stiffness of their environment. However this remains, so far, an open question. In this study, we investigated the role of micro RNAs (miRNAs) in the cellular response to matrix rigidity. Comparing miRNAs expression in fibroblast cultured on soft (1kPa) and stiff substrates (25kPa), we observed that ECM rigidity induced a decrease in miR‐625# and miR‐206 expression. Interestingly, inhibition of myosin II increased miR‐625# and miR‐206 expression, indicating that cell‐generated tension regulates miR‐206 and miR‐625# expression. Previous work have shown that fibronectin is a direct target of miR‐206, consistent with this we found that miR‐206 expression in cells cultured on stiff substrates decreased fibronectin expression. Additionally, we found that miR‐625# expression decreased cell spreading and survival on rigid ECM. These data indicate that miR‐206 and miR‐625# regulate the cellular response to ECM rigidity. We are currently pursuing this work to identify miR‐625# targets which impact cell adhesion and survival.
A Phosphoglycolate Phosphatase Virulence Factor from Staphylococcus aureus
Authors: Edwards Jasmine , Gehret Austin , O'Handley Suzanne
Published: June 2021

Methicillin Resistant Staphylococcus aureus (MRSA) is the bacteria responsible for staph infections; one of the most prevalent hospital acquired infections. Since its initial discovery, Staphylococcus aureus (S. aureus) has developed multidrug resistance making infection extremely difficult to treat. One avenue of pursuit in identifying new drug targets against staph infections might be found through the study of Haloacid Dehalogenase (HAD) superfamily phosphatases. A HAD phosphatase in S. aureus has been shown to serve as both a virulence factor and possess the ability to dephosphorylate 2‐phosphoglycolate. If not catabolized, 2‐phosphoglycolate accumulates in cells and inhibits triose phosphate isomerase (TPI). In S. aureus, TPI also serves as an adhesion molecule that can bind to host cells via sugar‐side chains. To confirm physiologically that the S. aureus HAD phosphatase is indeed a phosphoglycolate phosphatase (PGPase), the enzyme will be expressed in a PGPase Saccharomyces cerevisiae knockout. We have identified a potential growth phenotype for this PGPase knockout in response to hyperosmotic (1M NaCl) shock compared to wild‐type cells. We hypothesize that the S. aureus PGPase should be able to complement this growth phenotype if it is a true functional ortholog. Studies are currently underway to clone the S. aureus PGPase into a yeast expression vector for subsequent complementation work. This research was supported by an NIH AREA grant, RIT McNair Scholars Program, and RIT NTID and COS FEAD grants.
Chemoresistance‐Induced Epithelial‐Mesenchymal Transition of a Colorectal Cancer Cell Line
Authors: Martino Alice , San KayKay , Daniel Nalini , Ezekiel Uthayashanker
Published: June 2021

Transdifferentiation of epithelial cells to mesenchymal cells is called epithelial‐mesenchymal transition (EMT). EMT is a normal process during embryogenesis, where epithelial cells lose their characteristics and become more motile mesenchymal cells. Invasive and metastatic characteristics of carcinoma cells in primary tumors are mediated by EMT. Specifically, the primary tumor loses cell‐cell adhesion normally mediated by E‐cadherin and attains mesenchymal markers such as vimentin. Also, transitioning cells attain increased intercellular separation and elongated shape with pseudopodia. Studies have shown that chemotherapy and radiation therapy lead to formation of resistant cells by transdifferentiation into EMT. To study chemoresistance‐induced EMT, colon cancer (DLD‐1) cells were treated with increasing concentrations of oxaliplatin (an anti‐cancer drug used to treat metastasized colon cancer) to develop a chemo‐resistant cell line (DLD‐1 OxR). These resistant cells show properties of EMT by morphologic and cell migration measures. Previously, we showed that curcumin, a phytochemical derived from turmeric, inhibited DLD‐1 proliferation: its effect on DLD‐1 OxR suggests inhibition as well. Future studies of DLD‐1 OxR will include characterization of mesenchymal marker expression.
Enhancement of Glutathione Synthesis is Implicated in the Hepatopretective Effects of Silymarin against Acetaminophen
Authors: Kim Young , Kwon Do , Ahn Chul
Published: June 2021

Previous studies suggest that silymarin is capable of modifying the metabolism of sulfur‐containing substances in liver. Silymarin treatment was shown to up‐regulate cysteine β‐synthase while inhibiting cysteine dioxygenase, resulting in elevation of cysteine and its product, glutathione. To determine its pharmacological significance we evaluated the effects of silymarin on acetaminophen (APAP)‐induced hepatotoxicity. Male mice were treated with silymarin (200 mg/kg, po) every 12 h for a total of 3 doses prior to APAP challenge (500 mg/kg, ip). The liver injury was significantly attenuated in the silymarin‐treated mice as determined by elevation of plasma enzyme activities, hepatic protein‐nitrotyrosine adducts, and lipid peroxidation. Depletion of GSH was also relieved. Plasma concentrations of APAP, APAP‐glucuronide or APAP‐sulfate were not changed, but thiol conjugates of GSH such as APAP‐GSH, APAP‐cysteine and APAP‐N‐acetylcysteine were significantly elevated by silymarin. The enzyme activity and protein expression of CYP2E1, 1A2, or 3A were not induced, indicating that the increment in APAP‐thiol conjugates should be attributed to the increased GSH availability by silymarin. GST activity, reduced by APAP, was also recovered. It is concluded that in the mice pretreated with silymarin the increase in GSH generation plays a critical role in the alleviation of APAP hepatotoxicity by enhancing the detoxifying capacity of liver cell against the reactive metabolites generated during metabolic breakdown of this analgesic‐antipyretic.
Mechanism Based Inhibition of P‐glycoprotein
Authors: Brewer Frances , Follit Courtney , McClean Mindy , vogel Pia , Wise John
Published: June 2021

Multidrug resistances (MDR) pose a significant obstacle in the treatment of cancer and other diseases by decreasing the efficacy of therapeutics. MDR is often associated with the overexpression of ABC transporters like P‐glycoprotein (P‐gp) and has been linked to poor outcome in the treatment of cancers. Inhibiting these transporters could reverse the MDR phenotype, but so far clinical trials of inhibitors of P‐gp have only been moderately successful. We are employing a novel mechanism‐based approach in our search for P‐gp inhibitors to identify lead compounds that are specifically targeted at the energy transduction processes of the transporters. Using computational simulations of the catalytic transport cycle, drug docking and biochemical analyses, we identified four compounds capable of inhibiting ATP hydrolysis through interactions with the energy transducing structures in P‐gp. Three of these compounds reversed MDR phenotype in cancer cell culture models. The molecular mechanisms of P‐gp inhibition by these compounds was evaluated using biochemical and biophysical techniques. Kinetic analyses examined the modalities of inhibition and their drug‐like properties. Electron spin resonance spectroscopy using spin‐labeled nucleotides and site‐directed spin labeling evaluated the binding interactions of the inhibitors. Experiments designed to evaluate where in the catalytic mechanism the inhibitors block P‐gp's action are under way. This work is supported by NIH NIGMS [Grant 1R15‐GM094771‐01A1] to PDV and JGW, SMU Research Council, SMU Dean's Research Council, the SMU Center for Drug Discovery, Design and Delivery, and the Communities Foundation of Texas, Dallas.
Isolation of Lysyl Oxidase from Human Umbilical Cord
Authors: Pestov Nikolay , Korneenko Tatyana , Shidlovskaya Natalia , Petrukhin Vasily , Modyanov Nikolay
Published: June 2021

Five isoforms of human lysyl oxidases (LOX, LOXL1‐4) catalyze oxidative deamination of various primary amines and ε‐amino groups of lysine residues in proteins. LOX expression is up‐regulated by hypoxia in normal and cancer tissues. Usually, LOX is isolated from bovine aorta. However, authentic human enzyme should be preferred for pharmacological studies. We have optimized the aorta LOX protocol for human umbilical cord. The procedure includes homogenization, differential washes, urea extraction, absorption on hydroxyapatite and sulfoethylcellulose, followed by chromatography on DEAE‐cellulose and gel filtration. Simultaneously, semicarbazide sensitive amine oxidase (SSAO) can be also isolated using triton extraction followed by concanavalin A and ion exchange chromatography. Amine oxidase activities were measured as hydrogen peroxide release coupled to oxidation of 10‐acetyl‐3,7‐dihydrophenoxazine by horseradish peroxidase with methylamine as a SSAO substrate and free lysine or polyallylamine for LOX. In contrast with placenta, the cord lacks monoamine oxidase and diamine oxidase. In comparison with aorta, umbilical cord is poor in SSAO, whereas the yield of active lysyl oxidase is several folds higher. Immunoblotting using polyclonal antibodies against the five lysyl oxidase isoforms indicate that the isolated preparation contains predominantly LOX. In conclusion, the human umbilical cord is an excellent source of lysyl oxidase. Supported by RFBR grant 13‐04‐01413.
Expression And Purification of A Potential Antifolate Target, Dihydrofolate Reductase from B. malayi
Authors: Sanchez Karla , Gubler Ueli , Siekierka John , Goodey Nina
Published: June 2021

Brugia malayi is one of the three causative parasites of lymphatic filariasis, or elephantiasis, a parasitic disease causing swelling of extremities and lymphatic damage. Current literature shows antifolate drugs inhibit B. malayi, suggesting that dihydrofolate reductase (DHFR) is a potential drug target for the elimination of the parasite. Conditions for the expression and purification of B. malayi DHFR have not previously been investigated. As such, this study aims to establish ideal conditions for the expression and purification of Bm‐DHFR, to determine kinetic values for the enzyme (kcat, Km, Vmax), and to study existing antifolate compounds as possible inhibitors. Initial work was completed using a Bm‐DHFR gene whose sequence was obtained from the UniProt database (accession number A8QGA9). When compared to the Bm‐DHFR sequence entry from Wormbase (BM34009), the UniProt sequence was found to be missing a loop known to play a role in catalysis (FG loop) consisting of residues #113‐125 in BM34009. The Uniprot Bm‐DHFR was difficult to express in soluble form and exhibited no catalytic activity. These findings correlate with previous studies showing that point mutations in the E. coli DHFR FG loop result in markedly decreased catalytic activity. A new Bm‐DHFR construct is currently being used with the sequence obtained from BM34009 entry. This Bm‐DHFR was designed incorporating an N‐terminal His6‐tag and was obtained in the pUC57 vector. Current work includes subcloning the gene into pET25b an E. coli expression plasmid. Expression and purification conditions will be optimized and Ki and IC­50 values will be determined for a set of DHFR inhibitors including trimethoprim and pyrimethamine to validate Bm‐DHFR as a priority drug target.
Oligomeric Regulation of Ribonucleotide Reductase (RNR) by Antileukemic Nucleotides
Authors: Wisitpitthaya Somsinee , Fu Yuan , Li Minxing , Weiss Robert , Aye Yimon
Published: June 2021

Human RNR (hRNR) is the rate‐limiting enzyme for the de novo dNTP synthesis required for DNA replication and repair. hRNR activity is positively correlated with cancer cell proliferation and nucleotide analogs that can suppress hRNR activity such as gemcitabine and clofarabine (ClF) are proven effective in chemotherapy. However, the precise mechanisms by which many of the nucleoside prodrugs inhibit hRNR are unknown in most cases. The active hRNR is minimally an α2β2 holocomplex. Leukemia drug ClF is shown to inhibit hRNR through persistent hexamerization of α (α6) without requiring β. We hypothesize that α hexamerization is a universal mechanism by which antileukemic nucleotides inhibit hRNR, ultimately presenting a powerful small‐molecule approach to regulate hRNR. To test this hypothesis, I have sought to establish the mechanisms of hRNR inhibition by two additional clinically used nucleoside prodrugs—cladribine (CLA) and fludarabine (FLU). My studies have revealed that di‐ and triphosphates are active forms of both drugs that reversibly inhibit hRNR. CLA nucleotides [CLAD(T)P] are more potent than FLU nucleotides [FLUD(T)P] (Ki= 0.8, 0.1, 4.9 and 6.6 µM, respectively). In all cases, enzyme inhibition is coupled to α6 formation. We further validate in NIH‐3T3 cells stably expressing wild type α and hexamerization‐defective mutant α that hRNR inhibition by these nucleosides is directly correlated with oligomeric deregulation in cellular environment.
Anti‐Inflammatory Effects of Extracts from a Bioenergy Crop, Switchgrass, in Cultured Adipocytes
Authors: Garrison Richard , Scoggin Shane , Siriwardhana Nalin , Labbé Nicole , Ownley Bonnie , Gwinn Kimberly , D'Souza Doris , Moustaid‐Moussa Naima
Published: June 2021

Obesity is a low‐grade inflammatory condition that is associated with several metabolic diseases such as diabetes and cardiovascular disorders. Moreover, bioactive compounds from food and botanicals have been shown to reduce inflammation. However, anti‐inflammatory roles of switchgrass, a bioenergy crop is currently unexplored. Hence, we hypothesize that bioactives from switchgrass may have potential benefits on obesity‐related inflammation by reducing proinflammatory adipokines. Bioactives were isolated from chopped switchgrass using ethanol. To test our hypothesis, 3T3L1 preadipocytes were pretreated with the crude ethanol extracts or extracts fractionated into different organic solvents, followed by lipopolysaccharide (LPS) to determine if the extracts reduced LPS‐induced inflammation. Interestingly, some of these extracts reduced significantly and in a dose‐dependent manner, secretion of Monocyte Chemotractant Protein‐1 (MCP‐1) and Interleukin‐6 (IL‐6) from 3T3‐L1 cells. Furthermore, using MTT assays, some extracts significantly reduced cell viability while others did not. In summary, these findings demonstrate that switchgrass‐derived bioactives reduce adipocyte inflammation and provide the first evidence for an added value of switchgrass in obesity‐related inflammation. Additional studies are needed to characterize the specific phytochemicals responsible for the reduction in inflammation and their mechanism of action. In conclusion, bioenergy crops such as switchgrass are economically viable sources of anti‐inflammatory dietary supplements.
Improving the Uptake of L‐Telluromethionine by Escherichia coli DL41(DE3) Through the Use of Enhanced Chemically Defined Media
Authors: Cooper Brent , Sublett Veronica , Boles Jeffrey
Published: June 2021

In determining the three‐dimensional structure of a protein, hurdles of x‐ray crystallography can be overcome by incorporating unnatural amino acids into the protein of interest. Most naturally occurring atoms do not have sufficient mass to diffract electrons. incorporating unnatural amino acids containing a metalloid solves this problem. Bioincorporation of L‐Telluromethionine can provide atoms with sufficient mass for electron scattering when incorporated serendipitously. However, telluromethionine has cytotoxic effects which reduce cell growth and hinder uptake during protein synthesis. A proteomic analysis of telluromethionyl Escherichia coli was carried out using the methionine auxotrophe E. coli DL41 (DE3) (pCock) expression system. Both telluromethionyl and methionyl exposed cultures were analyzed with the objective of identifying differentially expressed proteins. The biological roles of differentially expressed proteins was elucidated and have been applied to improving methods for the bioincorporation of telluromethionine via the supplementation of chemically defined media with metabolites that ere negatively affected due to the exposure to L‐Telluromethionine.
CoREST in Peace: Dual Action Inhibitors of Histone Deacetylase and Lysine Specific Demethylase
Authors: Kalin Jay , Wu Muzhou , Hayward Dawn , Wang Liqing , Roberts Justin , Prusevich Polina , Hancock Wayne , Bradner James , Ryu Byungwoo , Alani Rhoda , Cole Philip
Published: June 2021

Histone deacetylases (HDACs) have been shown to act broadly on thousands of protein lysine acetylation sites to regulate cell growth and gene expression. The class I enzymes HDAC1 and HDAC2 can be found in various corepressor complexes in cells, including the CoREST complex, that also incorporates histone demethylase LSD1. Various combinations of HDAC inhibitors and LSD1 inhibitors have been reported previously to show additive or synergistic effects as anti‐tumor agents. In this study, we prepared several dual action HDAC/LSD1 inhibitors that integrate established pharmacophores targeting the active sites of these enzymes. We showed that several of these hybrid compounds can potently inhibit HDAC and LSD1 activity while maintaining the selectivity associated with the parent molecules. One of these compounds, 2‐68, derived from HDAC inhibitor MS‐275 (entinostat) and LSD1 inhibitor bizine, induced potent and predicted increases in histone H3 K9 acetylation and H3 K4 methylation. Interestingly, MS‐275 and 2‐68 showed anti‐proliferative effects across a panel of melanoma lines, but 2‐68 did not inhibit the proliferation of primary human melanocytes whereas MS‐275 did. Furthermore, we showed that, compared to the parent compounds, 2‐68 was superior in suppressing the function of regulatory T cells, suggesting that dual action CoREST inhibitors may offer advantages in enhancing tumor immune surveillance.
DUSP5 Functions in a Feedback Loop to Suppress Angiotensin‐Dependent Smooth Muscle Cell Proliferation and Pulmonary Arterial Hypertension
Authors: Ferguson Bradley , Demos‐Davies Kim , Cavasin Maria , Horita Henrick , Weiser‐Evans Mary , McKnight Robert , Lane Robert , McKinsey Timothy
Published: June 2021

Pulmonary arterial hypertension (PAH) is associated with marked remodeling of small pulmonary arteries (PAs) due to excess proliferation of fibroblasts, endothelial and smooth muscle cells (SMCs). Clinical and pre‐clinical studies have highlighted the renin‐angiotensin‐aldosterone system as a major contributor to pulmonary vascular remodeling in PAH. Indeed, angiotensin II (Ang II) is a potent stimulus for PASMC proliferation, in part through its ability to activate extracellular signal‐regulated kinase (ERK1/2). Here, we show that the ERK1/2 phosphatase, dual‐specificity phosphatase 5 (DUSP5), functions as a negative regulator of Ang II‐mediated SMC proliferation and PAH. DUSP5 is markedly upregulated in cultured SMCs in response to Ang II. Knockdown of DUSP5 leads to sustained ERK1/2 activation and enhanced cell proliferation in response to Ang II, and DUSP5 overexpression completely blocks Ang II‐mediated SMC proliferation. Remarkably, dusp5 null mice infused with Ang II develop profound PAH and right ventricular (RV) hypertrophy, in complete contrast to wild‐type mice exposed to Ang II. PAH in dusp5 null mice is associated with medial thickening of small PAs, stressing an in vivo role of DUSP5 as a negative regulator of Ang II‐dependent SMC proliferation. These data reveal a novel role for DUSP5 as an endogenous inhibitor of Ang II‐mediated ERK1/2 activation in SMCs, and demonstrate that disruption of this feedback loop leads to uncontrolled SMC proliferation and PAH.
Development of a Luciferase Reporter Gene Assay for Artemin‐induced RET Signaling
Authors: Chow Jennifer , Rauch Simone , Huestis Mikayla , Whitty Adrian
Published: June 2021

RET is a receptor tyrosine kinase that mediates the response of cells to stimulation with members of the glial cell‐line derived neurotrophic factor (GDNF) family of neurotrophins, which are important in the development and maintenance of a subset of neuronal cells as well as in other cell types and tissues. Improved methods to measure RET receptor activation and function are sought after to support the development of GDNF family ligands as potential drugs. Reporter gene assays are valuable tools to measure transcriptional and signaling pathway changes. Here, we report the development and validation of a stable luciferase reporter cell‐line for activation of RET by the GDNF family neurotrophin artemin (ART), using a previously established murine NB41A3 cell‐line expressing the GFRα3 co‐receptor. We observed a robust, dose‐dependent luciferase response upon stimulation of the cells with ART, which was quantitatively consistent with RET activation in the same cells measured using our previously reported RET Kinase Receptor Activation (KIRA) ELISA, or by measuring levels of activated ERK or Akt. Through inhibitor studies, we validated that RET activation of our reporter cell‐line is directly related to transcriptional changes induced by activation of the ERK/MAPK pathway. The reporter gene assay can be used to quantify the activation of RET by ART in a sensitive, convenient and robust way. We additionally report the use of this assay to investigate signaling dynamics and the efficiency of receptor‐effector coupling when RET is activated by a variety of different agonist proteins.
Carbonic Anhydrase Expression in TNBC Breast Cancer Cells and Human Tumor Grafts
Authors: Chen Zhijuan , Tu Chingkuang , Wilkinson Edward , Silverman David , Heldermon Coy , Frost Susan
Published: June 2021

Carbonic anhydrase IX (CAIX) has emerged as a potential target for triple negative breast cancer (TNBC). Our goal is to utilize the tumor graft model to demonstrate the efficacy of carbonic anhydrase (CA) inhibitors in reducing tumor load and metastasis. Here, we compare the expression of CAIX in TNBC human breast cancer cell lines with a bank of tumor tissues generated from subject‐derived breast tumor grafts in mice that retain characteristics of the original patient samples (Dr. Alana Welm). In our sampling of TNBC cell lines, CAIX expression was observed in four of the five lines. Two showed exclusive expression of CAIX relative to other cell surface CA family members, allowing us to evaluate catalytic activity. We observed that CAIX activity, measured by 18O exchange between H216O and H13C18O3−, was related to the level of CAIX expressed and inhibited by an impermeant sulfonamide (N‐3500). We then analyzed CAIX expression in tumor‐grafted tissue. Thirteen samples were available and represented the three major breast cancer phenotypes: estrogen and progesterone receptor‐positive (ER/PR), HER2‐positive, and TNBC. Of the six TNBC tissues, four expressed CAIX. No carbonic anhydrase XII (CAXII) was observed. Cryogenically preserved TNBC tissue was used to develop orthotopic tumors in nude mice. Immunohistochemistry showed high mitotic activity with CAIX expression adjacent to necrotic tissue. The tissue was negative for ER/PR and CAXII. These observations demonstrate that the tumor graft model will be useful in evaluating the efficacy of CA inhibitors for potential clinical use. We gratefully acknowledge funding from NIH (GM25253‐DNS and CA165284‐SCF), the UF Cancer Center (SCF), and the CSCRB, Inc (SCF).
Association of IDH1 Gene R132H Mutation and Increased Tumor HIF1‐α and Serum VEGF Levels in Primary Glioblastoma Multiforme
Authors: Aydin Hikmet , Yalaza Cem , Ak Handan , Cagli Mehmet , Ozgiray Erkin , Atay Sevcan
Published: June 2021

Glioblastoma multiforme (GBM) is the most common form of primary brain tumors. Although mutations in isocitrate dehydrogenase‐1 (IDH1) have been identified in a number of cancers, their role in tumor development has not been fully elucidated. In this preliminary study we aimed to investigate the association between IDH1 mutations, tumor tissue HIF‐1 alpha and serum VEGF levels in patients with primary GBM.

32 patients (mean age, years: 58 ± 14.0) diagnosed with primary glioblastoma multiforme were screened for IDH1 mutations (R132H, R132S, R132C and R132L) by direct sequencing. Serum VEGF and tumor tissue HIF1‐alpha levels were measured by ELISA. Associations between categoric variables were determined using chi‐square tests. Differences between two groups were compared with t test for continuous variables. Two patients were heterozygous for R132H mutation (6%). Tumor HIF1‐alpha and serum VEGF levels were found to be significantly increased in IDH1‐mutated tumor tissues.

Wild Type IDH1 (n=30)
Mutant IDH1 (n=2)
p

Serum VEGF (ng/ml)
0.189 ± 0.08
0.309 ± 0.016
0.0454

Tumor HIF1‐α (ng/ml)
0.710 ± 0.835
5.058 ± 3.75
< 0.0001

Our preliminary results suggest that mutated IDH1 may contributes to carcinogenesis via induction of HIF‐1 alpha pathway in primary GBM.
LOXL2 Expression in HCT116 Cells under Normoxia is Regulated by ROS and HIF Signaling
Authors: Okkelman Irina , Dmitriev Ruslan , Zhdanov Alexander , Papkovsky Dmitri
Published: June 2021

HIF1α is well known for its role in induction of metastasis leading to cancer progression.LOXL2 and LOX can induce epithelial‐to‐mesenchymal transition (EMT) of tumor cells and turn them into more aggressive phenotype. Recent findings show that LOX expression can be activated by both hypoxia and reactive oxygen species (ROS) via HIF1 pathway, suggesting complex regulation of LOX in EMT in vivo. To address this, we studied the mechanism of regulation of LOXL2 gene expression in human colon cancer HCT116 cells with impaired mitochrondrial respiration (SCO2‐/‐ mutant).

q‐PCR showed that under normoxic conditions (21% O2) expression of LOXL2 gene in SCO2‐/‐ cells, which have increased ROS production, is 8 times higher than in wild type. Culturing of SCO2‐/‐ cells for 1 week with ROS scavenger N‐acetylcysteine (NAC) or inhibitor of HIF‐α signaling FM19G11 under normoxia led to twofold decrease in LOXL2 expression, suggesting direct involvement of ROS/HIF1 axis in LOXL2 gene expression in this cell line. However, we saw no statistical difference in LOXL2 expression in SCO2‐/‐ cells treated with NAC under the hypoxic conditions (3% O2), confirming that ROS levels are decreased in hypoxia and are less involved in regulation of LOXL2. At the same time treatment of SCO2‐/‐ cells with FM19G11 led to twofold decrease in expression of LOXL2, thus demonstrating the active role of HIF pathway. Altogether these data demonstrate that different pathways can be involved in HIF1 dependent expression of LOXL2 under hypoxia and oxidative stress. This mechanism can play important role in vivo in cancer progression mediated by LOXs.

Supported by SFI.
Calcineurin Homologous Protein 2 Expression in Non‐Small Cell Lung Cancer
Authors: Tran Michelle , Cain Michael , Wallert Michael , Provost Joseph
Published: June 2021

Calcineurin Homologous Protein (CHP) is a Ca2+ binding protein that functions as an essential cofactor in pH regulation by directing the plasma membrane's Na+/H+ exchanger (NHE) activity. Both CHP1 and CHP2 bind to NHE in an unknown manner; however, CHP2 was previously identified to be expressed in select malignant cell lines (not determined in lung) while remaining below detection in non‐transformed cells. The relationship between CHP2 expression and function is still unknown. To better define a role of CHP2 in lung cancer, we examine the expression of CHP2 in both cultured cells and human lung tissues of Non‐Small Cell Lung Cancer (NSCLC) patients using two different detection methods. We first identified appropriate isotope specific CHP antibodies using recombinant CHP1 and CHP2 purified protein and then probed normal and malignant cultured cell lines using each CHP antibody. CHP1 was expressed in each cell line (normal or transformed) while CHP2 was not detected in normal cell cultured lysate. CHP2 expression was detected in adenocarcinoma, squamous cell carcinoma and malignant cultured lysates. Similar results were observed when blotting adjacent malignant/normal human lung tissue from NSCLC patients. We then used real‐time PCR (RT‐PCR) to quantify the expression levels of the same NSCLC cultured cell lines and human lung tissues. In both methods, all cultured cell lines and human lung tissues were analyzed again to confirm the expression of the house‐keeping gene Beta‐actin. This provides the first evidence of CHP2 expression in cultured human lung cells and in diseased tissues from NSCLC patients.