bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating putative key catalytic residues and uncoupled hydroperoxyflavin formation in the mechanism of 6‐hydroxynicotinate‐3‐monooxygenase, a decarboxylative‐hydroxylase in bacterial nicotinate catabolism
Authors: Perkins Scott W. , Snider Mark J. 
Published: June 2021

Agrochemicals, pharmaceuticals and personal care products (APPCPs) are contaminants of emerging concern that are ineffectively remediated by current methodologies. Many APPCPs contain N‐heterocyclic aromatic constituents (NHACs) that are persistent contaminants in aquatic environments and cause adverse effects in non‐target organisms. Understanding bacterial degradation of NHACs may assist future bioremediation techniques. Aerobic bacterial catabolism of nicotinic acid (NA) serves as a model for understanding the biodegradation of NHACs, and a key enzyme of this pathway is 6‐hydroxynicotinate 3‐monooxygenase (NicC). We recently published the first crystal structure and two postulated mechanisms for NicC. Both mechanisms involve acid‐base catalysis, in which active site residue His211 may serve as a key H‐bond donor. Additionally, the Cys202 active site residue may donate a thiol group, playing a role in one postulated mechanism. Kinetic characterization of the H211A and C202A variants shows that the apparent substrate affinity and catalytic turnover are largely unaffected by mutagenesis of these bases (H211A: KM of 6HNA increases 2.1–fold, KM of NADH increases 2.3–fold, kcat increases 30% compared to WT; C202A: KM of 6HNA increases 2.4–fold, kcat drops 15‐fold). These data suggest that the His211 and Cys202 residues are not critical for catalysis. 6‐Hydroxynicotinaldehyde (6‐HNAH), a competitive inhibitor of NicC with respect to 6‐HNA, exhibits tight binding (KD of 6HNAH = 36 ± 2 μM) compared to 6HNA (KD of 6HNA = 190 ± 25 μM), but shows weak inhibition (Ki of 6HNAH = 8.5 ± 2.4 mM). These results are consistent with the possibility that the formation of hydroperoxyflavin (FADHOOH) is uncoupled from the oxidative decarboxylation reaction, and subsequently produces hydrogen peroxide. Hydrogen peroxide production was detected for reactions of WT+6HNAH and H211A+6HNAH, but not detected for WT+6HNA and H211A+6HNA, suggesting that 6HNAH causes uncoupling and His211 is not a key catalytic residue in the mechanism of NicC.Support or Funding InformationCollege of Wooster Sophomore Research ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vitro Biochemical Studies of the Decarboxylase Domain of ArnA from Pseudomonas aeruginosa
Authors: de Miranda Rodger , Conrad Laura Miller 
Published: June 2021

Pseudomonas aeruginosa is an opportunistic, Gram‐negative pathogen that is resistant to many classes of antibiotics. One pathway leading to resistance to cationic antimicrobial peptides like colistin is upregulation of the arnBCADTEF operon. With the biosynthetic enzymes encoded in this pathway, P. aeruginosa modifies its outer membrane to decrease its net negative charge. This neutralization of charge lessens the driving force for electrostatic interactions with positively charged antibiotics such as colistin, hindering their entry into the cell. The first committed step of this pathway is catalyzed by the decarboxylase domain of ArnA in which UDP‐glucuronic acid undergoes a redox reaction with NAD+. Due to its role in the arnB pathway of antibiotic resistance, we seek to characterize the kinetics of ArnA using in vitro biochemical studies. To do this we measured the change in absorbance at 340 nm allowing us to calculate the reduction of NAD+ to NADH. These data were analyzed to produce the Michaelis‐Menten kinetics of ArnA from P. aeruginosa. Elucidating the kinetics of ArnA will enable analysis of small molecule inhibitors for changes in the activity of this enzyme. This knowledge will enable future work to design strategies to inhibit ArnA and thus block the resistance pathway.Support or Funding InformationNational Institutes of Health (NIH) Grant # 5SC3GM118199This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pomegranate phenolics inhibit type I collagen cross‐linking induced by glycative stress
Authors: Cai Ang , Liu Weixi , Ma Hang , Dombi George W. , Dain Joel A. , Seeram Navindra P. 
Published: June 2021

Collagen is a class of long‐lived structural proteins in the extracellular space in various fibrous tissues including skin, tendons, and ligaments. Collagen fibrils are subjected to metabolic stresses including oxidation and glycation, which lead to its structural modification including non‐enzymatic glycation and excess cross‐linking. These modifications are critical factors in microstructural cross‐link associated disorders including skin aging, Alzheimer's disease and cardiovascular disease. Many natural products including phenolics are regarded as promising inhibitors that prevent the formation of advanced glycation endrpoducts (AGE). Previously, we reported that hydrolyzable tannins including punicalagin (PA) and ellagic acid (EA) from pomegranate extract, along with their gut microbial metabolites, namely, urolithins, can inhibit the formation of AGE in a bovine serum albumin model. The beneficial inhibitory effects of these phenolics on the glycative stress‐induced collagen cross‐linking remain unknown. Therefore, in this current study, we examined the protective effects of pomegranate phenolics on collagen glycation. Pomegranate phenolics (at a concentration of 200 μM) including PA, EA, and urolithin A (UA) inhibited the fructose‐induced collagen glycation by 64.4, 15.0, and 17.3%, respectively, and the methylglyoxal (MGO)‐induced collagen glycation by 70.6, 17.6, and 14.4%, respectively. Furthermore, PA, EA, and UA (each at a concentration of 200 μM) inhibited the formation of Amadori products by 45.7, 31.5, and 40.0%, respectively. In addition, circular dichroism (CD) analysis showed that PA, EA, and UA exhibited protective effects on the secondary structure of collagen on the denaturing effect of glycation. The inhibitory effects of hydrolyzable tannins on collagen glycation warrants further investigation as they may provide a novel therapy to complications related to collagen glycation and crosslinking as in diabetes and hypertrophic scarring.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Expanding the Toolbox of Chemoenzymatically Synthesized Peptidoglycan Probes and Investigating their Interaction with Nucleotide Sugar Transporters”
Authors: Jones Zachary Scott , DeMeester Kristen Elizabeth , Liang Hai , Jensen Matthew , D'Ambrosio Elizabeth , Grimes Catherine Leimkuher 
Published: June 2021

IntroductionThe microbiome has gained increasing attention due to its relevance to human health, particularly in the intestinal tract. Bacteria possess a unique structure, peptidoglycan (PG), which is known to illicit an innate immune response by inflammation. Within the peptidoglycan, uridine diphosphate N‐acetyl muramic acid (UDP‐NAM) is the first committed step to PG biosynthesis. Previously, NAM probes with functional moieties on the 2‐position of the sugar have been used to label bacteria on the PG via copper catalyzed click chemistry. Using a chemoenzymatic synthesis method, a UDP moiety can be attached to achieve this first committed PG substrate.Glycan chemical synthesis of UDP sugar probes, has proven difficult to achieve synthetically. Currently, the goal is to optimize large‐scale quantities of UDP‐NAM and derivatives and to synthesize chemical probes with expanding functionalities in addition to fluorescent labelling for incorporation into the PG of bacteria. By tracking the dynamics of PG, specifically the breakdown of these chemical probes, innate immune reactions and responses may be better understood.MethodsThe method developed utilizes the recycling enzymes anomeric NAM/NAG kinase (AmgK) and uridylyltransferase (MurU) in order to chemoenzymatically synthesize UDP‐NAM derivatives. Due to the relaxed substrate specificity of these enzymes, the NAM derivatives are able to be accepted into the active site so that the muramyl lactol will be converted to the charged UDP sugar donor. AmgK and MurU were expressed and purified from Escherichia coli.Purification was carried out by high‐pressure liquid chromatography (HPLC) coupled to liquid chromatography mass spectrometry (LC/MS) on a hydrophilic liquid chromatography column. Fractions were collected and the products were characterized by LC/MS and nuclear magnetic resonance (NMR) imaging.Results and DiscussionCurrently, large scale synthesis, purification, and characterization has been completed of the natural UDP‐NAM substrate and approximately 8 derivatives on a high mg scale. Also, probes with a CF2H and a diazo moiety have been synthesized to be tested with the recycling enzymes. 2‐azido‐UDP‐NAM has successfully been incorporated into the PG of Lactobacillus acidophilus, but was unsuccessful in labelling other strains of gram positive bacteria.It is proposed that a nucleotide sugar transporter exists in L. acidophilus, and there have been several candidates found by BLAST sequencing. The presence of nucleotide sugar transporter proteins will be explored, and their homology and binding specificity to the UDP‐NAM derivatives will be examined.From these results, the library and functionality of UDP‐NAM probes has been expanded, and future work involves elucidating the mechanism by which the probes pass through the cell membrane.
Interleukin‐like EMT Inducer ILEI mediates breast cancer stem cell formation and tumorigenesis through LIF receptor signaling
Authors: Woosley Alec , Dalton Annamarie , Howe Philip 
Published: June 2021

During the epithelial‐mesenchymal transition (EMT), cells undergo a switch from a polarized, epithelial state to a more fibroblastic and mesenchymal phenotype. This process is associated with the formation of highly tumorigenic cancer stem cells (CSC). Our lab has identified a regulatory mechanism by which Transforming Growth Factor‐β (TGFβ) induces EMT at the translational step of gene expression. Among gene products controlled by this post‐translational mechanism is the cytokine Interleukin‐like EMT Inducer (ILEI), a secreted factor belonging to the FAM3 family that is involved in EMT induction and tumorigenesis. The biochemical mechanism of the ILEI signaling and identification of an ILEI receptor has remained elusive. Preliminary studies from our lab indicate that modulation of ILEI in several cell lines results in altered stemness potential, giving insight into the possible role of ILEI in the formation of CSCs. The basis of this study aims to investigate the mechanism of the ILEI signaling pathway and its contribution to CSC formation. In order to identify potential ILEI binding partners, we utilized yeast two‐hybrid screening and observed that ILEI interacts with the 190kD leukemia inhibitory factor receptor (LIFR). LIFR serves as a signaling platform for several cytokine ligands and forms intricate complexes with an array of other ligand‐specific co‐receptors during its activation. Our data indicates that ILEI functions through LIFR, inducing downstream CSC phenotypes by activating PI3k/Akt and Jak/STAT signaling pathways. Insight into the TGFβ/ILEI/LIFR pathway may serve as a platform for development of a novel targeted therapy against metastatic breast cancer.Support or Funding InformationNIH F31CA213627NIH R01CA555536This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Leptin alleviates the saturated fatty acid‐induced increase in BACE1 expression and Amyloid‐β production ‐ Relevance to Alzheimer's disease pathogenesis
Authors: Marwarha Gurdeep , Ghribi Othman 
Published: June 2021

Epidemiological studies implicate diets rich in saturated free fatty acids (sFFA) as a potential risk factor for developing Alzheimer's disease (AD). In particular, high plasma levels of the sFFA palmitic acid (palmitate) have been shown to inversely correlate with cognitive function. AD is histo‐pathologically characterized by the deposition of aggregated Amyloid‐β (Aβ) peptide as extracellular amyloid plaques and the intracellular accumulation of aggregated hyper‐phosphorylated tau protein as neurofibrillary tangles. Our recent studies have demonstrated that palmitate‐enriched diet increases Amyloid‐β (Aβ) genesis in the hippocampus by increasing the expression and activity of the aspartyl protease BACE1 which catalyzes the rate limiting step in Aβ (Marwarha et al. 2017, Ghribi & Marwarha 2017). We have also shown that palmitate‐enriched diet decreases the expression of leptin, an adipokine that functions as a key neurotrophic cytokine in the brain (Marwarha et al. 2016). However, the role of palmitate‐induced mitigation in leptin expression and dysregulation of leptin signaling in the palmitate‐induced increase in BACE1 expression and the ensuing Aβ genesis has not been examined. Herein, we determined the role of leptin in modulating the palmitate‐induced increase in BACE1 expression and the ensuing Aβ genesis in human neuroblastoma SH‐SY5Y cells. Concomitant treatment of the cells with exogenous recombinant leptin significantly decreased the exogenous palmitate‐induced increase in BACE1 expression and enzymatic activity that culminated into a reduction in intracellular and secreted Aβ levels. Additionally, human SH‐SY5Y neuroblastoma cells stably overexpressing endogenous leptin, exhibited a lower increase in BACE1 expression and enzymatic activity as well as the ensuing Aβ accumulation in response to exogenous palmitate. Our unprecedented study illuminates a novel role of leptin in the brain and provides a unique insight into the mechanisms that underlie palmitate induced effects on molecular players involved in the etio‐pathogenesis of Alzheimer's disease.Support or Funding InformationThis work was supported by a Grant from the NIH (R01AG045264) to Dr. Othman Ghribi.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Detection of Novel Rho GAP/GTPase Selectivities Using In Vitro Split‐luciferase Assays
Authors: Wilander Benjamin A. , Hamann Michael J. 
Published: June 2021

Rho GTPases regulate the assembly of cellular actin structures for a wide range of dynamic activities, including actin‐dependent migration, constriction/scission and transport‐related processes. These events are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase‐activating proteins (GAPs). Although most research has tended to focus on the role of GEF‐catalyzed GTPase signal activation, GAP inactivation of GTPase signaling pathways needs to be better characterized since inactivation is required to avoid prolonged and possibly detrimental cellular responses. To this end, the GTPase selectivity of several novel hematopoietically‐expressed RhoGAPS, specifically ARHGAP1, 9, 12, 17, 25, and 26, were tested by generating MBP fusion proteins of the GAP domains of these proteins and testing their activities with Cdc42, Rac1 and RhoA. Activities were assayed using a previously described in vitro split‐luciferase assay with bacterially‐expressed purified proteins, and GAPs with known selectivities were used as positive controls. These results will provide an important first step in linking these RhoGAPs to particular GTPase pathways and eventually linking them to cellular functions.Support or Funding InformationSupport was provided by the Nielson Foundation, Bemidji, MN and a grant from Regenerative Medicine Minnesota (RMM‐2017‐EP‐04)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
: Atypical G protein β5 promotes cardiac oxidative stress and fibrotic remodeling in response to multiple cancer chemotherapeutics
Authors: Chakraborti Sreemoyee , Pramanick Arnab , Maity Biswanath 
Published: June 2021

The clinical use of multiple classes of cancer chemotherapeutics is limited by irreversible, dose‐dependent, and sometimes life‐threatening cardiotoxicity. Though distinct in their mechanisms of action, doxorubicin, paclitaxel, and 5‐FU all induce rapid and robust upregulation of atypical G protein Gβ5 in the myocardium correlating with oxidative stress, myocyte apoptosis, and the accumulation of pro‐inflammatory and pro‐fibrotic cytokines. In ventricular cardiac myocytes (VCM) Gβ5 deficiency provided substantial protection against the cytotoxic actions of chemotherapeutics including reductions in oxidative stress and simultaneous attenuation of ROS‐dependent activation of the ATM and CaMKII pro‐apoptotic signaling cascades. In addition, Gβ5 loss allowed for maintenance of Δψm, basal MCU and mitochondrial Ca2+ levels, effects likely to preserve functional myocyte excitation‐contraction coupling. The deleterious effects of Gβ5 are not restricted to VCM, however, as Gβ5 knockdown also reduces chemotherapy‐induced release of pro‐inflammatory cytokines (e.g. TNFα), hypertrophic factors (e.g. ANP) and pro‐fibrotic factors (e.g. TGFβ1) from both VCM and VCF with the most dramatic reduction occurring in co‐cultured cells. Our experiments suggest that Gβ5 facilitates the myofibroblast transition, the persistence of which contributes to pathological remodeling and heart failure. The convergence of Gβ5‐mediated, ROS‐dependent signaling pathways in both cell types represents a critical etiological factor in the pathogenesis of chemotherapy‐induced cardiotoxicity. Indeed, intracardiac injection of Gβ5‐targeted shRNA allowed for heart specific protection against the damaging impact of chronic chemotherapy. Together, our results suggest that inhibition of Gβ5 might represent a novel means to circumvent cardiotoxicity in cancer patients whose treatment regimens include anthracyclines, taxanes or fluoropyrimidines.Support or Funding InformationRamalingaswami Fellowship (BT/RLF/Re‐entry/15/2013), Department of Biotechnology, and Department of Science and Technology, SERB (EMR/2016/006873), Government of India to Dr. B Maity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Blended Instruction in Introduction to Biochemistry Class
Authors: Nicholas Timothy , Reddy Neha , Srinivasan Rekha 
Published: June 2021

A challenge for biochemistry instructors is dealing with the volume and difficulty of concepts typically taught during semester‐long introductory biochemistry courses. Students struggle to grasp topics because they may not have enough in‐class instruction and practice time to process the new information. Blended learning strategies, such as the flipped classroom approach along with traditional lectures, may offer a solution to this concern. Herein we present the creation of a blended learning introductory biochemistry course. The course used online modules consisting of concept videos tied to active learning problem sets, followed by in‐class sessions of small groups to discuss the application of these concepts. Each topic was covered with a video presentation suited for display on mobile devices that focused on problem solving and an associated active learning project that encouraged practice and application of skills developed in that topic. Students were encouraged to take an online quiz after each set of core topics to assess their readiness in applying the concept. The initial implementation of this module was tested in the Introduction to Biochemistry class and the preliminary data is presented hereinSupport or Funding InformationNord Grant, UCITE, Case Western Reserve UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Triclosan Exposures on Interleukin 6 and Interleukin 1 Beta Secretion from Human Immune Cells
Authors: Wilburn Wendy JoAnne , Brooks Dylan , Whalen Margaret 
Published: June 2021

Triclosan (TCS) is an antimicrobial compound that is widely used in personal hygiene products such as mouthwash and toothpaste. TCS can be ingested or absorbed through the skin and has been found in human blood, breast milk, and urine. Interleukin (IL)‐6 and IL‐1 beta (IL‐1β) are important pro‐inflammatory cytokines produced by lymphocytes, monocytes, and other cells. Both regulate cell growth, tissue repair, and immune function, increased levels of each have been associated with a number of diseases including rheumatoid arthritis and certain cancers. TCS has been shown to inhibit the lytic function of human natural killer (NK) lymphocytes and to decrease expression of key cell surface proteins on NK cells. Here we examine whether TCS alters the secretion of IL‐1β and IL‐6 from human immune cell preparations. Human peripheral blood mononuclear cells (PBMCs) and monocyte‐depleted (MD)‐PBMCs were exposed to TCS at concentrations of 0–5 μM. Cytokine secretion was measured at 24 h, 48 h, and 6 days using enzyme‐linked immunosorbent assay (ELISA). Both IL‐1β and IL‐6 secretion were increased at one or more concentration of TCS at one or more length of exposure. These results indicate that TCS has the capacity to disrupt secretion of these two important pro‐inflammatory cytokines.Support or Funding InformationSupported by NIH grant U54CA163066This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.