bluuebunny/crossref_metadata_2025 huggingface dataset:
Ligand binding studies of a trimethoprim‐resistant dihydrofolate reductase by fluorine NMR
Authors: Gomez Gabriel Jose Fuente , Howell Elizabeth
Published: June 2021

Antibiotic resistance is a worldwide problem. The excessive use and misuse of antibiotics have resulted in the development of drug resistance. One example involves the antibiotic trimethoprim (TMP). TMP selectively inhibits the bacterial enzyme dihydrofolate reductase (DHFR) over its human counterpart. Inhibition of DHFR results in inhibition of DNA synthesis and ultimately in cell death. Unfortunately, extensive use and dissemination of TMP over the last forty years has resulted in the development of drug resistance in the form of highly transmissible plasmids. R67 dihydrofolate reductase is a plasmid‐encoded enzyme that confer resistance to TMP. Our lab has studied how R67 DHFR works in vitro using diluted conditions. However, most proteins function inside cells under crowded and heterogeneous environment. As 19F labeled proteins in complex mixtures can be characterized by NMR, we report our initial expression and characterization of R67 DHFR. To obtain such information, we have labeled R67 DHFR with fluorine at different positions in the indole ring and showed that fluorine incorporation into R67 DHFR does not affect the structure and function of the protein. We have characterized the 19F NMR spectra of apo R67 DHFR and optimized fluorine incorporation. Furthermore, fluorinated R67 DHFR shows different signals for binding of cofactor, NADP+, and substrate, dihydrofolate (DHF). We have obtained similar dissociation constants for the cofactor and substrate of R67 DHFR by both NMR and ITC. In our next step, we will measure dissociation constants in more complex solutions. We envision using fluorinated protein to quantify the effect of osmolytes and crowders on the binding affinity of substrate and cofactor towards R67 DHFR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular modeling of the cI repressor of bacteriophage ɛ34
Authors: Goodson Richardria , McGee Lauren , Gildea Logan , Brooks Ryan , Villafane Robert , Jackson Doba
Published: June 2021

Gene 46 of ɛ34 encodes a protein that is similar to other phage repressors. It has a C‐terminal protease domain which is about 80% identical to the repressors of phage λ (which infects E. coli), but its DNA binding N‐terminal portion is only about 50% identical to its closest known relatives in phages λ, P22, and Lex A. The N‐terminal DNA‐binding domain is homologous to the HTH_XRE family of DNA‐binding domains [1].

We specifically looked at the N‐terminal domain of the cI with its homology to the HTH_XRE DNA binding proteins (also called the Cro/cI type HTH domain). To this date, 19 unique structures have been determined and are deposited into the Protein Data Bank (PDB). The HTH_XRE family of proteins has not been reviewed in the past 10 years despite the historical significance of its members as being the earliest members of the conserved class of DNA binding domains. We used the SWISS‐MODEL server to generate a series of homology models with and without DNA of to investigate how this protein interacts with its cognate DNA sequence. Presented in another, study, our bioinformatic and experimental studies have determined the 17 bp asymmetric site that interacts with the cI repressor. Our preliminary models have revealed some unique protein‐DNA contacts of the cI repressor that are not found in other homologous proteins (cI of λ, Φ11, 434, SinR, CopR, and Cro). The goal is to eventually initiate a structure‐function study and determine the three‐dimensional structure of this repressor. This project was done as a collaborative venture between two laboratories, Huntingdon College (Dr. Doba Jackson) and Alabama State University (laboratory of Dr. Robert Villafane). This project is in line with Dr. Robert Villafane's research into the Genomic analysis of bacteriophage ɛ34 of Salmonella enteric serovar Anatum.

Support or Funding Information

Support for this project was provided by Alabama State University and Huntingdon College.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Toward Understanding the Functional State of the Grb7 Protein
Authors: Koirala Rajan
Published: June 2021

Human growth factor receptor bound protein 7 (Grb7) proteins are the family of adaptor proteins that include Grb7, Grb10 and Grb14. These proteins have a conserved topology that includes a proline rich N‐terminal domain, an RA domain, a PH domain and a C‐terminal SH2 domain. Grb7 proteins bind with activated receptor tyrosine kinase (RTK) receptors through their SH2 domain to mediate downstream signal transduction events. Previous studies in our laboratory have shown the Grb7SH2 domain exists in a dimeric form and this may control the dimerization of the full‐length protein (FLGrb7). In our current studies, we show a FLGrb7 phosphorylation mimic, Y492E‐FLGrb7, results in a monomeric form of the protein, by in vitro biophysical measurements. Specifically, size exclusion and analytical ultracentrifugation results demonstrate Y492E‐FLGrb7 is essentially monomeric, while under the same conditions wild‐type Grb7 (WTFLGrb7) exists as a dimer. Further, the size and shape of these proteins were determined through dynamic light scattering measurements. These studies suggest the phosphorylation mimic Y492E‐FLGrb7 may exist in a more open, as opposed to compact globular, conformational form. We interpret the above results with a view towards understanding the functional state of Grb7 protein, and its role in controlling cell signaling pathways via interaction with its binding partners.

Support or Funding Information

NIH5‐SC3‐6M116728

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing the NaK Ion Channel through Site‐Directed Mutagenesis
Authors: Luu Gina , Henzler‐Wildman Katherine
Published: June 2021

The NaK transmembrane protein is a non‐selective bacterial cation channel permeable to Na+ and K+ ions. This channel has structural similarities to mammalian channels and our studies of NaK may provide insight into the gating of bio‐medically important channels. It is hypothesized that the N‐terminal helix of NaK fits tightly against the base of the closed channel and mutations that disrupt this interaction increase the open probability. To test the hypothesis, we mutated residues that appear to form stabilizing interactions between the N‐terminus helix and the pore. If these mutants increase the open probability, they will then increase net ion flux through the channel. We used a bacterial complementation screen with the LB2003 K+ uptake‐deficient strain of Escherichia coli to identify residues that affect net flux through NaK. We focused on mutations to the hydrophobic cluster (F24A, F28A, W18A, F92A, and F94L) connecting the M0 helix to the channel pore, and the salt bridge (R10Q) connecting the M0 to the pore domain of NaK FL. The complementation assay demonstrated that the hydrophobic cluster mutations increased complementation more than the negative control but less than the positive control, while the salt bridge mutation increased complementation as much as the positive control. Our results suggest that the salt bridge is vital to stabilizing the closed state of NaK.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Effects of Altering the Nonpolar Core of Hemolysin A
Authors: Stuttgen Gage Michael , Weaver Todd Michael , Grilley Daniel Patrick
Published: June 2021

The protein structure and function paradigm is a foundational tenet of biomolecular science that underlies many infectious diseases. Hemolysin A (HpmA), a hemolytic protein produced by Proteus mirabilis, was used as a model to investigate the protein structure‐function paradigm. HpmA is a member of the two‐partner secretion (TPS) pathway, which is used by gram‐negative bacteria to export predominantly virulent proteins outside of the cell. Through this mechanism, the A‐component (HpmA) is translocated, folded, and activated by its cognate B‐component in the absence of high‐energy bond and electrochemical gradient dependency. All TpsA components are relatively large and can be further divided into two domains, the two‐partner secretion domain and the functional domain. Universally, all known TPS domains harbor a right‐handed, parallel b‐helix architecture, which has been shown critical for cognate TpsB‐dependent recognition and secretion. Conversely, functional domains provide TpsA diversification including, cytolysis, host cell adhesion, contact‐growth inhibition, and iron sequestration. A truncated version of hemolysin A (HpmA265) was implemented to define the contributions of the TPS domain toward TpsA structure and function. Recently, our group has further dissected HpmA265 into three sequentially folded structural units termed the polar core, non‐polar core, and carboxy‐terminal subdomains. This research project aims to expand upon our recent results and structurally map the role of the nonpolar core during TPS domain dependent secretion, folding, and function. Specifically, residues within the non‐polar core subdomain have been selectively targeted and modified. The structural and functional effects of these site‐selective modifications have been evaluated via chemical denaturation, protease sensitivity, and template‐assisted hemolytic activity. Site‐selective alterations within the non‐polar core subdomain shift four‐state b‐helix unfolding to a three‐state model, increased protease sensitivity, and decrease template‐assisted hemolysis. In contrast, site‐selective alterations on the exterior of the non‐polar core subdomain maintain four‐state unfolding and protease sensitivity, and increase template‐assisted hemolysis. Collectively, these results point toward TPS b‐helix stability as a requisite for robust template‐assisted hemolysis. Conservation of the TPS domain across gram‐negative bacteria makes development of antibiotics targeting b‐helix structure and function attractive.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamic Allosteric Biomolecular Design Using Artificial Intelligence
Authors: Thayer Kelly , Beveridge David , Langley David
Published: June 2021

Protein‐ligand binding mediated by an allosteric effector can be considered from the vantage of dynamic macromolecular structure. Recently, we have developed a methodology, MD‐based Markov State Models (MD‐MSMs) making use of this view. We envision gaining insight into the workings of allosteric systems, and applying those principles to allosterically engineer biomolecules as a new class of therapeutics. Such allosteric regulators hold promise for modulating currently “undruggable” targets. In our approach, Molecular dynamics simulations (MD) generate the Boltzmann ensemble of conformations of a system. Using machine learning to process trajectories, the number of conformational substates can be learned from the conformations of the system (Figure 1A). Network theory captures the dynamic interchange between conformations (Figure 1B). Our recent findings from the application of MD‐MSMs to allosteric proteins (Figure 1C) and principles for engineering allosteric effectors will be presented.

Support or Funding Information

NIH R15 GM128102 to KMT

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does SpNox directly produce hydrogen peroxide?
Authors: Scott James , Notice Brittany , Gonzalez Victoria , Thomas Sarah , Vermot Annelise , Petit‐Hartlein Isabelle , Fieschi Frank , Smith Susan ME
Published: June 2021

NADPH oxidases (NOXs) are a family of transmembrane enzymes that generate reactive oxygen species (ROS), which in humans acts both as a cytotoxic agent in autoimmunity and as a signaling molecule in many physiological processes. Human NOXes can produce different ROS species; for example, NOX2 isoform generates superoxide whereas NOX4 isoform produces mainly hydrogen peroxide. The ROS species produced affects downstream signaling, and misregulation produces disease states. A recently discovered prokaryotic NOX, SpNOX, provides an ideal model system to investigate controls on ROS product. SpNOX was previously shown to produce superoxide. Because superoxide spontaneously dismutes at a fast rate, assays that detect hydrogen peroxide cannot be used to assess direct hydrogen peroxide formation. Instead, as in the case of xanthine oxidase, we estimate total electron flux and also superoxide production; we assume that any difference between these values reflects direct hydrogen peroxide production by the enzyme. We estimate electron flux using three assays: i/ monitoring NADPH consumption using absorbance (340 nm); ii/ monitoring NADPH consumption using fluorescence (ex 345/em 454); iii/ monitoring cytochrome c reduction by the FAD of a dehydrogenase‐domain only construct of SpNOX. Superoxide generation from full‐length SpNOX is estimated by the superoxide‐dismutase‐inhibitable portion of cytochrome c reduction, monitored by absorbance at 550 nm. Here we present our findings regarding relative production of superoxide and hydrogen peroxide by SpNOX, as a first step toward manipulating ROS product in this model system.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Hydrogen Peroxide on Collagen
Authors: Valdes Diana , Tellez Jorge , Keenan Kelly
Published: June 2021

Americans spend billions of dollars every year on teeth whitening products including whitening strips; the active ingredient is hydrogen peroxide (H2O2). Collagen accounts for 95% of the protein found in the dentin layer of teeth, which lies beneath the enamel. Previous research done in our laboratory has indicated that treatment of teeth with over‐the‐counter whitening strips results in reduced amounts of collagen and hydroxyproline. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) was used on collagen extracted from teeth. For the untreated teeth, the extracted collagen showed the expected molecular weights (MW) on SDS PAGE but these bands were absent for the collagen extracted from treated teeth. For this reason, pure Type I collagen was used and treated either with water or with H2O2 for an hour before pepsin was added. Our results indicate that pre‐treatment of Type I collagen with low concentrations of H2O2 produced a lack of bands at the expected MW for collagen and these bands were present in collagen treated with water. This effect was seen as soon as 10 minutes after H2O2 was added. The impact of adding the protease pepsin was further assessed. These results suggest that hydrogen peroxide, even at the concentration used in the whitening strip, can damage the collagen in the dentin provided that the H2O2 can penetrate to the dentin in the teeth. This work was supported by Research and Professional Development Grant from Stockton University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of hydrogen sulphide in regulating invasion in trophoblast cells
Authors: Saxena Shobhit , Gupta Sunil Kumar , Arora Pallavi , Chetlapalli Pallavi , Sirohi Neerja , Sharma Arundhati , Dwivedi Sadananad , Luthra Kalpna , Bhatla Neerja , Dhingra Renu
Published: June 2021

Introduction

Hydrogen sulphide is recently being considered as endogenous gaseous mediator which has shown to be involved in regulating many physiological processes like cell proliferation migration, invasion and phenomena like oxidative stress, angiogenesis, vasoregulation and neuromodulation. However, its role in modulating invasion in trophoblast cells, the key component of placenta is not known. Hence, in the present study we sought to explore the effects of hydrogen sulphide (H2S) on invasion capacity of trophoblast cells using HTR‐8/SVneo cell line

Material and methods

The invasion potential of HTR‐8/SVneo cells was assessed by transwell invasion assay. The Matrigel invasion assay was performed at 37°C using 24 well transwell inserts (corning Greiner Bio‐One) coated with semi solid Matrigel 10% (Becton and Dickinson). The HTR‐8/SVneo cells received various treatments and then were transferred to the transwell(Boyden's) chamber. The cells were exposed to NaHS (sodium hydrosulphide:Hydrogen sulphide chemical mimic), its inhibitor AOAA (amino‐oxiacetate) in different doses for various time periods and hypoxia /reoxygenation. Cells (2500) suspended in 200ul of serum free medium were seeded onto the upper chamber. However,400ul of 10% FBS was added to lower chamber. Matrigel was removed from upper surface. Cells that migrated and invaded through the membrane to the under surface were methanol fixed and stained by 0.5% crystal violet. The cells were counted under Nikon Ti‐S Eclipse microscope using NIS‐AR software. The percentage invasion was calculated by the formula: mean number of cells invading the Matrigel membrane (test group) / mean number of cells invading through the insert membrane (control group) X100

Results

The Percentage invasion was much higher when the cells received hydrogen sulphide mimic i.e. NaHS 100mM for 10 minutes (220%) as compared to cells which did not receive any treatment (100%). The trophoblast cells which were subjected to hypoxia reoxygenation (two cycles) showed reduction in the percentage invasion (20%). However, the cells of hypoxia reoxygenation treatment group regained their invasion capacity (46%) after they were exposed to NaHS 100 μM. On the other hand, the treatment of cells using AOAA [Cystathionine beta synthase (H2S producing enzyme) inhibitor] treatment decreased the cell invasion capacity (32%)

Conclusion

The results from present study point towards the role of H2S in invasion of trophoblast cells, thereby predicting its role if any in placentation. These findings may also indicate that decrease in H2S may contribute to the pathogenesis of preeclampsia and also the other clinical outcomes associated with it like Fetal growth restriction (FGR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional Control of Fibroblast Plasticity
Authors: Small Eric
Published: June 2021

Heart disease is associated with the accumulation of resident cardiac fibroblasts that secrete extracellular matrix (ECM) and drive the development of pathological fibrosis and heart failure. However, the mechanisms underlying fibroblast proliferation and activation remain poorly defined. To address this gap in understanding we have utilized transcriptional profiling to characterize the cardiac fibroblast phenotype in health and disease. Cardiac fibroblasts exhibit dynamic changes in genes encoding cell cycle regulators and ECM proteins. Here, we report that the cardiac fibroblast cell cycle is in part controlled by Sprr2b, a small proline‐rich protein that is induced in heart disease and extinguished during exercise. Sprr2b is a novel regulatory component of the USP7/MDM2‐containing ubiquitination complex. Sprr2b stimulates MDM2‐dependent p53 degradation, thus facilitating the proliferation of pathological cardiac fibroblasts. These findings establish a paradigm of transcriptional regulation that governs cardiac fibroblast phenotypic plasticity and the development of pathological cardiac fibrosis.

Support or Funding Information

NIH / NHLBI R01‐HL133761 and R01‐HL120919

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mast Cells Augment Neuroinflammation and Neurodegeneration
Authors: Duraisamy Kempuraj , Premkumar Keerthivass , Selvakumar Govindhasamy Pushpavathi , Thangavel Ramasamy , Ahmed Mohammad Ejaz , Zaheer Smita A , Raikwar Sudhanshu P , Dubova Luliia , Iyer Shankar S , Zaheer Asgar
Published: June 2021

Activated glial cells, and neurons express several proinflammatory mediators that are implicated in neuroinflammation and neurodegeneration in neurodegenerative diseases including Parkinson's disease (PD), Alzheimer's disease (AD) and Multiple sclerosis (MS). Mast cells are immune and inflammatory cells that are implicated in neuroinflammation by releasing several prestored and newly synthesized multifunctional cytokines, chemokines and neurotoxic mediators. Protease activated receptor‐2 (PAR‐2) expression is increased in neuroinflammatory conditions. PD is characterized by neurodegeneration of dopaminergic neurons in the brain. Here, we have investigated how mast cells can contribute to neuroinflammation, and to the pathogenesis of PD. We demonstrate that inflammatory mediators such as interleukin‐1beta (IL‐1β), tumor necrosis factor‐alpha (TNF‐α) and the chemokine (C‐C motif) ligand 2 (CCL2) were lower in the brains of mast cell deficient (MC‐KO) mice and glia maturation factor‐deficient (GMF‐KO) mice when compared to wild type mice brains after acute 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) administration. Further, we found that reconstitution of mast cells in MC‐KO mice increases neuroinflammation in the MPTP‐injected mice brains as compared to the MC‐KO brains without mast cell reconstitution. Our immunofluorescence staining revealed that the PAR‐2 expression is increased in MPTP‐injected mice brains as compared to control wild type mice brains without MPTP administration. We also show significantly increased PAR‐2 expression in the cultured primary mouse astrocytes and neurons incubated with 1‐methyl‐4‐phenylpyridinium (MPP+), inflammatory protein GMF, mouse mast cell protease‐6 (MMCP‐6), and MMCP‐7. We conclude that mast cells augment neuroinflammation and neurodegeneration in neurodegenerative diseases including PD and inhibition of mast cell activation could be a therapeutic option in neuroinflammatory and neurodegenerative disorders.

Support or Funding Information

This work was supported by Veteran Affairs Merit Award I01BX002477 and National Institutes of Health Grants AG048205 & NS073670 to Prof. Asgar Zaheer

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interferon Induces Expression of the Immune Checkpoint PD‐L1 via Bcl3 Upregulation in Ovarian Cancer Cells
Authors: Zou Yue , Uddin Mohammad , Padmanabhan Sveta , Gaire Bijaya , Vancurova Ivana
Published: June 2021

Interferon‐g (IFN) induces PD‐L1 expression in ovarian cancer (OC) cells, thus promoting OC growth. In addition to inhibiting anti‐tumor T responses, the tumor‐expressed PD‐L1 has tumor‐intrinsic effects that include increased cancer cell survival and proliferation. However, the mechanisms by which IFN induces PD‐L1 expression in OC cells are unknown. Here, we show that IFN increases gene and protein levels of the proto‐oncogene Bcl3 in ovarian cancer SKOV3 and OVCAR3 cells, resulting in their increased survival, proliferation, and migration. The IFN‐induced Bcl3 expression then promotes IFN‐mediated PD‐L1 transcription in OC cells. The mechanism consists of an IFN‐induced, Bcl3‐ and p300‐dependent PD‐L1 promoter occupancy by K314/315 acetylated p65 NFkB. Since blocking PD‐L1 by neutralizing antibody reduces proliferation of OC cells overexpressing Bcl3, these results indicate that the pro‐proliferative effect of Bcl3 in OC cells is mediated by PD‐L1. Together, our work demonstrates that IFN induces Bcl3 expression in OC cells, identifies PD‐L1 as a novel target of Bcl3, and links Bcl3 to IFN signaling and PD‐L1‐mediated immune escape.

Support or Funding Information

Sponsor: Dr. Ivana Vancurova (ASBMB member ID: 32743).

Funding: NIH CA202775 grant (to Dr. Ivana Vancurova)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Semi‐Quantitative Analyses of Protein Expression in Polyphenon E‐Treated PC‐3 Cells
Authors: Murtagh Christopher Gerard , Carastro L. Michael
Published: June 2021

In the United States, approximately 1 in 9 men will be diagnosed with prostate cancer (PCa) during their lifetime. Furthermore, PCa is the second leading cause of malignant‐related death in the United States. Polyphenon E (Poly E) is a standardized, caffeine‐free, green tea extract reported to inhibit the progression of PCa. The chemopreventive mechanisms of action for Poly E are not fully understood. In an attempt to further understand the role of Poly E in PCa, we sought to identify its potential molecular targets. The aim of this study was to detect changes in protein expression levels in Poly E‐treated PC‐3 cells. We previously obtained DNA microarray and qRT‐PCR data from PC‐3 cells treated with increasing concentrations of Poly E (100, 200, 300 mg/L) to identify potential molecular targets. We extracted total cellular protein from Poly‐E treated PC‐3 cells and performed immunoblot analysis for RGCC, ATM, RB1, and beta‐tubulin. Immunoblotting data was used in semi‐quantitation analyses of protein expression to determine fold changes in protein levels compared to the reference sample (untreated control). Immunoblotting data was normalized against beta‐actin protein loading control. Data obtained from semi‐quantification of protein levels for selected genes was compared to qRT‐PCR data obtained previously. RB1 protein expression levels for exhibited dose‐dependent decreases upon increasing Poly E treatment. At the highest concentration of Poly E treatment (300 mg/L), RB1 protein expression decreased by 44% compared to untreated cells, and was consistent with qRT‐PCR data. RGCC protein expression levels did not exhibit dose dependency, with 1.2 and 1.1‐fold increases in protein expression upon 200 and 300 mg/L Poly E treatment, respectively, which was consistent with observed trends for the corresponding qRT‐PCR data. A dose‐dependent decrease in ATM protein expression was observed for the Poly E‐treated PC‐3 cells. At the highest concentration of Poly E treatment (300 mg/L), ATM protein expression decreased 2‐fold. However, qRT‐PCR data obtained for ATM indicated marginal changes in gene expression in Poly E‐treated PC‐3 cells. Our semi‐quantitative immunoblotting data for RGCC and RB1 protein expression in Poly E‐treated PC‐3 cells was generally consistent with our qRT‐PCR data. However, the dose dependent decreased ATM protein expression observed did not comport with the lack of gene expression level changes observed by qRT‐PCR.

Support or Funding Information

This work was supported by a University of Tampa David Delo Research Professor Grant (to: L. M. C.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recombinant Immunotoxins against Latent Epstein Barr Virus Infections
Authors: Zhu Yuyi , Emmanuel Muhsinat , Weldon John E.
Published: June 2021

Epstein Barr virus (EBV), commonly associated with acute infectious mononucleosis, is an oncogenic virus implicated in thousands of cancer‐related deaths every year worldwide. Estimates suggest that 90–95% of the population worldwide are infected with EBV, but acute infections are controlled with minimal symptoms by a healthy immune system. Like all herpesviruses, however, EBV persists in a latent state even after the acute infection has cleared. Studies have shown a definitive link between latent EBV infections and the development of certain cancers such as nasopharyngeal cancer and Burkitt's lymphoma, but there are currently no treatments for latent infections. A potential treatment for both latent EBV infections and EBV‐induced malignancies is antibody therapy targeted to virus‐specific membrane proteins. We have identified two EBV‐specific membrane proteins expressed on infected cells, LMP1 and LMP2, and are developing HEK293 cell lines constitutively expressing each of these proteins. We plan to develop recombinant immunotoxins (RITs) targeted to each of these proteins. RITs are fusion proteins comprised of an antibody and protein toxin designed to identify and kill specific populations of cells. RITs using the bacterial toxin Pseudomonas exotoxin A (PE) have been extensively studied. Several RITs are in clinical trials, and one (moxetumomab pasudotox) has recently received FDA approval for the treatment of Hairy Cell Leukemia. Using a previously developed anti‐LMP1 antibody, we have begun work to clone, express, and purify an anti‐LMP1 single‐chain variable fragment antibody joined recombinantly to a cytotoxic fragment of PE. We plan to evaluate this and other RITs as potential therapeutics for EBV‐related malignancies.

Support or Funding Information

This project was supported by the Towson University's Fisher College of Science and Mathematics.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Energy and Cellular‐Defense Systems are Target for Anti‐Senescence Activity of Methylene Blue
Authors: Atamna Hani
Published: June 2021

Methylene blue (MB) delays cellular senescence, induces complex‐IV, and activates Keap1/Nrf2. The molecular link of these effects to MB and cellular senescence is unclear. We investigated the effect of MB on specific factors that affect cell senescence. Since MB is redox‐active agent, we started by assaying its effect on NAD/NADH ratio. MB caused a transient increase in NAD/NADH. This increase triggered an investigation into the energy metabolism regulator AMPK. MB induced AMPK phosphorylation in a transient pattern, which was followed by the induction of PGC1α and SURF1: both are inducers of mitochondrial and complex‐IV biogenesis. As expected from previous results, MB‐treated cells doubled complex‐IV activity. In addition, a significant decline in cellular oxidants was measured in MB treated cells. The telomeres erosion rate was also significantly lower in MB‐treated cells. A previous research suggested that the pattern of AMPK activation (i.e., chronic vs. transient) determines AMPK's effect on cell senescence. We found that the anti‐senescence activity of MB (transient activator of AMPK) was 8‐times higher than that of AICAR (chronic activator of AMPK). We investigated the effect of MB on cell cycle. We found that MB lacked an effect on cell cycle, indicating that an MB‐dependent change to cell cycle is unlikely to contribute to the anti‐senescence activity of MB. The current findings in conjunction with the activation of Keap1/Nrf2 suggest a synchronized activation of the energy and cellular defense cellular systems as a possible key factor in MB's potent anti‐senescence activity.

Support or Funding Information

NIH

AFAR

Ames

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the NCoR1 complex and its association with the molecular control of the mitochondrial biogenesis process
Authors: Silveira Leonardo R , Oliveira André G , Lima Tanes I , Guimarães Dimitrius S.P. , Favero Bianca C , Araujo Hygor N , Araujo Rafaela
Published: June 2021

Metabolic homeostasis is regulated transcriptionally through the interaction between transcription factors, co‐regulators and proteins of the basal transcriptional complex. Nuclear co‐regulators work either as transcriptional repressors or as transcriptional activators and, therefore, are strictly regulated to synchronize metabolic pathways to environmental stimuli. Preliminary results from our laboratory have indicated that the NCoR1 co‐repressor plays an important role in the regulation of mitochondrial biogenesis and oxidative metabolism in muscle cells. During the differentiation process of C2C12 muscle cells, the expression of the NCoR1 repressor is reduced favoring the process of mitochondrial biogenesis. While, overexpression of NCoR1 negatively affects the oxidative characteristics of these cells. These observations suggest that NCoR1 plays a central role in binding the nuclear receptor to the transcriptional program of mitochondrial biogenesis. In this way, we are proposing here study to identify and characterize NCoR1 protein (s) involved in the regulation of mitochondrial biogenesis in MEF cells. Thus, we hope to identify potential therapeutic targets in the management of obesity and diabetes. The design will be performed through the following experimental steps: 1) Co‐immunoprecipitation/proteome of the NCoR1 complex; 2) In silico analysis of target candidates; 3) Identification of the target through functional/biochemical tests. Finally we intend to explore the target interface/NCoR1 as therapeutic potential in experimental models of insulin resistance/obesity. The overexpression of NCoR1 was confirmed by PCR and western blotting (Figure 1). After NCoR1 co‐imunoprecipitation (Fig. 2), cellular extract was submitted to mass spectrometry (LC‐MS/MS ) analysis and at least 15 targets were identified (Table 1). The identified proteins were listed according to (A) cellular components; (B) Molecular function; (C) Biological process (Fig. 3). Interactome analysis also demonstrated physical interaction among NcoR1 and indentified proteins including Hdac3, Tbl1xr1, GPS2 and Chd8 (Fig. 4). This analysis revealed potential candidates to control mitochondrial function/biogenesis in MEF cells.

Support or Funding Information

This work was supported by grant from Fundação de Amparo à Pesquisa do estado de São Paulo – FAPESP (2016/23008‐5), Capes and CNpq.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Suppression of NLRP3 inflammasome activation in LPS‐induced RAW264.7 cells by natural compound Lava Spirulina maxima extract
Authors: Chei Sungwoo , Song Ji‐Hyeon , Seo Young‐Jin , Lee Kippeum , Jin Heegu , Oh Hyun‐Ji , Oh Jae‐Hoon , Lee Boo‐Yong
Published: June 2021

Spirulina is microscopic filamentous cyanobacteria. Although the Spirulina was recently reported to exert beneficial effect such as reducing cholesterol or weight loss, the correlation of Spirulina and inflammatory response has not been confirmed. The interesting about effect of functional food or nutritional value of blue‐green algae has been increased. Spirulinal maxima was recently reported to exert beneficial effect which contains high amounts of protein, lipids, essential amino acids, minerals, vitamins, photosynthetic pigments, and biologically active substance such as phycocyanin, chlorophyll, beta‐carotene. We choose RAW264.7 cell line, murine macrophages cell to evaluate the effect of Spirulina maxima. We demonstrated that Lava seawater Spirulina maxima 70% ethanol extract (LSME) effectively inhibited the Lipopolysaccharide (LPS) ‐ induced inflammatory response through antioxidant activity. It is also decrease ROS production by down regulating oxidant. LSM is the source of antioxidant which can protect against oxidative damage. Tumor necrosis factor‐alpha (TNF‐α) and interleukin 1β (IL‐1β) is decreased the LPS‐induced overproduction by LSME. We demonstrated that reducing the NLRP3(Nucleotide‐binding oligomerization domain) inflammasome through inhibit MAPK pathway by LSME.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Cellular Pathways of Saturated Fatty Acid Toxicity
Authors: Nilsson Hannah , Brown Margaret , Inwards Emily , Stoeckman Angela , Listenberger Laura
Published: June 2021

Excess long‐chain saturated fatty acids cause cell dysfunction and death, which has been linked to obesity‐related health problems like heart disease and type II diabetes. In this study, we aimed to determine if the incorporation of long‐chain saturated fatty acids into phospholipids in the endoplasmic reticulum (ER) triggers ER stress and lipotoxic cell death. We treated AML12 and NIH3T3 cells with the saturated fatty acid palmitate under conditions that reduced phospholipid synthesis. While culturing cells in the absence of choline decreased phosphatidylcholine synthesis through the Kennedy pathway, choline depletion did not prevent palmitate induction of reactive oxygen species or mitochondrial dysfunction. We also examined palmitate toxicity in the presence of tubercidin, an inhibitor of the PEMT pathway of phosphatidylcholine synthesis and ATK, a phospholipase A2 inhibitor known to decrease phospholipid recycling through the Lands Cycle. Neither inhibitor prevented the changes in mitochondrial function indicative of palmitate toxicity. Therefore, these results do not support the hypothesis that long‐chain saturated fatty acids cause lipotoxicity by changing the cellular composition of phospholipid membranes. Additional experiments are needed to identify lipotoxic pathways.

Support or Funding Information

This work was supported by the St. Olaf College Office of Collaborative Undergraduate Research and Inquiry and a Cayman Chemical undergraduate research fellowship.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular mechanisms of STARD3 addiction in HER2 tumors
Authors: Voilquin Laetitia
Published: June 2021

Human Epidermal growth factor Receptor 2 (HER2) is overexpressed and amplified in approximately 15–20% of breast cancers. HER2 is a tyrosine kinase receptor belonging to the EGFR family. HER2 amplification and overexpression play a central role in initiation, progression and metastasis of common cancers like lung, breast and gastric cancer (Hynes and MacDonald, 2009). HER2 amplification is a marker of poor prognosis, especially in breast and gastric cancer (Slamon and Clark, 1988). Although anti‐HER2‐targeted therapies have shown significant clinical benefits, de novo and acquired resistance remain a considerable issue (Garrett and Arteaga, 2011). Genomic profiling has demonstrated that HER2 positive‐tumors have additional alterations, and more importantly they co‐amplify and co‐express several genes on the 17q12 locus (Bièche et al., 1996; Kauraniemi et al., 2001; Tomasetto et al., 1995). Among these genes co‐expressed with HER2, our team identified and characterized STARD3, a gene belonging to the HER2 amplicon (Alpy and Tomasetto, 2014).

STARD3 is a lipid transfer protein belonging to the START domain protein family (Alpy et al., 2001). This protein has two domains: the MENTAL domain anchors the protein in late endosomes and the cytoplasmic START domain allows cholesterol transport. Moreover, an FFAT motif present at the interface between the two domains of STARD3 interacts with VAP or MOSPD2 proteins localized in the endoplasmic reticulum (ER) (Alpy et al., 2013; Mattia et al., 2018). Thus, this interaction creates a membrane contact site between late endosomes and the ER allowing then the transfer of cholesterol towards late endosomes (Alpy et al., 2013; Wilhelm et al., 2017). Also, STARD3 has been functionally implicated in the survival of HER2‐amplified cells. Indeed, inhibition of STARD3 expression by RNA interference limits cell growth only in HER2‐amplified cells (Alpy and Tomasetto, 2014; Sahlberg et al., 2013). Thus, STARD3 belongs to the HER2 smallest region of amplification and its expression suggests that it contributes to HER2 tumor growth. We think that STARD3 cooperates with HER2 for tumor growth through an indirect molecular mechanism. Our results suggest that STARD3 acts on cancer cells by altering the normal cholesterol distribution and modifying the lipid composition of cellular membranes.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Assessments of Blood Lipids, Lipid Peroxidation, Antioxidant Status, and Fatty Acids in Women with Uterine Tumors during Menopausal Period
Authors: Kotrikadze Nanuli , Tufinashvili Tamar , Ramishvili1 Liana , Alibegashvili Manana , Nakashidze Irina , Nemsadze Gia , Gordeziani Manana , Khazaradze Ana , Sepiashvilli Bela , Ahmad Sarfraz
Published: June 2021

Objective

To study alterations in lipid spectrum, lipid peroxidation intensity, and fatty acid (FA) spectrum in blood of women with benign & malignant uterine tumors during menopausal period.

Methods

Blood samples from benign (myoma, n=15) and malignant (n=15) cases were collected, along with healthy menopausal (control, n=15). Spectrophotometric methods assessed lipid spectrum composition, antioxidant enzymes activities & lipid peroxidation intensity. Quantitative analyses of FA were performed by gas‐liquid chromatography.

Results

Saturated FA in malignant cases: decreased myristic acid (3‐fold), palmitic acid (~1.3 fold) compared to control, but stearic acid increased (1.13‐fold). The decreased ratio between palmitic/stearic acids (C16:0/C18:0) by ~1.5‐times was noted. Unsaturated FA in malignant cases showed a percent decrease in oleic (2.9‐fold), linolenic (1.5‐fold), eicosatrienoic (3‐fold), arachidonic (1.3‐fold) acid compared to control. The increased ratio between stearic/oleic acids (C18:0/C18:1) by 3.3‐times was noted in malignant cases. Increase in lipid peroxidation intensification was associated with lipid spectrum composition (hyperlipidemia, disruption in the exchange rate of phospholipids). The negative correlation between lipid peroxidation intensity & phospholipids was r=−0.96±0.04. Total cholesterol was higher in malignant cases. Total amount & activities of superoxide dismutase (SOD), catalase (CAT) decreased in malignant cases. The negative correlation between total cholesterol & SOD, CAT activities was r=−0.99±0.014. Amine‐containing phospholipids decreased but choline‐containing phospholipids increased with the severity of the disease.

Conclusions

Intensification of lipid peroxidation & alterations in lipid spectrum composition revealed hyperlipidemia, disruption of phospholipids' exchange rate and hypercholesterolemia. Thus, specific changes in these biochemical parameters can be useful in the differentiation of benign and malignant uterine tumors for menopausal women.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypolipidemic and anti‐atherogenic effect of omega‐3 enriched structured lipids from linseed oil (LSO) with refined palm olein oil (RPOO) on rats fed atherogenic diet
Authors: Dhavamani Sugasini , Thilagam Kasthuri , Prasanth Kumar PK , Shanmugam Kathiresan , Chidamabaranathan N , Kavitha B , Prabakaran K , Kanchana S
Published: June 2021

The anti‐atherogenic effect of omega‐3 enriched structured lipids prepared by lipase catalyzed enzymatic intersterification with refined palm olein oil (RPOO) and linseed oil (LSO) was studied in rats. LSO is a good source of short chain omega‐3 fatty acid (alpha linolenic acid, ALA, omega‐3 FA) whereas an RPOO contains a higher amount of palmitic acid (SFA) and oleic acid (MUFA). But it is lacking of omega‐3 fatty acids. Based on standardization with different ratios, the ideal combination was selected based on the fatty acid composition and TAG molecular species of oils. The structured lipids (SL) of RPOO with FXO (40:60) has obtained an ideal SFA:MUFA:PUFA ratios near to 1:1:1 and enriched with omega‐3 fatty acids. This redisposition of fatty acids boost the functional properties and beneficial for cholesterol lowering properties. The feeding experiment conducted on rats fed on normal and atherogenic diet containing 10% omega‐3 SL (experimental) and RPOO(control‐native oil) indicated no adverse effects on growth and food intake. Rats fed Omega‐3 enriched structured lipids showed a decrease in cholesterol as compared to RPOO in atherogenic diets. However, the omega‐3 structured lipids potentially lowered plasma, hepatic and aortic lipids, predominantly cholesterol, LDL +VLDL cholesterol and triglycerides. Feeding of an omega‐3 enriched structured lipids resulted in the accretion of long‐chain omega‐3 PUFA and decrease in the long chain omega‐6 PUFA in plasma, hepatic and aortic lipids. The lipid accumulation observed in the aorta of rats fed atherogenic diet with Omega‐3 enriched structured lipids was considerably reduced as compared to natural oil of RPOO. However, the Omega‐3 enriched Structured lipids exhibited better incorporation of omega‐3 fatty acids and beneficial for cardiac function and reduce the risk for cardiovascular diseases.

Support or Funding Information

none

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Pasteurization Types on the Content and Isomerization of Conjugated Linoleic Acid in Milk
Authors: Hurst Sarah , Bunde Terry
Published: June 2021

The world is currently facing an epidemic of giant proportions – diet‐induced obesity (DIO) and associated co‐morbidities (type 2 diabetes, hypertension, certain cancers, etc.). While diet and exercise are the gold standards for treatment and prevention of DIO, isomers of conjugated linoleic acid (CLA) (cis‐9/trans‐11 and trans‐10/cis‐12) have been shown to reduce body fat mass and increase lean tissue mass, and more importantly, aid in the treatment of a host of co‐morbidities. Apart from supplementation, meat and dairy products from ruminants (cows, sheep, and goats) are the major source of CLA for humans. However, CLA composition ranges greatly in these products as processing i.e. cooking, pasteurization, etc. alters the content of CLA as well the isomers themselves. As the extend of these changes are unknown, the effects of time and temperature on CLA isomerization in milk through varying pasteurization types was investigated. First, raw milk samples were obtained from a local farm, and subjected to three different types of pasteurization – vat, ultra, and high temperature, short time pasteurization. Results show that the ultra‐high temperature pasteurized sample had more trans/trans isomerization than the raw sample, while the other samples were comparable to the raw. Therefore, as temperature increased, the extent of CLA isomerization also increased. Further experiments using thermochemistry and kinetics should be conducted to identify the times and temperatures at which each isomer is converted or formed. This knowledge could then be used to produce more beneficial CLA within a given sample and to decrease the undesired trans/trans CLA.

Support or Funding Information

Support for this work was provided by the Department of Chemistry, Maryville College.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.