bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Roles of the Essential Proteins Dib1, Prp31, Prp6 and the U5 snRNA During Splicing
Authors: Embry Addie , Potts Camille , Jamison Danielle , Turner Caitlyn , Maeder Corina 
Published: June 2021

Pre‐mRNA splicing is a conserved biological process essential to all eukaryotes. Splicing defects lead to a variety of disorders in humans including Retinitis Pigmentosa and Burn‐McKeown Syndrome, defining the importance of splicing in health science fields. Pre‐mRNA splicing requires complex machinery composed of five small nuclear RNAs (snRNAs) complexed with ~90 proteins that form small nuclear ribonucleoproteins. Spliceosome assembly occurs over several dynamic steps to form a pre‐catalytic B complex before splicing reactions can occur.Splicing cannot begin until the pre‐mRNA and U5 snRNA can interact. Analysis of cryo‐EM structures (Plaschka, et al. 2017) as well as biochemical and genetic studies reveal three essential proteins in the B complex blocking a pre‐mRNA‐U5 snRNA interaction. At the center of the catalytic site sits Dib1, a 17‐kDa protein with a thioredoxin‐like (TRXL) domain. Prp6 is a large, 102‐kDa U4/U6.U5 triple‐snRNP protein whose N‐terminal region appears to interact with the hydrophobic pocket located on one side of the Dib1 TRXL domain. Prp31 is another large, 56‐kDa protein that appears to interact with a basically charged region of Dib1 opposite of the hydrophobic pocket via acidic residues on its extended loop region. Crosslinking studies (Dix, et al. 1998) revealed that the U5 snRNA loop 1 interacts with a 16‐kDa protein and cryo‐EM studies of B complexes revealed U5 loop 1 may interact with Dib1.Prp6, Prp31, and Dib1 must all depart the spliceosome before splicing can occur. The interactions between these proteins and the U5 snRNA may serve to stabilize the spliceosome and prevent the premature splicing of pre‐mRNA. Here, we aim to identify temperature sensitive (ts) growth mutants of Prp6, Dib1, Prp31 and U5 snRNA that inhibit or abolish splicing and identify the mutants' roles in the assembly of the B complex and the U4/U6. U5 triple snRNP. Studies are performed in S. cerevisiae, an excellent model to study splicing. Our findings of key residues in Prp6, Prp31 and U5 snRNA that are important for interactions with Dib1 will be presented as well as the effects of these novel mutants on splicing and spliceosome assembly.Support or Funding InformationNIH‐NIGMS R15GM120720 and the Welch FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Influence of PIP2 on IDE and UCP2 in RIN‐5F Cells”
Authors: Clemons Tameka Arlene 
Published: June 2021

Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) is present on the plasma membrane of cells and is the precursor to inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG). Although PI(4,5)P2 has traditionally been considered the substrate for phosphatidylinositol 3‐kinase (PI3Kinase) or phospholipase C (PLC), PI(4,5)P2 has recently been shown to bind Insulin Degrading Enzyme (IDE). IDE is a cytosolic protein that has been shown to be anchored by PI(4,5)P2 until it is ready to perform its function as a protease. IDE serves as a protease for several proteins including insulin and amylin. Insulin and amylin are two proteins that are co‐secreted from pancreatic β cells after a rise in cellular glucose levels. The degradation of insulin and amylin occur by IDE after the two proteins have completed respective actions, most notably the lowering of glucose levels and the increase in satiety. A rise in glucose is known to lead to a rise in insulin and amylin levels in order to maintain metabolic homeostasis. Uncoupling Protein 2 (UCP2) is a mitochondrial membrane protein that is known for its impact on the oxidative phosphorylation process. The presence of UCP2 on pancreatic β cells has previously been shown to have an inverse relationship with insulin. Here we conducted IDE, amylin, and UCP2 ELISA assays before and after inhibition of PLC or PI3Kinase under varying concentrations of glucose in RIN5F cells, which is a rat pancreatic β cell line, in order to investigate the impact of IDE on the activity level of amylin and UCP2 and to establish a possible relationship between amylin and UCP2. In this study, we measured IDE, amylin and UCP2 levels with the PLC inhibitor, U73122, and PI3kinase inhibitor, wortmannin, in comparison to the negative controls. The initial data shows that a decrease in IDE occurs in the presence of the inhibitor and suggests a possible link between IDE, UCP2, and amylin.Support or Funding InformationNational Science Foundation Research Initiation Award# 24430This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MAPK Signaling Influenced by Oxygen Environments
Authors: Forsberg Kersten , Chrestensen Carol 
Published: June 2021

Mammalian cells thrive in a variety of different environments. In tissue culture there are many variables cells are exposed to; the impact of oxygen gas is one that is not well understood in relation to signaling pathways. In tissue culture, cells are commonly grown in atmospheric oxygen (~21%), yet the environment of many cells in vivo is considerably less (1% – 14% depending on the cells type). The increased oxygen content can cause higher levels of reactive oxide species (ROS). When ROS overwhelm the cell, normal cellular responses are affected. Extracellular kinase 1 and 2 (ERK 1/2) are proteins know to be directly modified by ROS, sometimes resulting in kinase inhibition. In this study we examined responses of the MAPK pathway to epidermal growth factor (EGF) in normoxic (6% O2) and atmospheric (21% O2) conditions. Baby hamster kidney (BHK‐21) or Human Dermal Epithelial (HMEC‐1) cells were grown and maintained in atmospheric conditions. The cells were then seeded and either kept under atmospheric conditions (normal tissue culture conditions) or moved to 6% oxygen in a Bactrox™(with all other conditions remaining constant). These cells were grown for two days until approximately 80–90% confluent and then they were serum starved and treated with EGF (0–30 minutes) in the two environments. They were harvested on ice in their respective environments. The lysates were then removed and frozen. After lysis the two conditions were treated in atmospheric oxygen. We hypothesized that the ERK 1/2 pathway responses would be different under the conditions of 6% versus 21% oxygen. ERK 1/2 phosphorylation status was assessed in parallel with kinase activity. Also explored was the difference in cell growth between the two environments. Our results show subtle differences in the peaks of phosphorylation and in the threshold of responses as well as differences in activity of the harvested ERK. Our work will lead to an enhanced understanding of how cellular signaling pathways are impacted by oxygen levels.Support or Funding InformationNIH grant # R15 GM110634‐01A1 to C.A.C., and Kennesaw State 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.
Re‐polarization of tumor‐associated macrophages via reconstituted high‐density lipoprotein nanoparticles
Authors: Dossou Akpedje Serena , Sabnis Nirupama , Raut Sangram , Dasseux Jean‐Louis , Lacko Andras G. 
Published: June 2021

Phenotypic plasticity of macrophages allows them to assume different functional profiles as they are called upon to respond to biological events. For example, the M1 phenotype of macrophage potentiates pro‐inflammatory and anti‐tumoral properties whereas the M2 phenotype is associated with immuno‐regulatory, tissue repair and pro‐tumoral properties. Tumor‐associated macrophages (TAMs) initially present anti‐tumoral behaviors via their “M1” phenotype. Under the stimulation of factors secreted by the malignant tumor, TAMs progressively take on a pro‐tumoral role via polarization to an “M2” phenotype. The M2 phenotype which supports immune‐dampening activities helps sustain tumor proliferation, immune system evasion, angiogenesis, tissue invasion and metastasis. TAMs, thus, make attractive targets for an anti‐cancer immunotherapy strategy. Considering the whole‐body distribution of macrophages, and their critical role in immune response, we propose to pursue the repolarization of M2‐type macrophages in the tumor microenvironment toward an M1 phenotype, utilizing a targeted drug delivery carrier.Lipoproteins are natural transporters of hydrophobic substances in the blood, including lipids, hormones and drugs. Our lab developed a biocompatible reconstituted high‐density lipoprotein (rHDL) nanoparticle with properties highly similar to the native HDL. Dynamic light scattering analysis and transmission electron micrographs have confirmed a sub‐50 nm size for our particles and a spherical shape. In addition to siRNA molecules, hydrophobic drugs such as paclitaxel and valrubicin were successfully encapsulated by the rHDL nanoparticle. Photophysical studies showed that as a carrier, the rHDL holds its cargo at its core. Receptor blocking studies showed that scavenger receptor, class B type 1 (SR‐B1) expressed on the surface of the target cell mediate the intracellular delivery of the nanoparticle's payload by interacting with ApoA1, a major protein component of the nanoparticle. Also, protein expression analysis showed that M2‐type macrophages exhibit higher levels of SR‐B1 compared to M1‐type macrophages. The rHDL nanoparticles can transport hydrophobic small molecule inhibitors or non‐coding RNAs often used in subtype reversal/re‐polarization (M2 to M1) strategies. Most importantly, the SR‐B1 expression profile of M2‐type macrophage indicates that the rHDL is particularly suited for selective targeted delivery of these agents to TAMs.Support or Funding InformationCerenis TherapeuticsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The CH2 Domain of Pseudophosphatase MK‐STYX Regulates Neurite Outgrowth
Authors: Reed Kirstin , Zhang Ashley , Whitehurst Wesley , Hinton Shantá D. 
Published: June 2021

Pseudoenzymes are emerging as key regulators in signal transduction cascades. More specifically, pseudophosphatases have been implicated in diseases such as Alzheimer's disease, cancer, metabolic disorder, and obesity, highlighting their importance in cellular processes. The unique catalytically inactive member of the mitogen‐activated protein kinase phosphatases (MKP) family, MK‐STYX (mitogen activated kinase phosphoserine/threonine/tyrosine binding protein), is a regulator in the stress response, apoptosis, and neuronal differentiation. We previously reported that MK‐STYX increases neurite outgrowths in PC‐12 cells through the RhoA signaling pathway. Recently, we found that MK‐STYX overexpressing cells produce more growth cones than control cells. Furthermore, immunoblots show that MK‐STYX overexpressing cells have increased actin expression, which is sustained in the presence or absence of nerve growth factor. These MK‐STYX‐induced neurites have post‐synaptic and pre‐synaptic properties when stained with the classical axon and dendrite markers, Tau‐1 and MAP2 (microtubule‐associated protein 2) respectively. Furthermore, a dopamine assay demonstrated that MK‐STYX‐induced neurites are dopaminergic. In primary neurons, MK‐STYX causes morphological changes, such as axons and dendrites being visibly indistinguishable. MK‐STYX also elicits a significant increase in the number of neurites per neuron in hippocampal primary neurons. Thus, it is apparent that MK‐STYX has a role in neuronal differentiation. To investigate the molecular mechanism that MK‐STYX utilizes to cause neurite formation, PC‐12 cells expressing GFP‐tagged truncated versions of MK‐STYX rhodanese (cdc 25 homology; CH2) or dual specificity phosphatase (DSP) domain and stimulated with NGF were analyzed for neurite outgrowths. Cells expressing the CH2 domain formed a significant number of neurites, whereas cells expressing only the DSP domain produced no neurites. Furthermore, cells expressing the DSP domain of MK‐STYX had significantly fewer neurites compared to cells expressing wild‐type MK‐STYX. Taken together, these data provide evidence that MK‐STYX is a regulator in neurite formation and that the CH2 domain is key for regulating neurite differentiation while the DSP domain may be the accessory domain. It also highlights that this unique member of the MKP subfamily potentially has a major role in neuronal signaling.Support or Funding InformationGrace Blank Award; The Mary E. Ferguson Memorial Research GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PUFA supplementation may confer enhanced metabolic capacity in stimulated skeletal muscle cells
Authors: Symanietz Sarah , Conley Emily , Blair Jennifer , Petros Mera , Schlater Amber E 
Published: June 2021

Skeletal muscle is an energetically demanding tissue that catabolizes a variety of different substrates, a phenomenon that is largely activity‐dependent. Lipids are an energetically‐rich macromolecule and, in this light, are advantageous for skeletal muscle performance and maintenance. It has long been established that lipids have dichotomous lasting effects on the body that appear to be, at least in part, species‐specific. While lipid‐related pathologies are largely associated with species such as saturated fatty acids, diacylglycerols, and ceramides, other lipid species, such as various unsaturated fatty acids, confer adaptive changes and may actually protect against diseases. Lipids have previously been linked to increased aerobic capacity in skeletal muscle, though the role of fatty acid quality in this context (e.g., degree of saturation) is less clear. In the present study, we used C2C12 cells, an immortalized line of mouse skeletal muscle cells, to better understand how polyunsaturated fatty acids (PUFAs), in combination with exercise, affect metabolism and protein concentration in skeletal muscle. Following standard proliferation protocols, mature myotubes were differentiated for 7 days in either control media or media supplemented with a 5% addition of linoleic acid (PUFA); additionally, to stimulate contractile activity, a subset of cell cohorts from each media group were exposed to caffeine for 20 minutes per day during differentiation days 4–7. All cells were harvested for protein on differentiation day seven. Protein concentration across cohorts was measured using a Bradford protein assay. Metabolic changes were characterized by running functional enzyme assays for lactate dehydrogenase (LDH) and citrate synthase (CS) to compare anaerobic and aerobic changes, respectively. Preliminary data show that non‐exercised cohorts have higher protein concentration when supplemented PUFA, and that exercise, both with and without PUFA supplementation, decreases protein concentration as compared to respective non‐exercised cohorts. Regarding enzyme activity, PUFA supplementation appears to decrease LDH activity in non‐exercised cells, while PUFA supplementation appears to increase LDH activity in exercised cells; CS activity also appears to change in PUFA supplemented cohorts. Collectively, these preliminary data suggest activity‐induced adaptive changes in skeletal muscle metabolism are contingent upon specific substrate availability, whereby PUFA availability may advantageously prime skeletal muscle for a higher overall metabolic capacity.Support or Funding InformationMcNair Scholars Program and The College of Saint ScholasticaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Generation of a tetracycline‐inducible NKCC2 expressing MDCKI cell line
Authors: Rosenbaek Lena Lindtoft , Rizzo Federica , Staub Olivier , Fenton Robert 
Published: June 2021

In the kidney, the sodium chloride potassium cotransporter NKCC2 of the thick ascending limb (TAL) of the loop of Henle plays an important role in regulation of body salt and water homeostasis. Due to a lack of a suitable polarized epithelial cell line model, the majority of functional and cell biological studies of NKCC2 have been performed in isolated TAL tubules or following expression of NKCC2 in xenopus oocytes. The aim of this study was to generate a MDCKI cell line with stable and tetracycline‐inducible expression of NKCC2. Our previous studies using this approach have demonstrated that this is an excellent approach to study another member of the same protein family, the NaCl cotransporter NCC. FRT‐MDCKI cells transfected with the A‐isoform of human NKCC2 (hNKCC2‐A) had robust NKCC2 mRNA expression after 24 h of tetracycline induction. Enhanced NKCC2 protein expression was achieved by additional treatment of cells with the histone deacetylase inhibitor, valproic acid in conjunction with tetracycline. PNGase F treatment of cell lysates determined that NKCC2 was expressed as highly glycosylated monomers and dimers. NKCC2 was observed both intracellularly and at the apical pole of MDCKI‐hNKCC2‐A cells using immunofluorescence labeling and confocal microscopy. Cycloheximide chase studies determined that the mature form of NKCC2 has a half‐life of around 12 hours, whereas the immature, non‐glycosylated form of NKCC2‐A has a much shorter half‐life. NKCC2 levels in the apical membrane increased with forskolin or hypotonic low chloride stimulation, which were accompanied with significantly increased phosphorylation at Ser126 or Thr96, respectively. We conclude that the MDCKI‐hNKCC2 A cell line is an excellent model for studying NKCC2 function and regulation.Support or Funding InformationDFF ‐ Danish Council for Independent Research, Medical SciencesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluorescence resonance energy transfer (FRET) based methods to study DNA topology and topoisomerases
Authors: Leng Fenfei 
Published: June 2021

Previously, we reported the synthesis of a type of unique fluorescence‐labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET) (Gu et al, Scientific Reports, 2016, 6:36006). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 or FL924 carrying a 42 nt. AT sequence with fluorescein and dabcyl or TAMRA and BHQ2 labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905 or pAB1_FL924. Since the fluorescence intensity of pAB1_FL905 and pAB1_FL924 is dependent on its supercoiling status, these DNA molecules are powerful tools to study DNA topology and topoisomerases by FRET. Recently, we are able to use these unique plasmid DNA molecules to study kinetics of different DNA topoisomerases and find that all DNA topoisomerases follow the classical Michaelis‐Menten kinetics and have unique steady‐state kinetic parameters, KM, Vmax, and kcat. We also developed high throughput screening (HTS) assays to identify inhibitors/activators targeting different DNA topoisomerases including bacterial DNA gyrase and topoisomerase I.Support or Funding Information1R21AI125973‐01A1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Links Between the Autophagy Machinery and Cellular Homeostasis
Authors: Levine Beth , Liu Yang , Fernandez Alvaro Fernandez 
Published: June 2021

The autophagy machinery is best‐known for its roles in targeting unwanted cargo for lysosomal degradation and other intracellular membrane trafficking events. In this talk, I will discuss our work identifying two novel nodes of molecular cross‐talk between the autophagy protein, Beclin 1, and cellular metabolism and homeostasis. Beclin 1 is a scaffold protein that regulates the lipid kinase activity of class III phosphatidylinositol 3‐kinase complexes that function in autophagy initiation and autophagosomal/endosomal maturation. Recently, we found that the function of Beclin 1 in cellular metabolism and homeostasis extends beyond autophagy and endocytosis. We identified an interaction between an innate immune sensor and Beclin 1 that regulates exercise‐induced activation of AMPK, a master regulator of cellular energy homeostasis, AMPK. We also identified a role for an interaction between Beclin 1 and the Na+,K+‐ATPase pump in the regulation of life and death decisions of the cell during tissue ischemic injury. Together, these findings suggest that autophagy proteins play a multifunctional role in cellular energy regulation and homeostasis.Funding Sources:National Institutes of HealthCancer Prevention Research Institute of TexasFondation LeducqSupport or Funding InformationNational Institutes of Health Cancer Prevention Research Institute of Texas Fondation LeducqThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydralazine Improves Mitochondrial Function via SIRT1/SIRT5 Activation and Protects against Glucose Toxicity
Authors: Mirzaei Hamid Harrison , Dehgan Esmaeil 
Published: June 2021

Mitochondrial dysfunction is one of the hallmarks of aging. Improving mitochondrial activity has been a prime target for improving organismal fitness and longevity and in many cases a potential treatment for age‐related diseases. In this study, we demonstrate that hydralazine, an FDA approved antihypertensive drug, improves mitochondrial function in a SIRT1/SIRT5‐dependent manner. We show that hydralazine treatment alters central carbon metabolism, increases TCA cycle metabolites, and improves respiration inC. elegans. Our data suggest that hydralazine‐induced mitochondrial activation is essential for the drug's prolongevity effect in C. elegans. We demonstrate that under glucose stress, hydralazine treatment restores locomotor performance, mitochondrial health, and lifespan in C. elegans. These new insights into hydralazine's ability to alter the mitochondrial activity and metabolic homeostasis deepen our understanding of hydralazine's mechanism of action and its potential health benefits and side effects.Support or Funding InformationThis work was supported by the National Institutes of Health (grant R03AG045504 to H.M.), Robert A. Welch Foundation (grant I‐1850 to H.M.) and the Cancer Prevention and Research Institute of Texas (grant R1121 to H.M.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.