bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uridylated Histone mRNA Degradation Intermediates Bind SLBP and 3′hEXO Individually and Together
Authors: Harris Sarah E. , Mihailescu Mihaela‐Rita , Lackey Patrick E. 
Published: June 2021

Histone mRNAs are a tightly cell‐cycled regulated group of mRNAs that are strongly expressed at the beginning of S‐phase and rapidly degraded when DNA replication ends. This cell cycle regulation is controlled by histone mRNA's unique 3′ end; instead of the traditional poly(A) tail, histone mRNA instead ends with a 3′ stem‐loop. This stem‐loop is an important factor in controlling the expression and degradation of histone mRNA during S‐phase, along with two key protein binding partners: the stem‐loop binding protein (SLBP) and the human 3′ exonuclease (3′hEXO). At the beginning of degradation, 3′hEXO trims 2 to 7 nucleotides from the 3′ end of the message. These nucleotides are then often replaced by a short uridylation that preserves the length of the stem‐loop. The exact function of these uridylated intermediates and whether they affect histone mRNA metabolism is currently unknown. We characterized the structure of a stem‐loop with seven nucleotides trimmed from the 3′ end and replaced with five uridines using nuclear magnetic resonance (NMR) to show that a stem‐loop still forms, despite this trimming‐and‐replacement mechanism that turns the six‐membered stem into a nine‐membered stem. We also characterized interactions between SLBP, 3′hEXO, and the trimmed and uridylated RNA using electrophoretic mobility shift assays (EMSA) and found that the RNA interacts with both proteins individually and in a ternary complex. As SLBP must dissociate from the stem‐loop before total degradation can progress, the formation of a degradation intermediate that binds SLBP raises new questions about the nature of the interactions and molecular conformation of the uridylated stem‐loop during binding. Additionally, ternary complex formation with the uridylated stem‐loop raises questions as to how these protein/stem‐loop interactions will change upon the addition of known degradation intermediates. Understanding the interactions between a uridylated histone mRNA degradation intermediate and binding proteins will help us better understand the nature of these unique mRNA molecules.Support or Funding InformationThe National Science Foundation CHE‐1659821 (REU)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Molecular Model of the Alpha Globin/eNOS Complex
Authors: Keller T.C. Stevenson , Columbus Linda , Isakson Brant E 
Published: June 2021

Nitric oxide (NO) signaling is a primary mechanism of vasodilation that has therapeutic potential for hypertensive pathologies. A key regulator of vascular wall nitric oxide production is the expression of alpha globin in the endothelium. We have previously shown that a small peptide mimicking alpha globin binds directly to the oxygenase domain of endothelial NO synthase (eNOS), competes with the full‐length protein, and thereby decreases NO scavenging by alpha globin. A molecular model of this interaction informs the mechanism of NO signaling inhibition by alpha globin and provides a framework for continued development of anti‐hypertensive therapies.The residues on alpha globin that determine its interaction with eNOS are known, but the binding site on eNOS is yet undescribed. To determine the motif on eNOS that interacts with alpha globin, a map of the interface is described via crosslinking mass spectrometry with proteolytic cleavage. Differential cleavage patterns after orthogonal protease treatments show eNOS peptides that are modified by alpha globin crosslinking. The sequence of proteolysis products modified by crosslinking are identified by high‐resolution LC‐MS, and the interaction model is reconstructed in silico. Using disulfide trapping, we can validate the orientation of the molecular model with full‐length proteins in an overexpression culture system using mammalian and primary cell lines. Overall, the molecular understanding of this complex informs the mechanism of alpha globin inhibition of NO signaling in the endothelium.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 the effects of FATP2 gene deletion on fatty acid‐responsive gene expression in liver
Authors: Gabell Jeffrey T , Perez Vincent , Black Paul , DiRusso Concetta 
Published: June 2021

Fatty Acid Transport Protein 2 (FATP2) is highly expressed in the small intestine, liver, kidney, pancreas, and placenta. FATP2 has two activities: [1] it facilitates the uptake of long chain fatty acids into the cell and [2] it activates very long chain fatty acids destined for incorporation into cellular lipids or β‐oxidation. Previous work from this laboratory implicates FATP2 activity in the promotion of lipotoxicity that may lead to cell death. The present studies employ mice lacking the FATP2 gene (FATP2−/−) to more fully understand the function and effects of FATP2‐dependent fatty acid uptake on liver metabolism and lipotoxic disease. We hypothesize a reduction in uptake and metabolism of fatty acids dependent upon FATP2 may lead to alterations in expression of genes that are responsive to exogenous fatty acids. Gene expression was measured in wild type and FATP2−/− livers using quantitative PCR and validated using western blots to assess the impact of genotype, diet, and fasting period. Preliminary data shows that expression of FABP1, CD36, Cyp4A10, and ACOX are elevated in livers of FATP2−/− mice independent of dietary fat levels. In contrast, expression of NPC1L1 was decreased. Differences between diets alone resulted in an increase in FABP2 and FABP4, FAS, Serpinb6 and ApoB expression, while FABP1, CD36, and ACOX expression was dependent on both genotype and diet. These results further suggest deletion of FATP2 increases PPARa signaling, perhaps through increases in a native fatty acid ligand. Overall, these studies further demonstrate an important role for FATP2 in liver lipid homeostasis during high fat dietary challenge.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Potential Specificity Code for B. subtilis SsbA and SsbB Quaternary Structures
Authors: Nguyen Thanh Phuong , Dubiel Katarzyna , Keck James L 
Published: June 2021

Bacterial single‐stranded (ss) DNA‐binding proteins (SSBs) bind and protect ssDNA intermediates formed during genome maintenance reactions and help to recruit DNA replication and repair proteins to their sites of action through interactions mediated by their C‐terminal tails. While SSB has been studied extensively in E. coli, much less is known about the paralogous SSBs, SsbA and SsbB, that are found in many gram‐positive bacteria such as B. subtilis. Similarly to E. coli SSB, B. subtilis SsbA and SsbB both bind ssDNA through an oligosaccharide/oligonucleotide binding (OB) domain and function as homo‐tetramers. However, SsbB lacks the critical C‐terminal tail responsible for protein‐protein interactions, leading to differences in functionality. Although the OB domains of B. subtilis SsbA and SsbB are 63% identitical to one another, native gel analysis indicates that mixtures of SsbA and SsbB do not exchange monomers, but instead maintain strictly homo‐tetrameric quaternary structures. A recent crystal structure of B. subtilis SsbA revealed a unique hydrogen‐bonding interface between SsbA monomers that was not observed in the SsbB structure. We hypothesize that these interface differences could prevent SsbA/SsbB hetero‐tetramer formation. To test this hypothesis, we engineered a B. subtilis SsbA interface variant that change a key OB domain interface residue to the corresponding residue in SsbB (Asp74Ser). This variant failed to allow hetero‐tetramer formation. However, since Asp74 is only one residue in a more complex interaction network, we are now testing our hypothesis using a double variant of SsbA (Asp74Ser, Arg4Gln) that better mimics the SsbB interface network for mixed SsbA/SsbB tetramer formation. If a variant that is capable of forming mixed SsbA/SsbB tetramers is discovered, we plan to use it to test the effects of mixed‐tetramer formation in vivo and to further probe the functions of SSBs in gram‐positive bacteria.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 TY52156 on Lipopolysaccharide‐induced Inflammatory Responses in Murine Peritoneal Macrophages
Authors: Son So‐Eun , Heo Jae‐Yeong , Im Dong‐Soon 
Published: June 2021

BackgroundSphingosine 1‐phosphate (S1P), a bioactive lysolipid, acts as a ligand for five G protein‐coupled receptors, S1P1–5. Up‐regulation or activation of sphingosine kinase 1 and subsequent increased generation of S1P have been observed in many cell types, including macrophages and microglia, in response to inflammatory stimuli. Functional roles of S1P1 and S1P2 have been reported in macrophage recruitment during inflammation and atherosclerosis. However, pro‐inflammatory function of S1P has not been fully elucidated at the receptor molecular level.METHODSIn order to assess the functional roles of S1P and S1P3, murine peritoneal macrophages were stimulated with lipopolysaccharide to evoke inflammatory response.RESULTSS1P3 receptor and sphingosine kinase 1 were induced by stimulation of lipopolysaccharide. Lipopolysaccharide treatment induced inflammatory genes, such cyclooxygenase‐2, interleukin‐1b, interleukin‐6, inducible nitric oxide synthase, and tumor necrosis factor‐a. Presence of TY52156, an antagonist of S1P3 suppressed the inductions of inducible nitric oxide synthase and interleukin‐1b in a concentration dependent manner. Suppression of inducible nitric oxide synthase induction was further confirmed by Western blotting and nitric oxide measurement. Suppression of interleukin‐1b induction was also confirmed by western blotting and enzyme‐linked immunosorbent assay. Caspase 1, which is responsible for interleukin‐1b production, was similarly induced by lipopolysaccharide and suppressed by TY52156.CONCLUSIONThe present findings suggest that S1P3 functions as a pro‐inflammatory receptor in primary mouse macrophages.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myocardial infarction‐relevant MMP‐12 interactions identified by correlation analysis
Authors: Gonzalez Osvaldo Rivera , Lindsey Merry L 
Published: June 2021

Myocardial infarction (MI) occurs as a result of prolonged coronary artery occlusion that blocks the blood supply to the left ventricle. Matrix metalloproteinases (MMPs) degrade extracellular matrix proteins and play important roles in MI remodeling. MMP‐12 inhibition after MI in mice increases inflammation and exacerbates LV dysfunction and wall thinning, indicating a potential protective role. Here, we used the Mouse Heart Attack Research Tool 1.0 (mHART 1.0) database to correlate MMP‐12 expression over the MI time course (days 0, 1, 3, 5, and 7 MI) with expression of 165 ECM and cytokine genes in the LV infarct. A total of 39 records in the database had complete information on gene array data, echocardiography, and necropsy. There were a total of 42 genes that correlated with MMP‐12 (all p<0.05). The most significant correlations were between MMP‐12 and MMP‐10 (r= 0.92; p= 1.38E‐16) or Ctnna2 (r=0.92; p= 1.50E‐16), followed by TIMP‐1 (r=0.90; p= 6.42E‐15). Through Enrichr analysis for transcription factor profiling, Yap 1 was the most enriched (combined score=10.27, p value=0.002), indicating strong Hippo signaling. Combined, our results reveal novel candidate interactors with MMP‐12 and indicate Hippo signaling through Yap1 may be a key regulated MMP‐12 feature in the MI left ventricle.Support or Funding InformationNIH HL075360, NIH GM104357, GM114833, GM115428, HL051971, HL129823, and VA 5I01BX000505This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effect of Royal Jelly on Satellite Cell Activation in Aged Monkey Muscle Cells
Authors: Sato Shuichi , Boudreaux Seth P , Bellar David 
Published: June 2021

While global average life expectancy has been consistently increasing in recent year, a gap between healthy life expectancy and the average life expectancy has not been filled yet. Sarcopenia, which is characterized as age‐associated decline of skeletal muscle mass, is one of the most common causes of functional decline and loss of independence, impeding healthy life style in the elderly. Satellite cells are myogenic progenitors and sarcopenia is predicted to be the result of reduced satellite cell number and/or function that leads to a loss of capacity for skeletal muscle mass maintenance. Royal jelly (RJ) produced by worker honeybees is known to have an antioxidant effect and a previous study demonstrated that royal jelly treatment was of benefit on revitalizing aged satellite cells in vitro and muscle regenerative capacity after injury in mice. However, the mechanism by which RJ stimulates the activation of satellite cells is not known. We aimed at further investigating if RJ can attenuate the loss of satellite cell number and/or function by improved satellite cell proliferation using the cells isolated from aged nonhuman primates, which can closely simulate humans. Muscle biopsy from quadriceps was performed then primary muscle cells were isolated from untreated rhesus macaques (young: u up to 10 years old, aged: 18~20 years old). The cells were cultured in either 6‐well or 12‐well plates in growth medium (F‐12 with 10% FBS, 1% Pen/Strep, and hEGF) at least 48 hours. For treatment group, protease‐treated RJ (Institute for Bee Products & Health Science, Okayama, Japan) was dissolved in PBS and added to the culture medium at different concentrations (250, 500, or 1000 μg/mL). After 48 hours of treatment, the cells were harvested and biochemical analyses were conducted. Flow cytometry analysis revealed that the levels of satellite cell activation/proliferation were decreased in aged monkey muscle cells compared to those of young ones (17.0±16.3% vs. 87.4±3.7%, respectively, p<0.05). qRT‐PCR analysis showed an elevation of mRNA levels of PAX7 (1.00±0.14 for control vs. 1.76±0.43 for RJ treatment group, p<0.05) in aged cells with RJ treatment. Enzyme activity assay demonstrated that 48‐hour RJ treatment increased catalase enzymatic activity by 24% (p<0.05). However, the levels of PAX7+/MyoD+ cells, indicative of activated/proliferating satellite cells, were comparable between two groups, which were confirmed by flow cytometry. In conclusion, RJ administration may stimulate satellite cell activation by moderately reducing oxidative stress.Support or Funding InformationSupported by Yamada Research Grant (No. 0221).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ER Stress Induced Apoptosis of Smooth Muscle in Pulmonary Hypertension is Regulated by the Sodium‐Hydrogen Exchanger
Authors: Huetsch John , Yun Xin , Jiang Haiyang , Shimoda Larissa 
Published: June 2021

ObjectiveAltering the apoptosis‐resistant phenotype of pulmonary arterial smooth muscle cells (PASMCs) may be key to reversing pathologic pulmonary vascular remodeling in pulmonary hypertension (PH). Endoplasmic reticulum (ER) stress and sodium‐hydrogen exchanger (NHE) activity have each been implicated in vascular remodeling in PH. We studied the effect of NHE activity upon ER stress‐induced apoptosis in PASMCs isolated from the SU5416‐hypoxia (SuHx) rat model of PH.MethodsPH was induced in male Wistar rats via exposure to the vascular endothelial growth factor receptor inhibitor SU5416 plus 3 wk of 10% oxygen followed by 2 wk of normoxia (SuHx model), while control rats were exposed to vehicle and 5 wk of normoxia. PASMCs were isolated from SuHx and control rats and expanded in culture. Apoptosis was measured using Hoechst stain. Thioflavin fluorescence was quantified as a measure of ER stress. Protein levels of the ER stress‐induced transcription factor, CCAAT/enhancer‐binding protein homologous protein (CHOP), were measured via immunoblot. To measure NHE activity, PASMCs were incubated with pH‐sensitive fluorescent dye and the Na+‐dependent recovery of intracellular pH following acidification by ammonium pulse challenge was measured via fluorescence microscopy. Cell stress was induced with 250 μM H2O2. NHE was pharmacologically inhibited with 10 μM ethyl‐isopropyl amiloride.ResultsER stress, a known stimulus of apoptosis, was increased by H2O2 in control PASMCs and was elevated at baseline in SuHx PASMCs. H2O2 exposure increased apoptosis in control PASMCs. Despite increased ER stress, SuHx PASMCs exhibited low levels of apoptosis both at baseline and in response to H2O2. CHOP protein expression, which is induced by ER stress and triggers the apoptotic cascade, was increased by H2O2 in control cells, but remained low in H2O2‐treated SuHx PASMCs. NHE activity was increased in PASMCs from SuHx rats, and pharmacologic NHE inhibition in SuHx PASMCs increased both basal and H2O2‐induced CHOP expression, and also increased apoptosis.ConclusionsPH PASMCs are resistant to ER‐stress induced apoptosis due to decreased CHOP expression. NHE inhibition increases CHOP expression in PH PASMCs and restores ER‐stress induced apoptosis, suggesting a novel mechanism to control vascular remodeling in PH.Support or Funding InformationNIH K08HL133475 & R01HL073859This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Zinc binding to Muscle LIM Protein regulates its structure and interaction with binding partners
Authors: Boateng Samuel , Atkins Jennifer , Hawanatu Sesay , Sorensen Thomas , McGuffin Liam 
Published: June 2021

Muscle LIM protein (MLP) is a member of the cysteine‐rich protein family, localising predominantly to the Z‐disk of the myofilaments in cardiac myocytes. MLP is a mechanosensor and functions through its translocation to the nucleus from the myofilaments and through protein‐protein interactions in the cytoplasm. Mechanosensing enables the heart to adapt appropriately to alterations in hemodynamic load, whilst mutations in the MLP gene lead to cardiomyopathy and heart failure. MLP contains two LIM domains each consisting of a double zinc‐finger motif. Rather than interacting with DNA, LIM domains mediate protein‐protein interactions associated with cellular signalling.The aim of this investigation is to determine how zinc binding regulates the structure of MLP and determine the impact on its interaction with binding partners. The analysis was performed using a combination of circular dichroism (CD), bioinformatics tools and online servers. CD is the difference in absorption between left‐handed and right‐handed circularly polarised light, occurring when a molecule contains light‐absorbing groups. It can be used to study the secondary, tertiary and quaternary structure of proteins, as well as studying its conformational stability under different conditions.In this study, we used CD to determine structural changes to MLP in the presence and absence of zinc. MLP is predominantly helical (~36%) with a small percentage of beta‐strands of ~8%, 34% of unordered and 21% of turns. In the presence of excess zinc, the helical content was reduced to ~19% with a gain of beta‐strand component to 30%. The presence of EDTA caused MLP to lose some helical conformation to 22% with an increased in the beta‐strand content to 16%. These data suggest that zinc increases the structural folding and organization of MLP.The full length wildtype MLP sequence was submitted to a variety of servers along with the C58G mutation known to reduce zinc binding in the first LIM domain. This mutation in humans leads to heart failure. Heart failure is defined as a progressive condition in which the heart is unable to pump enough blood to meet the body's needs for oxygen and nutrients. Bioinformatic protein‐protein analysis indicated that the C58G mutation inhibits interaction with NRAP, TCAP, alpha‐actinin and cofilin 2 which would interfere with MLP binding to the z‐disk and the myofilaments within cardiac myocytes. This would have a significant negative impact on mechanosensing in the heart and explains why the C58G mutation in MLP leads to heart failure.Support or Funding InformationUniversity of Reading and Diamond Light SourceThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proliferation and osteogenic differentiation of human mesenchymal stem cells on cross‐linked polypeptide multilayer films
Authors: Yuan Chia‐Sui , Hung Yu‐Han , Yoneda Takashi , Cheng Yung‐Hsin , Lo Chun‐Min 
Published: June 2021

Polyelectrolyte multilayer film is an emerging method for substrate coating to regulate cellular behaviors. This material allows for modulation of thickness, stiffness, and adhesiveness. In this study, human mesenchymal stem cells (hMSCs) were cultured on polypeptide multilayer films built up by the alternate deposition of cationic poly‐L‐lysine (PLL) and anionic poly‐L‐glutamic acid (PLGA), cross‐linked with EDC/sulfo‐NHS, and coated with type I collagen. The in‐vitro degradation of the cross‐linked PLL/PLGA films in the presence or absence of hMSCs were followed by fluorimetry after labeling of the outermost PLL layer with FITC. Cell proliferation was measured by CyQuant proliferation assay and osteogenic differentiation was evaluated by q‐PCR of osteogenic specific genes such as ALP, OCN, and OPN. The results showed that these cross‐linked films, with or without hMSCs on top of them, were quite stable in the culture medium and not subjected to degradation at least for a few weeks. While hMSCs maintained similar attachment and spreading behavior regardless of layer number (1–6 layers) of films, they demonstrated better proliferation rate on even number layer of films, where the top layers are PLGA films. In addition, compared with cells cultured on polystyrene culture wells and treated with osteogenic induction medium, hMSCs cultured on 6‐layer cross‐linked PLL/PLGA films showed a significant increase in the osteogenic differentiation rate. Our observations indicate a feasible way to manipulate stem cell behavior on polypeptide multilayer films by changing the number of layers, the outermost polypeptide layer, and the concentration of cross‐linkers.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.