bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological inhibition of HuR improves survival and reduces adverse cardiac remodeling following left‐ventricular pressure overload
Authors: Anthony Sarah R. , Green Lisa C. , Lanzillotta Lindsey , Wu Xiaoqing , Nieman Michelle L. , Blaxall Burns C. , Lorenz John N. , Rubinstein Jack , Xu Liang , Tranter Michael
Published: June 2021

Human antigen R (HuR) is a widely expressed RNA binding protein that has been implicated in numerous human diseases including cancer, neurological disorders, and cardiovascular disease. We have previously shown that HuR is both necessary and sufficient for induction of hypertrophic signaling pathways in isolated primary myocytes. In addition, data from our lab is the first to suggest that HuR expression and activation is increased in failing human hearts. To determine the role of HuR in hypertrophic signaling in vivo, we created an inducible cardiomyocyte‐specific HuR deletion mouse (iCM‐HuR−/−), and showed that genetic deletion of HuR reduces pathology using a transverse aortic constriction (TAC) model of pressure‐overload‐induced hypertrophy, adverse cardiac remodeling, and heart failure. In this work, we sought to recapitulate this reduction in pathology using KH‐3, a novel pharmacological inhibitor of HuR, to determine the translational potential of HuR inhibition as a viable therapeutic target.

Twenty wild‐type mice were randomized to either vehicle or KH‐3 at 4‐weeks post‐TAC, a time‐point consistent with substantial development of cardiac hypertrophy, and monitored via serial echocardiography for a further 7 weeks. Our results show that treatment with KH‐3 increased survival relative to vehicle controls. In addition, as compared with vehicle, KH‐3 treatment significantly abated the continued progression of left ventricular (LV) hypertrophy. This was accompanied by a significant preservation of LV ejection fraction and reduction in LV chamber dilation in KH‐3 treated mice. Importantly, KH‐3 had no effects on systemic blood pressure and no adverse reactions were observed. In conclusion, these results suggest that inhibition of HuR is a promising therapeutic approach to treat pathological LV hypertrophy.

Support or Funding Information

This work was partially funded by an AHA Scientist Development Grant (MT) and NIH R01 HL132111 (MT).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MiR‐486a‐5p Alters Cell Survival in Ischemia‐Reperfusion Induced Myocardial Injury
Authors: Phan Anh , Luther Kristin , Haar Lauren , McGuinness Michael , Jones Walter K.
Published: June 2021

Previous work in our lab has shown that mesenchymal stem cells (MSC)‐derived exosomes protect cardiomyocytes from ischemia‐reperfusion (I/R) induced myocardial injury both in vitro and in vivo. We found that microRNA‐486a‐5p (miR‐486) was highly and selectively expressed in MSC‐derived exosomes (1686‐fold relative to MSC (p<0.001)). The role of miR‐486 in cell survival is still debated and miR‐486 has been reported to act as either a tumor suppressor or an oncogene. We hypothesized that miR‐486 upregulation will protect cardiomyocytes from I/R injury. We searched for miR‐486 gene targets using miRanda and Targetscan and found 1687 gene targets. We focused upon targets involving in the cell death and survival pathways, leaving 104 gene targets. Of these 104 targets, program cell death 4 (PDCD4), a tumor suppressor and PTEN, which negatively regulates the PI3K/Akt survival pathway, were chosen for further study based upon considerations of scores for mRNA‐miRNA interaction and the predicted targets. Luciferase reporter gene assays indicated that the 3′‐UTR of PDCD4 and PTEN are both targeted by miR‐486. A mimic and inhibitor (antagomir) of miR‐486 were used to assess its ability to modulate PDCD4, and PTEN expression and Akt activity. Although the mRNA levels of PDCD4 and PTEN were not altered by treatments, immunoblotting results showed a 0.5‐fold reduction in PDCD4 protein expression with miR‐486 mimic and no change in expression with antagomir (n=3). We did not observe a change in PTEN protein levels, and we also saw no change in pAkt/Akt ratio which is regulated downstream of PTEN. Simulated ischemia/reperfusion (sim I/R) was performed on H9c2 cells treated with miR‐486 mimic or scrambled control to measure the cytoprotective effect of miR‐486. H9c2 cells treated with the mimic of miR‐486 showed a significant increase in cell survival by 28–43% (p<0.001, n=4–6) and a reduction of 20% in LDH release compared to control cells (p<0.05, n=6). Western blot results with cell lysate after sim I/R showed a marked decrease in levels of PDCD4, cleaved‐caspase 3 and Bax in the group treated with miR‐486 mimic compared to control cells (p<0.05, n=3). These findings support that miR‐486 upregulation is anti‐apoptotic. Additional studies are needed to discern whether miR‐486 upregulation will reduce cell injury using rodent models, and how much exosomal miR‐486a‐5p contributes to the effects of exosomes and stem cells. Such efforts will lead to a new therapeutic approach for prevention damage of I/R injury.

Support or Funding Information

NIH 5R01HL091478 (WKJ)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Down‐Regulation of Gamma‐Adducin Disrupts the Actin Cytoskeleton in FHH rats and May Contribute to the Development of Hypertension‐induced Renal Injury
Authors: Wang Shaoxun , Travis Olivia , He Xiaochen , Fan Fan , Roman Richard
Published: June 2021

Fawn Hooded Hypertensive (FHH) rat develops chronic kidney disease after onset of hypertension. We previously reported that the expression of Add3 is decreased in various tissues, including kidney and renal vessels in FHH rats. More recently, we demonstrated that Add3 dysfunction contributes to renal vascular dysfunction in FHH rats, and they exhibit enhanced glomerular injury, interstitial fibrosis and proteinuria after the development of hypertension. Add3 is an actin‐binding protein that regulates actin capping and polymerization. The tail of the dimerized adducin protein interacts with actin and spectrin. However, it remains to be elucidated whether Add3 dysfunction alters actin cytoskeleton and contributes to renal microvascular dysfunction in FHH rats. The present study examined the role of Add3 on the regulation of the actin cytoskeleton in a vascular smooth muscle (VSMC) cell line treated with an Add3 DsiRNA, and in primary VSMCs isolated from FHH and FHH. Add3 transgenic rats. Downregulation of Add3 disrupted the actin cytoskeletal structural integrity in VSMC cells transfected with an Add3 DsiRNA (20 nM) in comparison to untreated or scramble siRNA treated cells. F‐actin distribution was significantly reduced in VSMCs isolated from the renal vasculature of FHH (2.4 ± 0.3%/cell, n = 6) vs. FHH. Add3 transgenic rats (11.4 ± 1.7%/cell, n = 6) consistent with disruption of the actin cytoskeleton. In addition, we observed the formation of an F‐actin net in primary renal VSMCs isolated from FHH rats, and reduced expression of the myosin regulatory light chains (MYL12A/B) that regulate actin polymerization and vascular contraction upon phosphorylation. These results suggest that down‐regulation of Add3 disrupts the actin cytoskeleton and the expression of MYL12A/B protein in renal VSMCs. These effects combine to impair the myogenic response of the Af‐art and autoregulation of renal blood flow. This increases transmission of pressure to the glomerulus, which contributes to the development of chronic kidney disease induced by hypertension in the FHH rat.

Support or Funding Information

This study was supported by grants HL36279 (RJR) and DK104184 (RJR), AG050049 (FF), P20GM104357 (RJR, FF ) from the National Institutes of Health; 16GRNT31200036 (FF) from the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced Hydrogen Peroxide and renin release in Primary cultures of Juxtaglomerular (JG) cells from diabetic mice
Authors: Mendez Mariela
Published: June 2021

Diabetes mellitus and its complications represent an increasing burden for healthcare. A large fraction of patients with diabetes mellitus develops diabetic nephropathy. The Akita mice strain is one of the few mouse models of diabetes mellitus that shows features of the human disease including hypertension and kidney damage. Previous reports have shown increased levels of reactive oxygen species in the kidneys of Akita mice. We previously showed that hydrogen peroxide is a potent stimuli for renin release from the juxtaglomerular cells, increasing plasma renin and blood pressure in mice. Thus, we hypothesized that hydrogen peroxide levels are higher in JG cells from Akita mice leading to increased renin release in this strain. Using Western Blot, we found that the NADPH oxidase isoforms NOX1 and NOX4 are expressed, but only NOX1 being upregulated in primary JG cells isolated from Akita mice, compared to JG cells from the control (C57) strain (C57=100%. Akita/NOX1= 147%±10; n=4; p<0.05). Confocal imaging showed the expression of NOX1 and NOX4 in JG cells where they both co‐localized with renin immunolabeling in renin‐containing granules. In addition, the expression of Catalase, which scavenges H202, was reduced in JG cells from diabetic Akita mice (C57=100%; Akita= 72%±7.2 of C57; n=4; p<0.05). We measured H202 levels by transducing JG cells with the fluorescent protein sensor Hyper under control of the renin1 promoter. We found that JG cells isolated from Akita mice had higher baseline levels of H202 (C57= 100, Akita=117±7%, p<0.05). Since we previously found that H202 stimulates renin release, we studied whether JG cells from Akita mice had higher total renin activity or release. For this, we measured baseline renin release to the media during 2 hours from primary JG cells cultured from Akita or control mice. JG cells were then lysed and total renin content measured. We found that in the absence of any stimuli, JG cells from Akita mice had ~2 fold higher renin release (C57= 100%, Akita=219.2±26%; p<0.04, n=6). We did not observe a significant difference in total renin content between strains. Transduction of replicate plates with adenovirus encoding Catalase (AD‐Cat) decreased baseline renin release by 35±8% (p<0.05) in JG cells from Akita mice whereas it did not affect renin release in control JG cells. We conclude that renin cells from diabetic Akita mice have higher endogenous H202 leading to increased renin release. The NADPH isoform NOX1 is enhanced in Akita mice and may be responsible for higher levels of H202 and renin release from JG cells. Our data suggest that direct stimulation of renin release by H202 could be potentially be involved in renal damage in diabetes mellitus.

Support or Funding Information

NIH/NIDDK R03DK105300‐02

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Seven Consecutive Days of Remote Ischemic Preconditioning Improved Cutaneous Vascular Reactivity Induced by Post Occlusive Reactive Hyperemia
Authors: Kim Jahyun , Vianna Lauro , Franke Warren , Lang James
Published: June 2021

Objective

Remote ischemic preconditioning (RIPC), induced by brief periods of ischemia followed by reperfusion, elicits strong cardioprotection from ischemia reperfusion injury in animal models. However, recent clinical trials in humans have yielded mixed results that may be explained by coexisting conditions and other experimental factors. Using human skin as an alternative model to study RIPC, we hypothesized that cutaneous vascular reactivity to post‐occlusive reactive hyperemia (PORH) will be increased following repeated RIPC.

Methods

Young healthy participants (26 ± 1 years; 4 male, 5 female) performed RIPC for seven consecutive days. Each daily RIPC session consisted of 4 repetitions of 5 minutes of upper arm blood flow occlusion interspersed by 5 minutes non‐occlusion. Pre‐ and 24 hours post‐RIPC training, an index of skin microvascular blood flow (i.e., cutaneous red blood cell flux) was measured from the ventral forearm using laser speckle contrast imaging. Flux was measured continuously during baseline, 5 minutes of arm blood flow occlusion with 200 mmHg of cuff pressure, and during 5 minutes of PORH. Peak flux during hyperemia, Max (peak after subtracting baseline), Time to peak (Tp) (index of vascular resistance), Max/Tp (indicator of early vascular changes), and area under curve (AUC) were analyzed from collected data. Data are presented as cutaneous vascular conductance (CVC), which was calculated as flux/mean arterial pressure.

Results

Seven consecutive days of RIPC increased Max/Tp (Pre: 0.10 ± 0.02 vs. Post: 0.12 ± 0.02 CVC/sec, p<0.05). Although not statistically different, other indices of PORH were increased; AUC (Pre: 89.7 ± 13.5 vs. Post: 107.2 ± 10.4 CVC·sec, p=0.10), Peak (Pre: 2.18 ± 0.02 vs. Post: 2.28 ± 0.20 CVC, p=0.33), Max (Pre: 1.68 ± 0.20 vs. Post: 1.79 ± 0.20 CVC, p=0.18), and Time to peak (Tp) (Pre: 18.6 ± 1.1 vs. Post: 17.0 ± 1.3 sec, p=0.11).

Conclusions

Seven consecutive days of RIPC augmented cutaneous microvascular reactivity to PORH. Collectively, these data support repeated RIPC as a means to improve microvascular function in humans.

All experimental procedures were approved by the Institutional Review Board at Des Moines University and conformed to the standards set by the Declaration of Helsinki (IRB#: 08‐15‐05)

Support or Funding Information

Funding source: Des Moines University, IOER #03‐14‐01 (PI: J. Lang)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering a VEGF fusion protein for use with an artificial extracellular matrix with programmable binding affinities
Authors: Elliott Robert , Barkas Aleeza , Balog Eva Rose
Published: June 2021

We are interested in novel protein biomaterials that recapitulate the natural chemical and physical signals employed by the body to manage and inform blood vessel regulation. Despite great advances in artificial extracellular matrices (aECMs) for controlled presentation and release of Vascular Endothelial Growth Factor (VEGF), these materials still fall short of the incredibly complex dynamic signaling of the natural extracellular matrix. Here we show the molecular cloning and bacterial expression of a VEGF fusion protein composed of a soluble VEGF isoform and the protein domain Src Homology 3 (SH3). When combined with an aECM possessing SH3 binding peptides of variable affinity, this novel fusion protein will allow us to study the effects of ECM‐binding on VEGF signaling. Toward this goal we have also designed and synthesized a mini‐suite of elastin‐like polymers (ELPs) with various SH3‐binding sites for incorporation into 2‐D or 3‐D aECMs.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Amino Acid Prodrugs of Cytarabine
Authors: Slaughter Syara , Cooper Anthony , Modi Shujal , Parihar Harish , Wang Desuo , Wu Zhiqian
Published: June 2021

Cytarabine is widely used in chemotherapy for different types of leukemia due to the targeting effects on cells in the S phase of cell cycle. However, its use is limited by its poor absorption and fast degradation to biologically inactive forms and elimination. Amino acid (isoleucine and valine) prodrugs of cytarabine were synthesized to improve uptake of cytarabine. The prodrugs may also reduce the proportion of the drug that was degraded and increase bioavailability. Stability and permeability studies performed with cytarabine prodrugs demonstrated improved uptake. Cytotoxic studies utilizing K562 cell lines revealed 4.5% greater growth inhibition at 24 hours incubation and 19% greater growth inhibition at 72 hours incubation with the cytarabine isoleucine prodrug than with cytarabine. The results indicated that the prodrug may work more efficiently than the parent drug.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altering mitochondrial bioenergetic efficiency
Authors: Neufer Darrell
Published: June 2021

Mitochondrial oxidative phosphorylation (OXPHOS) accounts for the vast majority of daily energy expenditure, making the efficiency at which this process occurs a potential attractive target for treating diseases associated with positive energy balance. Organic cations are a class of small carbon molecules that carry a formal charge on a nitrogen or phosphorus atom and/or contain an aliphatic amine group which protonates at physiological pH. Well studied examples from each class include the anti‐diabetic compounds berberine (+1 charge) and metformin (+2 charge at pH of 7). These compounds, like all cations, are naturally drawn to the mitochondrial matrix by the negative potential across the inner membrane. According to Nernst equilibria, the accumulation of a positively charged compound into the mitochondrial matrix is predicted to reduce the net proton motive force (Δp) available to drive ATP synthesis. In an in vitro assay of respiratory capacity, the reduced Δp will manifest as a decrease in the maximal rate at which ATP can be synthesized and therefore the maximal rate at which O2 is consumed. However, in vivo where the interplay between energetic driving forces and flux continuously adjusts to demand (e.g., ATP turnover rate), a decrease in Δp is predicted to decrease the efficiency at which those demands can be met, requiring a greater flux rate through OXPHOS and therefore energy expenditure per unit of time. Testing of >15 different organic cations in isolated mitochondria thus far has revealed remarkably similar, dose‐dependent decreases in NAD‐linked ADP‐stimulated respiration, H2O2 emission, and the Δp under different rates of clamped respiration. Additional data will be presented on the dose‐dependent impact of acute and chronic administration of organic cations in vivo in both mice and rats. Together, the data highlight a novel therapeutic model by which organic cations mitigate obesogenic conditions by altering the interplay among energetic driving forces to decrease OXPHOS efficiency. Organic cations may serve as a potential novel class of drugs for the prevention and/or treatment of diseases stemming from chronic metabolic imbalance.

Support or Funding Information

National Institutes of Health Grants R01 DK096907

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 structure of apoA‐II on HDL reveals novel insights into its regulation of lipoprotein composition and function
Authors: Melchior John T , Street Scott E , Jerome W. Jay , Hart Rachel C , Heinecke Jay W , He Yi , Davidson W. Sean
Published: June 2021

High‐density lipoproteins (HDL) are compositionally and functionally diverse. HDL can host at least 95 different proteins and studies suggest many of these interactions are governed by specific conformations of HDL's primary scaffold protein, apolipoprotein (apo)A‐I. Previously, we showed that the presence of HDL's second most abundant scaffold protein, apoA‐II, impacts particle composition and function; presumably through interactions with apoA‐I. The goal of this study was to determine the structural conformation of apoA‐II on HDL to understand how its presence impacts HDL metabolism.

We used chemical cross‐linking and mass spectrometry to derive spatial information on homogenous populations of recombinant HDL (rHDL) generated in vitro. The rHDL was created using a mixture of wild‐type and isotopically‐labeled apoA‐I to unambiguously identify the molecular span of the cross‐linked peptides; i.e. within a molecule of apoA‐I (intramolecular) or between two molecules of apoA‐I (intermolecular). Two rHDL populations were studied: (i) rHDL that contained only apoA‐I (LpA‐I) and (ii) rHDL containing both apoA‐I and apoA‐II (LpA‐I/A‐II).

Cross‐linking and SDS‐PAGE showed both populations contained two molecules of apoA‐I while the LpA‐I/A‐II rHDL contained a single dimer of apoA‐II. Analysis by negative stain electron microscopy revealed both populations were discoidal in shape with no differences in size. The intermolecular cross‐linking pattern between apoA‐I molecules were consistent with the “double‐belt” model of apoA‐I on HDL and, surprisingly, the presence of apoA‐II didn't appear to perturb the pattern. However, we observed strong cross‐links between apoA‐II and the C‐terminus of apoA‐I.

In a parallel approach, native LpA‐I and LpA‐I/A‐II HDL was isolated from human plasma using immunoaffinity and size exclusion chromatography. Shotgun proteomics and cross‐linking revealed accessory proteins were mostly associated with LpA‐I HDL and interactions were primarily localized to the N‐ and C‐termini of apoA‐I. As with the rHDL, we found apoA‐II specifically interacts with the C‐terminus of apoA‐I in the LpA‐I/A‐II particles. Taken together, this suggests that apoA‐II's compositional and functional effects on HDL are exerted via its interaction with the C‐terminus of apoA‐I possibly limiting its availability to interact with the accessory proteins. We have gone on to generate preliminary all‐atoms models of apoA‐II on HDL to illustrate this structural mechanism.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Both type I TGFβ Receptors ALK‐1 and ALK‐5 Promote Inhibition of Endothelial Cell Proliferation Mediated by TGFβ1
Authors: Hawker James , Duffy Iain
Published: June 2021

Control of angiogenesis is critical in diseases such as cancer, heart disease, or wound repair. Growth factors such as transforming growth factor beta (TGFβ) and fibroblast growth factor 2 (FGF‐2) contribute to angiogenesis. We have examined the effects of TGFβ and the TGFβ family signaling type I receptors Activin‐Like Kinase 1 Receptor (ALK‐1) and ALK‐5 and their effects on endothelial cell growth. We report that TGFβ inhibits both DNA synthesis and also Cyclin A promoter‐luciferase expression stimulated by FGF‐2 or serum. We also show data that TGFβ also decreases cyclin A and cyclin D1 protein expression in endothelial cells (EC). In previous work it has been shown that FGF‐2 is tranlocated to the nucleus as a part of its positive stimulatory growth of EC and other cell types. We also show that TGFβ blocks nuclear translocation of FGF‐2 but not FGF‐2 stimulation of Mitogen Activated Protein Kinase (MAP) Kinase activity. TGFβ can bind and activate both ALK‐1 and ALK‐5 in endothelial cells. Transfection of either activated ALK‐1 or activated ALK‐5 QD (kinase activated) plasmids into endothelial cells increased inhibition of DNA synthesis mediated by TGFβ. This data indicates that the growth inhibitory effects of TGFβ are mediated through both ALK‐1 and ALK‐5 type I receptors in endothelial cells. We have also shown that ALK‐1 is also involved in endothelial tube formation or angiogenesis. Other ligands such as Bone Morphogenetic Protein 9 (BMP‐9) and Activin A may also be involved in endothelial cell function. Thus we are also looking at expression of various ALK‐1 ligands and co‐receptors in endothelial cells under various conditions and we are generating mutants of ALK‐1, ALK‐5, and endoglin to further test their roles in angiogenesis.

Support or Funding Information

Research support was from AHA and Dept Veteran's Affairs grants (JH) and funds from Saint Leo University (ID).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GDNF‐induced intestinal epithelial barrier stabilization is RET‐dependent
Authors: Schlegel Nicolas , Kannapin Felix , Meir Michael , Flemming Sven , Waschke Jens , Burkard Natalie
Published: June 2021

Glial cell line‐derived neurotrophic factor (GDNF) stabilizes the intestinal epithelial barrier under basal and inflammatory conditions. These effects were previously shown to be mediated by a p38MAPK‐dependent stabilization of the desmosomal protein desmoglein2 (DSG2). However, the receptor underlying GDNF‐induced effects on enterocytes still requires verification. We therefore tested whether the RET receptor is critically involved in GDNF‐induced epithelial barrier stabilization in enterocytes.

RET receptor and GFRα isoform expression was first confirmed in primary human and mouse enterocytes as well as in Caco2 cells. Stimulation of enterocytes with GDNF resulted in increased phosphorylation of the RET receptor at Tyr905 which is a recognized necessary step for further RET‐dependent downstream signaling. Unexpectedly, complete deletion of the RET receptor in Caco2 cells using CRISPR/CAS9 significantly arrested cell growth. Using siRNA as an alternative approach to achieve partial knock‐down of the RET receptor in Caco2 monolayers levels were reduced by 20 ± 8% compared to controls. This prevented GDNF‐induced redistribution of desmosomal protein Desmoglein2 (DSG2). In untransfected Caco2 cells and those with partial knock‐down of the RET receptor, application of TNFα significantly augmented epithelial permeability as revealed by measurements of 4 kDa FITC‐dextran flux and transepithelial electrical resistance (TER). Application of GDNF blocked TNFα‐induced loss of intestinal epithelial barrier function in untransfected Caco2 cells but not in those with partial knock‐down of the RET receptor. Preliminary data further revealed that partial knock‐down of the RET receptor results in increased phosphorylation of p38MAPK under basal conditions. Accordingly, under these conditions, GDNF did not prevent TNFα‐induced phosphorylation of p38MAPK.

In summary these data show that GDNF‐induced effects on intestinal epithelial barrier function are RET‐dependent. Furthermore, GDNF‐dependent signaling via RET appears to be important for cell proliferation which confirms an additional role of GDNF in supporting intestinal epithelial wound healing.

Support or Funding Information

DFG SCHL1962/5‐1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification and Proteomic Analysis of Major Whey Proteins in a Nutritional Supplement: A Six‐Week Undergraduate Independent Project
Authors: Sterne‐Marr Rachel , Havlicek Elizabeth E. , Kolonko Kristopher J.
Published: June 2021

We sought to create an undergraduate laboratory experience to introduce the field of proteomics and the associated mass spectrometric (MS) techniques alongside classic protein purification methodologies. Students were tasked with the purification and identification of one of the major proteins found in a whey‐based nutritional supplement with the ultimate goal of having students become the drivers of their independent projects. Two major challenges were 1) to complete the experiments in 4‐hour blocks over several weeks in a typical semester and 2) to train sixteen students per lab period on a single mass spectrometer. A six‐week project was developed where students spent four lab periods performing either ion‐exchange or gel filtration chromatography and SDS‐PAGE, and two lab periods gathering bottom‐up and top‐down proteomic data with a matrix‐assisted laser desorption ionization (MALDI) mass spectrometry. The resulting data, peptide mass fingerprints and tandem mass spectra, were analyzed via database searching (i.e. MASCOT) during these weeks or outside the laboratory periods. The abundance of literature on the major whey proteins in terms of the biology, purification, and even proteomic analyses allowed students to determine their mode of protein purification and conditions for MALDI‐MS. This project is fairly low cost and widely applicable to any institution with appropriate facilities.

Support or Funding Information

This work was funded in part by a grant from the Siena College Committee on Teaching and Faculty Development (RSM) and the Siena College School of Science (EEH).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Signaling by the Epidermal Growth Factor Receptor regulates DNA repair
Authors: Boras Ivan , Nasser Rawan , Sabatinos Sarah , Antonescu Costin N.
Published: June 2021

The Epidermal Growth Factor (EGF) Receptor (EGFR) is a receptor tyrosine kinase that when deregulated can drive tumor growth and can also contribute to drug resistance. Upon binding its ligand EGF, EGFR triggers the activation of many signaling pathways including phosphatidylinpositol‐3‐kinase (PI3K)/Akt, Ras‐Erk, signal transducer and activator of transcription (STAT), and phospholipase C γ1 (PLCγ1). EGFR may also control DNA repair mechanisms, although this phenomenon this remains poorly understood. Control of DNA repair by EGFR may be particularly relevant in the context of action of and resistance to anti‐cancer drugs that cause DNA damage (e.g. cisplatin). We examined how acute activation (10–30 min) of EGFR by ligand stimulation regulates DNA damage and repair responses induced by chronic (16 h) cisplatin treatment. To do so, we examined various markers of DNA damage and repair such as γH2AX and 53BP1. We observed that as little as 10 min of EGF stimulation is sufficient to elicit remodelling of DNA damage and repair markers such as γH2AX in chronic cisplatin‐treated cells. This indicates that acute EGFR activation triggers signaling pathway(s) that control the DNA damage response and/or DNA repair. Using these methods, we dissected the contribution of various EGFR signaling pathways and membrane traffic phenomena to this EGFR‐dependent control of DNA repair. This work may reveal new ways to enhance the efficacy of existing chemotherapies such as cisplatin for cancer treatment.

Support or Funding Information

This work was supported by a Project Grant and a New Investigator Salary Award from the Canadian Institutes of Health Research (CIHR) to C.N.A

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

The 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

Background

Sphingosine 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.

METHODS

In order to assess the functional roles of S1P and S1P3, murine peritoneal macrophages were stimulated with lipopolysaccharide to evoke inflammatory response.

RESULTS

S1P3 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.

CONCLUSION

The 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 Information

NIH HL075360, NIH GM104357, GM114833, GM115428, HL051971, HL129823, and VA 5I01BX000505

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 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 Information

Supported 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.