bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationNIH/NIDDK R03DK105300‐02This 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

ObjectiveRemote 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.MethodsYoung 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.ResultsSeven 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).ConclusionsSeven 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 InformationFunding 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 InformationNational Institutes of Health Grants R01 DK096907This 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 InformationResearch 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 InformationDFG SCHL1962/5‐1This 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 InformationThis 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 InformationThis work was supported by a Project Grant and a New Investigator Salary Award from the Canadian Institutes of Health Research (CIHR) to C.N.AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.