bluuebunny/crossref_metadata_2025_split huggingface dataset:
ARB treatment ameliorates triacylglycerol accumulation during insulin‐resistant conditions in the liver of OLETF rats
Authors: Godoy‐Lugo Jose Arquimides , Lee Debby , Thorwald Max Andrew , Nakano Daisuke , Nishiyama Akira , Hui David , Ortiz Rudy M. 
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is a growing concern globally, and is associated with obesity, hyperlipemia, hypertension, insulin resistance, and diabetes; all traits linked to metabolic syndrome, which affects 20% of the US population. While angiotensin receptor blockers (ARB) effectively ameliorate the hypertension associated with metabolic disorders, its potential benefits on other aspects of metabolism during such conditions are not fully appreciated. ARB treatment may reduce overproduction and accumulation of hepatic triacylglycerols (TG), but it remains unknown whether ARB treatment has favorable effects in fatty livers associated with insulin resistance. To evaluate the benefits of the ARB treatment on lipid‐related genes, three groups (n=4–6) of 25‐week‐old rats were used: (1) LETO (lean strain‐control), (2) insulin resistant OLETF, and 3) OLETF + ARB (10 mg olmesartan/kg/d × 8 wks). An oral glucose tolerance test (oGTT) was performed to mimic a high glucose challenge. Liver and plasma were collected at T0, T30, and T60 min of the oGTT. We measured gene expression of hepatic PPARα, SREBP1c, FASN, and CPT1 along with TG and cholesterol levels in liver and plasma. PPARα expression was 1.6‐fold lower at T0 in OLETF+ARB compared with OLETF, but no changes were detected for other genes evaluated. Hepatic TG content was 1.5‐, 3.4‐ and 1.5‐fold lower at T0, T30, and T60, respectively, in OLETF+ARB compared to OLETF, along with 1.2‐fold lower plasma TG at T30. These changes suggest that AT1 signaling contributes to stabilizing lipid metabolism especially during a glucose challenge, and contributes to maintaining lower hepatic TG content that translates to reduced plasma TG during a glucose challenge.Support or Funding InformationM. Thorwald was supported by NIH NCMHD9T37MD001480. R.M. Ortiz was partially supported by NIH NHLBIK02HL103787. Research was partially funded by NIH NHLBIR01HL091767.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingosine 1 Phosphate Regulates Store‐Operated Calcium Entry through binding to STIM1
Authors: El‐Shewy Hesham M. , Parnham Stuart , Fedarovich Dzmitry , Bullesbach Erika , Luttrell Louis M. 
Published: June 2021

A growing body of evidence suggests that sphingosine kinase 1 (SK1)/ sphingosine 1‐phosphate (S1P) pathway contributes to vascular dysfunction through dysregulation of Ca2+ mobilization. We demonstrated recently that intracellular S1P triggers store‐operated Ca2+ (SOC) entry and independent of G protein‐coupled S1P receptors suggesting SOC as intracellular target for S1P. However, the mechanism by which S1P modulates SOC entry is not well defined. SOC is composed of two molecular components; stromal interaction molecules (STIM), and Orai proteins. While STIM function as Ca2+ sensors within the ER, the Orai proteins constitute the pore‐forming unit of SOC in PM. We first investigated the role of SK1/S1P on STIM1/Orai1 assembly by confocal microscopy. Pretreating transiently transfected HEK293 cells with SK1 specific inhibitor inhibited angiotensin‐induced colocalization of GFP.STIM1 and mCherry Orai1. Similarly, using BRET assay, inhibition of SK1 decreased NET BRET values of STIM1‐FlAsH and Orai1‐Rluc in transiently transfected HEK293 cells stimulated with GPCR agonists. Our molecular modeling simulation predicted direct binding of S1P to EF/SAM1 domain of STIM1 molecule. This is initially assessed by in vitro binding assay revealed binding of biotinylated S1P to STIM1 in the membrane fractions of vascular smooth muscle cell lysates. We then used circular dichroism spectroscopy (CD) to measure the thermostability of the EF/SAM1 protein in the presence or absence of S1P. Addition of S1P to EF/SAM1 induced a consistent left shift indicative of lower melting temperature (Tm) compared to vehicle control. To further validate direct binding, we prepared 15N‐labeled EF/SAM1 for the NMR spectroscopy. A five point titration series with S1P caused perturbations of residue specific chemical shift due to binding interactions between EF/SAM1 and S1P using 2D TROSY (Transverse Relaxation Optimized Spectroscopy). Collectively, our data provide evidences of intracellular role for S1P in regulation of SOC through direct binding to STIM1.Support or Funding InformationAmerican Heart Association 17GRNT33700222 (HME)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the Function of Apolipoprotein A‐I Using Chimera Proteins
Authors: Patel Nairuti , Weers Paul M. M. 
Published: June 2021

Human apolipoprotein A‐I (apoA‐I) is a 243 residue, 28 kDa protein that consists of an N‐terminal (NT) helix bundle domain and a less structured C‐terminal (CT) domain. ApoA‐I is the major protein component in high‐density lipoprotein (HDL) and is known to play an important role in reverse cholesterol transport. HDL promotes the removal of excess cholesterol from peripheral tissues to the liver. The CT domain of apoA‐I has a high affinity for phospholipids and initiates lipid binding, whereas the NT domain harbors the site of lecithin‐cholesterol acyltransferase activity. A recent study showed that removal of residues 231–243 led to a decrease in lipid binding affinity. Other studies have suggested that segments in the NT domain, residues 1–43 and 44–65, initiate lipid binding. Thus, conflicting data exists regarding the location of high‐affinity lipid binding in apoA‐I. A chimera protein was recently designed in which the CT domain of apoA‐I was attached to insect apolipophorin (apoLp‐III). The insect apolipoprotein acquired apoA‐I like properties, including high affinity lipid binding. Thus, this system allows for the identification of apoA‐I segments that have a high affinity for lipid surfaces. To identify potential lipid binding regions within apoA‐I, three chimeric constructs were designed using L. migratoria apoLp‐III. Residues 1–43 or 44–65 of apoA‐I were attached to the NT end of apoLp‐III. In addition, residues 231–243 of apoA‐I were attached to the CT end of apoLp‐III. The three DNA constructs were inserted into the pET‐20b (+) expression vector through restriction enzymes HindIII and NdeI. Site‐directed mutagenesis was conducted to introduce two cysteines into apoLp‐III at positions Thr‐20 and Ala‐149. The two cysteines lock the protein in an inactive state under oxidizing conditions to prevent lipid binding. This enables to study the lipid binding properties of the attached apoA‐I segments in the chimera proteins. Proteins are currently being expressed in Escherichia coli BL21(DE3) pLysS cells and will be purified through nickel‐affinity and size‐exclusion chromatography. The lipid binding properties will be assessed by the ability to solubilize phospholipid vesicles.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Roles of the Juxtamembrane Cysteine and Glutamine Residues in Mucin 1 (MUC1) Dimerization
Authors: Li Edwin , Herrera Roberto , Cani Ksandros , Freeman Christina 
Published: June 2021

Mucin 1 (MUC1) is an O‐glycosylated transmembrane protein involved in forming protective mucous barriers on epithelial cells that line the mucosal surfaces of different tissues including breast, stomach, pancreas, and lung. When this protein is overexpressed, MUC1 dimerizes through the formation of disulfide bonds. A MUC1 fragment is then cleaved and transported to the nucleus, where it regulates apoptotic genes that allow for tumorigenesis. The CQC motif, located in the juxtamembrane domain of MUC1, has been shown to be important for dimerization to occur. However, the importance of the position of the CQC motif and the roles of the glutamine and each cysteine residues remain unclear. To answer these questions, we have been using the ToxR assay to measure beta‐galactosidase activity as a reporter of transmembrane dimerization. In this assay, we use a chimeric protein that contains the transmembrane domain and the juxtamembrane CQC motif of MUC1. To investigate the relevance of the position of the cysteines with respect to each other within the CQC motif, we have inserted an alanine before the glutamine (CAQC). To determine the role of the glutamine residue in dimerization, we have exchanged it for an alanine (CAC). Furthermore, to study the contribution of each cysteine, we have substituted the cysteine residues with alanine (CQA, AQC, and AQA). By providing additional information about the role of the CQC motif in dimerization we will further our knowledge about the mechanism of the MUC1 pathway that is involved in cancer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
COQ9 Membrane Association and Its Role in Coenzyme Q Biosynthesis
Authors: Von Bank Helaina C. , Lohman Danielle C. , Aydin Deniz , Smith Robert , Bingman Craig , Peraro Matteo Dal , Pagliarini David J. 
Published: June 2021

Coenzyme Q is a ubiquitous and hydrophobic lipid essential in cellular respiration. The de novo eukaryotic synthesis of coenzyme Q involves at least nine different proteins (COQ1–9), several of which have undiscovered functions. Many of these proteins are peripherally associated with the inner mitochondrial membrane as part of the biosynthetic complex Q. How these proteins bind the membrane and interact with coenzyme Q lipid intermediates remains unresolved. Filling these gaps in knowledge provides potential for improving the treatment of diseases associated with Coenzyme Q deficiency, such as Parkinson's disease and cardiomyopathy. Recently, we discovered that one member of complex Q, COQ9, can associate with mitochondrial membrane lipids. Our biochemical, crystallographic, and computational data reveal that a structurally unique amphipathic helix of COQ9 drives membrane association. Collectively, this work provides mechanistic insight into how a lipid‐binding protein associates with biological membranes and has important implications toward understanding its role in coenzyme Q biosynthesis.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myocardin‐Related Transcription Factor Plays a Role in Myofibroblast Transdifferentiation of Retinal Pigment Epithelial Cells
Authors: Ueda Shunichiro , McDonald Kevin , Tamiya Shigeo 
Published: June 2021

Retinal pigmented epithelial (RPE) cells have been implicated in fibrotic scarring of the retina. Past studies have shown transforming growth factor (TGF)‐beta plays a critical role in this process by inducing myofibroblast transdifferentiation. Myocardin‐related transcription factor (MRTF) has been implicated to play a key role in TGF‐beta initiated signaling in several fibrotic diseases, and thus, we examined its role in an in vitro model using small molecule inhibitors.Primary cultured porcine RPE cells were cultured for 3 days in media containing TGF‐beta2 (10ng/ml) in the presence or absence of the following inhibitors: TGF‐beta receptor inhibitor SB431542 (10 micro M), MRTF signaling inhibitors CCG100602 and CCG203971 (25 microM), and CCG1423 (10 microM). Staining of actin cytoskeleton and alpha‐smooth muscle actin (SMA) were performed using phalloidin and anti‐alpha‐SMA antibody, respectively. For the collagen matrix contraction assay, an in vitro model for fibrosis, cells were cultured three days on type 1 collagen gels, which were subsequently released from 24 well plates and photographed to evaluate contraction by measuring gel size. These same gels with attached cells were then lysed by RIPA buffer and used for Western blot analysis of alpha‐SMA, and tropomyosin1.TGF‐beta treatment induced myofibroblast transdifferentiation as evidenced by expression of alpha‐SMA incorporated prominent stress fibers and the associated significant increase in contractility. This phenotypic change was completely blocked by the TGF‐beta receptor inhibitor SB431542. Similarly, MRTF inhibitors CCG100602, CCG203671, and CCG1423 prevented cytoskeletal changes as well as the enhanced collagen matrix contraction induced by TGF‐beta. Interestingly, MRTF inhibitors did not significantly alter alpha‐SMA expression, but reduced expression of tropomyosin 1, a MRTF target that has been implicated to play a role in alpha‐SMA incorporation into stress fiber.These data strongly implicate that MRTF plays a key role in myofibroblast transdifferentiation of RPE cells downstream of TGF‐beta receptor activation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pericyte Cd248 Promotes Fibrosis Through Induction Of Pro‐Fibrotic Macrophages During Kidney Injury
Authors: Pai Chen‐Hsueh , Tsai Yu‐Fei , Yen Ching‐Tzu , Lin Shu‐Rung , Lin Shuei‐Liong , Lin Shu‐Wha 
Published: June 2021

CD248, also known as endosialin or tumor endothelial marker 1, is a type I transmembrane glycoprotein that is expressed in stromal cells of normal kidneys and uterus after birth. Using lacZ knock‐in (KI/+) mice we have shown that Cd248 expression decreases in most organs but increases and persists in postnatal kidneys, specifically in glomerular mesangial cells and pericytes/perivascular fibroblasts. In human chronic kidney disease, upregulated expression of CD248 is shown in myofibroblasts, which is negatively associated with renal survival. Although limited experimental data suggest that CD248 is involved in the cross‐talk with the post‐receptor signalling and cell proliferation of fibroblasts activated by platelet‐derived growth factors, the role of CD248 in kidney fibrosis is still unclear. Many studies including ours have identified CD248+ pericytes as the major source of precursors of scar‐producing myofibroblasts during progressive kidney fibrosis. Our data showed that expression of Cd248 was upregulated in the fibrotic kidney myofibroblasts of mouse model induced by unilateral ureteral obstruction (UUO). Cd248 knockout mice (Cd248−/−) showed less fibrosis and cell numbers of macrophages in the UUO kidneys. Moreover, UUO kidney macrophages expressed lower levels of chemokinde (C‐C motif) ligand 17 (Ccl17) and Ccl22 in Cd248−/− mice, suggesting CD248 deficiency led to a decrease of pro‐fibrotic macrophages. In vitro functional polarization assay of macrophages induced by co‐culture with wild‐type or CD248‐deficient myofibroblasts confirmed that CD248 played a role in the differentiation of pro‐fibrotic macrophages. Our results suggest that pericyte CD248 promotes fibrosis through induction of pro‐fibrotic macrophages during kidney injury.Support or Funding InformationThis study is supported by MOST (105‐2314‐B‐002 ‐096 ‐MY3 and 106‐2319‐B‐002‐001) and the Mrs. Hsiu‐Chin Lee Kidney Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Frutapin can interact with TLR4 and increase IL‐6 production by fribroblasts
Authors: Moreira Renato Azevedo , Sousa Felipe Domingos , Silva Ayrles Fernanda Brandão , Xu Shiwen , Owen James S. , Abraham David J. , Monteiro‐Moreira Ana Cristina Oliveira 
Published: June 2021

ObjectiveFrutapin (FTP) is a glucose/mannose‐binding lectin derived from Artocarpus incisa L. seeds which is a wide spread plant, common in pan‐tropical regions, popularly known as “fruta‐pão” (breadfruit) and it is consumed cooked, by the local Brazilian population. FTP is the second most abundant lectin in A. incisa seed extracts, but proved difficult to purify with very low yields and contamination with Frutalin, a multiple binding lectin, frustrating its characterization. Herein, we assess the effect of rFTP, obtained by heterologous production in Escherichia coli, on fibroblast biology.MethodsrFTP was successfully isolated in its biologically active form using SUMO‐tag strategy. Human skin fibroblasts (HSF) were used to perform cell viability and migration assay. Mitomycin C was used as an anti‐mitotic agent to assess whether FTP induces migration in scratch assay. The ability of rFTP to interact with TLR4 was evaluated using HEK‐Blue™‐hTLR4 cells. The effect of rFTP on IL6 levels was estimated by ELISA. Also, the effect of this lectin in HSF protein expression was evaluated by western blot.ResultsrFTP did not show cytotoxicity in a concentration range of 0.001 – 1mg/mL. HSF were cultured with media supplemented with rFTP to perform migration assay. By 24 h, samples treated with FTP had completely repaired the scratch, migrating across the entire denuded area effecting efficient wound closure. rFTP was able to potently stimulate TLR4 pathway in a manner similar to that observed with LPS. Also, rFTP stimulation increased reaching a plateau after five hours. High levels of IL6 produced by HSF were observed when compared to untreated cells (p<0.0001). The protein expression levels of pERK 1/2 and MyD88 were significant higher when compared to untreated cells during 24 h of treatment with rFTP concentration (p<0.05).ConclusionsIn conclusion, this study showed that rFTP was non‐cytotoxic to human fibroblasts. This lectin contributes substantially to cell migration and increase IL6 production by these cells, also stimulating TLR4 receptor. Given that fibroblasts play an important role in tissue repair, rFTP may represent a potential therapeutic biomolecule for wound healing and other related skin diseases.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MAST CELL NUMBERS OF RAT LUNGS IN AN ACUTE MODEL OF FAT EMBOLISM ARE REDUCED BY ALISKIREN AND LOSARTAN BUT NOT BY CAPTOPRIL
Authors: Colson Jordan , Arif Dauod , Voelker Dayne , Spaedy Abigail , Hamidpour Soheila , Poisner Alan , Pour Mohammad , Monaghan Paula , Khalafi Farnaz , Molteni Agostino 
Published: June 2021

IntroductionFat embolism (FE) induced by iv injection of triolein (T) causes vasculitis, septal inflammation and fibrosis in rat lungs at 48 hrs that is mitigated by two drugs acting on the renin‐angiotensin system (RAS): captopril and losartan (1). The histopathological changes found at this time are also ameliorated by treatment with the renin inhibitor aliskiren as the associated increase in mast cell numbers (2). This study examines whether captopril and losartan also prevent the increase in mast cell number at this time.MethodsSprague‐Dawley rats (280–300g) were treated with 0.2 ml iv (T) n=31 or saline n=7. One hour later 0.2 ml of saline (n=7) ip, aliskiren (50 mg/kg n=6), (100 mg/kg n=6), captopril (150 mg/kg n=6) or losartan (10 mg/kg n=6) were given. Saline treated rat (n=7) receive ip, one hour later, the same dose of saline. 48 hours later all the rats were necropsied after isofluorane anesthesia, lungs fixed in 10% formalin and H&E stained for morphology, Masson trichrome for fibrosis and CD 117 for mast cells. Two pathologists unaware of slides identity took at random 10 photographs of all the slides at 400× and separately counted the mast cells.ResultsAs previously reported, 48 hours after triolein the lungs showed severe histopathological damage that was prevented by all three of the RAS drugs (1–3) and was associated with an increase in the number of mast cells (3). Aliskiren treatment reduced their numbers confirming previous findings (3,4). A significant reduction was also induced by losartan but not by captopril despite its protective effects on lung histopathology.ConclusionsMast cells may be involved in the pathological damage found after FE as suggested by their increase in number in the lungs after T and their reduction during protective treatment with aliskiren. Aliskiren and losartan also reduced their number. However, the lack of effect of captopril on mast cell number at this acute time period may suggest that some other pathways are involved in the time course of mast cell recruitment, such as angiotensin (1–7).Support or Funding InformationSupported by the Catherine T Geldmacher Foundation, St. Louis, MOThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gas6/AXL Modulates Increased Inflammatory Cytokines During Preeclampsia in Rats
Authors: Hall Parker D. , Caskey Bryson , Hirschi Kelsey M. , Reynolds Paul R. , Arroyo Juan A. 
Published: June 2021

Preeclampsia (PE) is a complicated obstetric complication characterized by increased blood pressure and the production of inflammatory cytokines. The growth arrest‐specific 6 (Gas6) protein is known to induce dynamic cellular responses and is elevated in PE. Gas6 binds to the AXL tyrosine kinase receptor and AXL‐mediated signaling is implicated in proliferation and migratory mechanisms in several tissues. Our laboratory utilized Gas6 to induce preeclamptic conditions in pregnant rats. Our objective was to determine the role of Gas6 and AXL signaling in the release of cytokines during the development of PE in the rat. Briefly, pregnant rats were divided into 3 groups that received daily ip injections of PBS, Gas6, or Gas6 + R428 (an AXL inhibitor). Treatment was done from embryonic day 7.5 (E7.5) to E18.5, at which time they were euthanized and serum was harvested for Multiplex cytokine determination. Fetal and placental weights were recorded. Blood pressure and proteinuria were determined. We observed: increased proteinuria and elevated blood pressure in Gas6 treated animals, but each were inhibited by R428; a 12‐fold increase of serum interferon gamma was reduced by AXL inhibition; a 12‐fold increase in serum IL1‐alpha was inhibited by AXL inhibition; a 45‐fold increase in serum IL‐2 was reduced by AXL inhibition; a 10‐fold increase in serum IL1‐beta was reduced by AXL inhibition; a 29‐fold increase in serum IL4 was decreased by AXL inhibition; a 13‐fold increase in serum IL5 was reduced by AXL inhibition; an 24‐fold increase in serum IL6 was reduced by AXL inhibition; and a 20‐fold increase in IL12p70 was reduced by AXL inhibition. These data suggest roles for Gas6/AXL in cytokine elaboration coincident with PE and offer possible treatment avenues that may alleviate PE symptoms.Support or Funding InformationThis work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.