bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peptides derived from extruded amaranth (Amaranthus hypochondriacus) improved the anti‐inflammatory effect in LPS‐induced human THP‐1 and mouse RAW 264.7 macrophages by preventing the activation of NF‐κB pathway (1045.3)
Authors: Montoya‐Rodríguez Alvaro , De Mejia Elvira , Dia Vermont , Reyes‐Moreno Cuauhtémoc , Milán‐Carrillo Jorge 
Published: June 2021

The objective was to compare the anti‐inflammatory potential of unprocessed and extruded amaranth pepsin/pancreatin hydrolysates in lipopolysaccharide (LPS)‐induced human THP‐1 and mouse RAW 264.7 macrophages. THP‐1 and RAW 264.7 cells were activated with LPS and treated with unprocessed and extruded amaranth hydrolysates (0.25 to 1.00 mg/mL). Peptides in extruded amaranth hydrolysates displayed antioxidant capacity, angiotensin converting enzyme‐inhibitor activity, antithrombotic and dipeptidyl peptidase‐IV inhibitor activity. Extruded amaranth (1 mg/mL) significantly reduced TNF‐α secretion in THP‐1 and RAW 264.7 cells by 36.5% and 33.5%, respectively; with concomitant reduction in PGE2 (15.4% and 31.4%, respectively), and reduction in COX‐2 (38.1% and 67.6%, respectively). Phosphorylation of IKK‐α was significantly reduced by 52.5% and 88.2% leading to reduced phosphorylation of IκB‐α (86.1% and 66.2%, respectively); resulting in reduction in the expression of p‐65 NF‐κB subunits in the nucleus by 64.2% for THP‐1 cells and 70.7% for RAW 264.7 cells. In conclusion, amaranth hydrolysates inhibited LPS‐induced inflammation in human and mouse macrophages by preventing activation of NF‐κB signaling. Extrusion improved the anti‐inflammatory effect of amaranth hydrolysates in both cell lines, which might be attributed to the production of bioactive peptides during processing.Grant Funding Source: University of Sinaloa Research Grant
Resveratrol compounds are potent inhibitors of human holocarboxylase synthetase and cause a lean phenotype in Drosophila melanogaster brummer mutants (1045.39)
Authors: Cordonier Elizabeth , Ajam Riem , Camara Teixeira Daniel , Onur Simone , Zbasnik Richard , Döring Frank , Schlegel Vicki , Zempleni Janos 
Published: June 2021

Acetyl‐CoA carboxylases (ACC) 1 and 2 are key enzymes in fatty acid (FA) synthesis and repression of FA degradation, respectively. Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of the essential coenzyme biotin to ACCs. We hypothesized that dietary inhibitors of HLCS cause a loss of biotinylated ACCs, leading to a lean phenotype. The PEKISH library of natural compounds was screened for HLCS inhibitor activity in vitro; the screen suggested that resveratrol and its metabolites piceatannol and piceid inhibited HLCS by >90% at nutritionally relevant low micromolar levels (P<0.05 vs. vehicle, by ANOVA &amp; posthoc, n=3). When NIH3T3 fibroblasts were treated with 25 µM piceatannol the levels of biotinylated ACC 1 and 2 decreased by 22% compared to controls. When Drosophila melanogaster brummer mutants, which are genetically predisposed to accumulate body fat, were fed diets supplemented with resveratrol compounds, body fat mass decreased by 50% (P<0.05, n=4) in male and female flies. We conclude that resveratrol compounds are potent inhibitors of HLCS‐dependent biotinylation of ACC 1 and 2, thereby contributing toward body fat loss in Drosophila melanogaster brummer mutants. Future studies will include mammalian models.Grant Funding Source: Supported by ARD Hatch, NIH, and BMBF.
Matrix metalloproteinase‐19 deficient fibroblasts display a profibrotic phenotype (1046.4)
Authors: Pardo Annie , Jara Paul , Calyeca Jazmin , Romero Yair , Placido Luis , Yu Guoying , Maldonado Vilma , Selman Moises 
Published: June 2021

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually lethal interstitial lung disease of unknown etiology characterized by aberrant activation of epitelial cells that induce the migration, proliferation and activation of fibroblast/myofibroblasts. The resulting distinctive fibroblastic foci are responsible of the excessive extracellular matrix production. We have recently found that Mmp19‐/‐ mice develop an exaggerated bleomycin‐induced lung fibrosis but the mechanisms are unclear. In this study we explored the effect of MMP19 deficiency on fibroblast behavior. Compared with wild type mice, Mmp19‐/‐ lung fibroblasts showed a decreased expression of Th1‐1 (p<0.01), and alpha 1 (I) collagen gene and collagen protein production at baseline and after TGF‐β treatment (p< 0.05). Likewise, they showed a significant increase in growth rate (p< 0.01), and in transmigration over Boyden chambers coated with type I collagen or with Matrigel (p< 0.05). Microarray analysis revealed the upregulation of several profibrotic pathways in Mmp19‐/‐ lung fibroblasts. These findings suggest that the absence of MMP19 induces in lung fibroblasts a profibrotic phenotype.Grant Funding Source: CONACyT 177641
The role of platelet activation in the expression and function of platelet hyaluronidase‐2 (1046.7)
Authors: Albeiroti Sami , Ayasoufi Katayoun , de la Motte Carol 
Published: June 2021

Background: The deposition of hyaluronan (HA), which is a major glycosaminoglycan component of the extracellular matrix, increases during the course of inflammation. The inflammatory HA matrix, which can promote leukocyte binding and recruitment, can be fragmented be the hyaluronidase enzymes, specifically HYAL1 and HYAL2. Our lab has recently shown that human platelets express HYAL2 and that they can degrade HA from the surfaces of intestinal cells into smaller fragments capable of promoting inflammatory cytokine production. Objective: Define the mechanism by which platelets degrade HA and assess the role of platelet activation. Methods: Platelet degradation of purified HA in solution, immobilized HA and cell surface HA was assessed by carbohydrate gel electrophoresis, hyaluronidase assay, and HA ELISA‐like assay, respectively. HYAL2 expression was assessed by immunoblotting and flow cytometry. Results: 1) Platelets degrade purified HA only under acidic pH; 2) Platelets degrade cell surface HA in a HYAL2 dependent manner; 3) Activation of platelets results in increased surface HYAL2 expression and activity; 4) Platelet activation causes an increase in surface expression of HA binding proteins. Significance: Platelets circulate in an activated state during inflammatory diseases. Increased HYAL2 on activated platelets may lead to higher levels of HA fragments that perpetuate inflammation.Grant Funding Source: Supported by the NHLBI
Cyclin A2 as a regulator of DNA strand stability (1047.6)
Authors: Gygli Patrick , Catacutan Fay , Gokozan Hamza , Schmidt Theresa , Stephens Julie , Palanichamy Kamalakannan , Otero Jose 
Published: June 2021

Cyclin A2 regulates replication origin firing during S‐phase. It binds to Cdk1 and Cdk2 to direct phosphorylation of proteins for DNA synthesis. The goals of this study were to determine the role of Cyclin A2 in the prevention of DNA double strand breaks (DSB). We deleted Cyclin A2 in mouse neural stem cells (NSC) using a cre/lox approach. Cyclin A2‐null NSC showed increased BrdU incorporation, as well as increased staining for the mitotic marker pH3. However, the growth rate in these Cyclin A2‐null cells was slowed. To explain this discordance, we examined the DSB marker γH2AX. Cyclin A2‐null NSC show increased levels of γH2AX as compared to Cyclin A2‐preserved cells. When a DSB forms, the cell cycle is arrested until the break is repaired. Rad51, a marker of homologous recombination repair, and 53BP1, a marker of non‐homologous end joining, both localized to the site of DSB in Cyclin A2‐null cells. This indicates that Cyclin A2‐null NSC can repair these breaks. Our results suggest that Cyclin A2 does not function in the repair of DSB, but instead plays a role in DNA strand stability. Because NSC display similarities to glioma initiating cells, our results may be applied to development of a treatment of glioblastoma, which is currently treated by a combination of temozolomide and radiation. Novel therapies for this fatal brain tumor are needed, and Cyclin A2 is a potential target for radiosensitizing therapy for glioblastoma.Grant Funding Source: 1R21EB017539‐01A1, 8UL1TR000090‐05
Aquaporin‐1 translocation and degradation mediates the water transportation (1062.2)
Authors: Li Xuejun , Zhang Jianzhao , An Yu 
Published: June 2021

Acetazolamide (AZA), a prototype of carbonic anhydrase (CA) inhibitor, acts as a diuretic agent with its site of action on renal proximal tubules. These nephron segments also contain aquaporin‐1 (AQP1) water channels, which is required for the formation of concentrated urine. In the present study we examined the role of AQP1 on the diuretic effect of AZA. By using in vivo and in vitro models, we found that the AZA increased the urine volume from 8 hrs to 14 days. However, total CAs activity was decreased within 8 hours and then gradually recovered to the normal level and the CAs protein expression was increased at the beginning of AZA treatment. AQP1 was significantly reduced after AZA administration for 7 days. When combined with NaHCO3, AZA had a more pronounced diuretic effect. But it didn’t induce CA isozymes expression and the CAs activity anymore. However, AQP1 expression was still inhibited from day 7. AZA increased AQP1 ubiquitination in the HK‐2 cell membrane followed by the degradation induced by proteasome. By using the coimmunoprecipitation and the surface plasmon resonance (SPR) methods, we found that AZA facilitated AQP1 translocation onto cell membrane by promoting the interaction of AQP1 and myosin heavy chain (MHC). And this process was mediated by ERK and myosin light chain kinase (MLCK) pathway. These findings indicate that AZA promotes AQP1 translocation and degradation, which may partially mediates the effect of AZA.Grant Funding Source: the National Natural Science Foundation of China (No. 91129727, 81020108031, 30973558)
Transient knockdown of SULT4A1 induces upregulation of phototransduction proteins (1064.19)
Authors: Crittenden Francis , Ethen Cheryl , Wu Zhengliang , Chen Dongquan , Kraft Timothy , Parant John , Falany Charles 
Published: June 2021

Since it’s identification in 2000 by Falany et al, sulfotransferase (SULT) 4A1 has presented an enigma to the field of drug metabolism. The SULTs are a superfamily of enzymes responsible for the sulfation of a wide variety of endogenous and exogenous compounds. They catalyze a Phase II conjugation reaction in which a sulfonate group is transferred onto the substrate primarily for conjugation to hydroxyl groups, leading to increased water solubility and excretion. Key structural features shared between SULT4A1 and other SULTs include the KXXFTVXXXE dimerization domain, active site histidine, and conserved PAPS binding site. SULT4A1 is primarily expressed in neurons of the central nervous system and is highly conserved in vertebrates, suggesting an important, conserved function within the nervous system. A function or enzymatic activity for SULT4A1, however, has yet to be elucidated. The goal in this study is to identify a function for this highly conserved orphan SULT. Human SULT4A1 and zebrafish SULT4A1 exhibit 88.4% sequence identity, making zebrafish an excellent model organism for genetic manipulation and functional characterization of this protein. Splice blocking morpholino oligonucleotides (MOs) were used to suppress SULT4A1 expression in zebrafish larvae over the first several days of development. Knockdown (KD) efficacy was demonstrated by qPCR and immunoblot analysis. RNA‐seq was used to investigate the effects of the inhibition of SULT4A1 expression on the transcriptome of the KD larvae. Analysis of the RNA‐seq data using Ingenuity Pathway Analysis showed the phototransduction pathway to be significantly dis‐regulated by SULT4A1 KD. A total of 13 genes known to be involved in phototransduction were shown to be up‐regulated by SULT4A1 knockdown. qPCR was used to validate up‐regulation of selected genes identified by RNA‐seq. This study is the first to identify a cellular process whose regulation appears to be associated with SULT4A1 expression.Grant Funding Source: Supported by NIH grant R21MH095946
Glycosylation of protease‐activated receptor‐1 regulates G12/13 versus Gq signal pathway bias (1066.13)
Authors: Smith Thomas , Soto Antonio , Chen Buxin , Trejo JoAnn 
Published: June 2021

The objective of this study was to examine the role of N‐linked glycosylation in protease‐activated receptor‐1 (PAR1) biased signaling. Thrombin cleaves the N‐terminus of PAR1, generating a new N‐terminal domain that functions as a tethered ligand by binding intramolecularly to extracellular loop 2 (ECL2) to elicit signaling through multiple G protein subtypes including Gq, G12/13 and Gi. Activated PAR1 displays biased signaling in response to different activating proteases. We discovered that N‐linked glycosylation of PAR1’s ECL2 dictates differential coupling of PAR1 to Gq versus G12/13 signaling. Thrombin activation of PAR1 WT caused robust G12/13‐dependent RhoA activation and stress fiber formation, whereas these responses were diminished in a PAR1 mutant lacking glycosylation. Accordingly, activated PAR1 WT associated more robustly with G12/13 proteins than did the PAR1 NA ECL2 glycosylation mutant. In contrast, activated PAR1 NA ECL2 mutant exhibited a greater capacity to associate with Gq, elicit Gq signaling, and promote Gq‐dependent cellular proliferation compared to WT receptor. These findings suggest that N‐linked glycosylation at ECL2 is critical for stabilizing different PAR1 active conformations that facilitate coupling to distinct G protein subtypes. Thus, we hypothesize that N‐linked glycosylation at ECL2 is critical for regulating PAR1 biased signaling.Grant Funding Source: Supported by R01 GM090689 and F31 HL116187‐01A1
Ubiquitination of PAR1 nucleates a non‐canonical p38 signaling pathway to regulate thrombin‐induced vascular leakage (1066.15)
Authors: Grimsey Neil , Smith Thomas , Le Philip , Trejo JoAnn 
Published: June 2021

Thrombin activation of the protease‐activated receptor‐1 (PAR1), a GPCR, triggers inflammation, endothelial barrier permeability and vascular leakage. Multiple signaling pathways contribute to vascular leakage including activation of the p38 MAP kinase. However, the precise mechanism of PAR1‐stimulated p38 activation is not known. In this study, we examined the role of PAR1 ubiquitination in the activation of p38 signaling and endothelial barrier dysfunction. We demonstrate that thrombin activation of PAR1 stimulates p38 signaling via a novel ubiquitin/TAB1/TAB2‐induced p38 autophosphorylation pathway. Thrombin‐induced ubiquitination of PAR1 is mediated by the activation and recruitment of the E3 ubiquitin ligase NEDD4‐2. The adaptor protein TAB2 then binds to ubiquitinated PAR1 and recruits another adaptor TAB1. TAB1 also binds and triggers p38 activation through autophosphorylation and not the MKK3/MKK6 pathway. This pathway is conserved for ADP‐dependent activation of the P2Y1 purinergic receptor. We further demonstrate that thrombin activation of PAR1 requires non‐canonical p38‐signaling pathway to regulate barrier permeability of cultured human endothelial cells. Collectively, these data suggest that a subset of GPCRs utilize ubiquitin to assemble and activate a TAB2 and TAB1 non‐canonical p38 signaling complex that regulates endothelial barrier permeability and vascular leakage.Grant Funding Source: Supported by NIH R01 HL073328 and Western States Affiliate AHA Postdoctoral Fellowship
Understanding the mechanism of biased agonism at chemokine receptors (1066.17)
Authors: Rajagopal Sudarshan , Bassoni Daniel , Campbell James , Gerard Norma , Gerard Craig , Wehrman Tom 
Published: June 2021

Chemokines display considerable promiscuity with multiple ligands and receptors shared in common. Their receptors are G protein‐coupled receptors (GPCRs), which are capable of signaling with different efficacies to their multiple downstream pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands and the biologic importance of such signaling is unclear. To assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a pharmacologic analysis of a set of chemokine receptors using assays for G protein signaling, β‐arrestin (βarr) recruitment and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein‐ or βarr‐mediated signaling or receptor internalization. Moreover, some chemokine receptors display significant bias for all their ligands, acting as “biased receptors”. Ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.Grant Funding Source: Supported by HL07101‐34 (SR); R21AI092388, R21AI097468 (JC); R01AI039795‐14, 5R01HL51366‐15 (CG)
The ubiquitin ligase Deltex‐3L regulates endosomal sorting of the G protein‐coupled receptor CXCR4 (1066.18)
Authors: Holleman Justine , Marchese Adriano 
Published: June 2021

G protein‐coupled receptor (GPCR) sorting into the degradative pathway is important for attenuating signaling. Upon agonist activation of the chemokine receptor CXCR4, a GPCR, it is rapidly ubiquitinated, internalized to endosomes and sorted to lysosomes via the endosomal sorting complex required for transport (ESCRT) pathway. ESCRT‐0 ubiquitination is linked to the efficiency with which CXCR4 is sorted for lysosomal degradation, however mechanistic insight is lacking. Here, we show for the first time that the RING‐domain E3 ubiquitin ligase Deltex‐3‐like (DTX3L) mediates CXCR4 sorting from early endosomes to lysosomes. Using several biochemical and immunochemical techniques including fixed cell confocal immunofluoresence microscopy, co‐immunoprecipitation and in vitro ubiquitination assays, we show that DTX3L localizes to early endosomes upon CXCR4 activation and interacts directly with and inhibits the activity of the E3 ubiquitin ligase atrophin‐1 interacting protein 4 (AIP4). This serves to limit the extent to which ESCRT‐0 is ubiquitinated and is able to sort CXCR4 for lysosomal degradation. Therefore we have defined a novel role for DTX3L in GPCR endosomal sorting and propose that DTX3L may play a broad role in endosomal sorting. In addition, our data reveal an unprecedented link between two distinct E3 ubiquitin ligases to control the activity of the ESCRT machinery.Grant Funding Source: GM075159
CGRP release from perivascular capsaicin‐sensitive sensory nerves regulates vascular function through pannexin‐1 channel opening (1075.4)
Authors: Gaete Pablo , Lillo Mauricio , Poblete Inés , Figueroa Xavier 
Published: June 2021

Resistance arteries are inervated by capsaicin‐sensitive sensory nerves (CSSNs) that release calcitonin gene‐related peptide (CGRP). CGRP is a vasodilator, but its participation in the regulation of vascular signaling has not been explored. We assessed if CSSNs regulate the expression of key proteins involved in the control of vascular function. The isolated mesenteric arterial bed of rat was perfused with 1 µM capsaicin for 20 min. Protein expression was evaluated by Western blot and cell permeability through hemichannels such as pannexin (Panx) channels was assessed by ethidium uptake. Expression of eNOS and Panx‐1, but not connexin (Cx) 32, Cx43 or Panx‐2, was reduced 3 h after capsaicin treatment. CGRP8‐37, a CGRP‐1 receptor blocker, or probenecid, a Panx‐1 channel inhibitor, prevented the reduction of eNOS and Panx‐1 levels. Capsaicin and CGRP (100 nM) increased ethidium uptake in mesenteric arteries, but only CGRP in cultured endothelial cells. Capsaicin‐ or CGRP‐induced ethidium uptake was prevented by CGRP8‐37, probenecid, KT‐5720 (PKA inhibitor) or NG‐nitro‐L‐arginine (NOS blocker). These data suggest that transient activation of CSSNs leads to down‐regulation of eNOS and Panx‐1 expression by a Panx‐1 channel‐dependent pathway. Panx‐1 channel opening depends on CGRP‐1 receptor activation, which initiates a PKA‐mediated signaling and NO production.Grant Funding Source: Supported by FONDECYT 1100850 and AT 24121388.
LRRC26 is a functional auxiliary γ subunit for BKCa channels in arterial smooth muscle cells (1077.1)
Authors: Evanson Kirk , Bannister John , Leo M , Jaggar Jonathan 
Published: June 2021

Smooth muscle cell (myocyte) large‐conductance calcium (Ca2+)‐activated potassium (BKCa) channels are functionally significant modulators of arterial contractility. Leucine‐rich repeat containing (LRRC) proteins are novel auxiliary γ subunits that were recently identified to elevate the voltage‐sensitivity of recombinant or LNCaP cell BKCa channels. Here, we investigated the expression and physiological functions of LRRC26 in arterial myocytes. RT‐PCR and Western blotting detected LRRC26 mRNA and protein in cerebral artery myocytes. Biotinylation, immunofluorescence and immunofluorescence resonance energy transfer microscopy indicated that LRRC26 was primarily plasma membrane‐localized and in close spatial proximity to BKCa α subunits. LRRC26 knockdown using RNAi reduced arterial LRRC26, but did not alter BKCa α or β1 protein. Patch‐clamp electrophysiology demonstrated that LRRC26 knockdown reduced BKCa channel voltage‐ and apparent Ca2+‐sensitivity and transient BKCa current frequency and amplitude in arterial myocytes. LRRC26 knockdown increased myogenic tone over a range (40 ‐ 100 mmHg) of intravascular pressures and reduced functional responses to iberiotoxin, a BKCa channel inhibitor. In summary, we show that LRRC26 is expressed, associates with BKCa α subunits, and elevates channel voltage‐ and Ca2+‐sensitivity in arterial myocytes to induce vasodilation.Grant Funding Source: Supported by NHLBI/NIH (HL67061 and HL110347 to J.H.J and HL116175 to K.W.E)