bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulator of G‐protein signalling 4 (RGS4) KO mice show increased susceptibility to atrial fibrillation
Authors: Cifelli Carlo , Dissanayake Kaveesh , Zhang Hangjun , Liu Jie , Nattel Stanley , Backx Peter H , Heximer Scott 
Published: June 2021

Susceptibility to atrial fibrillation (AF) depends strongly on parasympathetic activity. Since RGS4 inhibits parasympathetic signaling in the sinoatrial node, we explore whether changes in RGS4 levels altered the susceptibility of atrial fibrillation. Consistent with this possibility, RGS4 levels were decreased in atria of tachypaced dogs prior to their development of chronic AF. Moreover, in vivo ECG recordings of anaesthetized mice showed greater susceptibility to AF while optical mapping of isolated atrial preparations using a voltage‐sensitive dye revealed greatly increased susceptibility to rotor formation when rgs4 was ablated. Consistent with altered parasympathetic signaling in the myocardium of rgs4−/− mice, acetylcholine‐sensitive potassium currents (IKACh) evoked by carbachol application were greater in isolated atrial myocytes from rgs4−/− mice. These IKACh changes were, as expected, associated with marked enhancements of action potential duration shortening in response to parasympathetic activation, but not to slower conduction velocities. Together, our findings establish that RGS4 protects atrial tissues from excess parasympathetic signaling that predispose to atrial fibrillation.Technical and financial assistance for this work was provided by the Heart and Stroke Foundation of Ontario Grant‐in‐Aid Program grants T6799, and Canadian Institutes of Health Research (CIHR) Canada Graduate Scholarship Program.
Is b‐arrestin‐2 a determining factor in PAR‐2 stimulated allergic asthma?
Authors: Richards Heddie L , Wilson Emma , DeFea Kathryn 
Published: June 2021

PAR‐2 exhibits pro‐ and anti‐inflammatory effects in the airway and lungs. From a molecular standpoint, the anti‐inflammatory effects signal via PAR‐2/Gαq resulting in mobilization of intracellular calcium and prostaglandin production. Pro‐inflammatory effects include leukocyte migration and cytokine production. Our lab has demonstrated that PAR‐2 promotes leukocyte migration in a β‐arrestin‐dependent pathway as β‐arrestins scaffold the actin filament severing protein, cofilin, with its upstream activator, CIN at the leading edge. This brings CIN and cofilin into proximity leading to cofilin dephosphorylation and activation and is independent of classic Gαq signaling. We believe the PAR‐2 pro‐inflammatory effects are mediated by β‐arrestin‐dependent actin reorganization and chemotaxis, and will be abolished in mice lacking β‐arrestins.These studies illustrate β‐arrestin‐2 mediation of an allergic asthma inflammatory response to intranasal administration of allergen and PAR‐2 activating peptides. Wild type mice exposed to allergen and treated with PAR‐2 activating peptide have increased leukocyte recruitment to BAL and lung epithelium; while β‐arrestin‐2−/− mice do not. These data suggest that β‐arrestins facilitate PAR‐2 stimulated cell migration by recruitment of actin machinery to the leading edge. This pathway is important for PAR‐2 stimulated inflammatory responses in vivo.
Post‐endocytotic trafficking of ETB receptors in cardiomyocytes: effects of pressure‐overload hypertrophy on their subcellular distribution.
Authors: MERLEN Clemence , FARHAT Nada , Villeneuve Louis R. , Gillis Marc‐Antoine , Thorin Eric , Allen Bruce G. 
Published: June 2021

Endothelins are implicated in both cardiac function and pathophysiology via their actions on ETA (selective for ET‐1>>ET‐3) and ETB (non selective) receptors. To date, the cellular signaling evoked by ET‐1 has been studied by activating receptors at the cell surface. However, ETB are primarily located on the nuclear membrane in adult cardiac ventricular myocytes (ACVMs). The present study was to determine the origin of these receptors. In ACVMs, which express ETA and ETB, fluorescently‐labeled ET‐1 was internalized and localized to lysosomes plus perinuclear structures. ET‐3 was transported soley to lysosomes. In rat aortic endothelial cells, which express ETB, endocytosed ET‐1 and ET‐3 were transported to lysosomes. To determine if the subcellular distribution of ETRs was altered during hypertrophy, mice were subjected to transverse aortic constriction (TAC) for 7 days and the distribution of ETRs examined by immunocytofluorescence. Whereas ETB immunofluorescence was associated with the nuclear membrane in sham animals, additional perinuclear staining was observed in TAC hearts. The distribution of ETA immunofluorescence was unaffected. Hence, localization of ETB to the nuclear membrane is not a result of post‐endocytotic ligand/receptor complex trafficking and the subcellular distribution, and possibly trafficking, of ETB is altered during hypertrophy.(Supported by the CIHR, FICM and HSFC)
Enzymology of DHHC‐mediated protein palmitoylation
Authors: Jennings Benjamin Carey , Linder Maurine E 
Published: June 2021

Protein S‐palmitoylation is a post‐translational modification in which a 16‐carbon fatty acid is thioester linked to a cysteine residue. It is mediated by a family of enzymes with a conserved Asp‐His‐His‐Cys sequence (DHHC). Incubation of DHHC proteins with palmitoyl‐Coenzyme A (palm‐CoA) both in the presence and absence of a protein substrate results in the DHHC protein itself being acylated. Enzyme autoacylation is a prerequisite for substrate palmitoylation. Using two approaches, we are testing the hypothesis that DHHC enzymes use a two‐step transfer mechanism where the autoacylated species represents an acyl‐enzyme intermediate.Mutation of the DHHC cysteine residue results in loss of both enzyme and substrate acylation. Enzyme acylation is hydroxylamine‐sensitive, suggesting palmitate is attached via a thioester linkage to cysteine. In our first approach to directly monitor transfer a DHHC protein is reacted with [3H]palm‐CoA and then purified from any free [3H]palm‐CoA. The charged [3H]palm‐DHHC is then incubated with a protein substrate to determine if the radioactive acyl group is transferred. Our second approach is to use steady‐state kinetics to determine if DHHC proteins use a Bi‐Bi (ping‐pong) mechanism. A fluorescent peptide, HPLC‐based protein acyltransferase assay has been developed to achieve the higher substrate concentrations needed for kinetic analysis. Funding was provided by a pre‐doctoral fellowship from the American Heart Association (BCJ) and NIGMS (ML).
Sumoylation impacts the transcriptional activity of Nrf2 and is necessary for its localization to promyelocytic leukemia nuclear bodies
Authors: Theodore Melanie , McIntosh Deneshia , Kawai Yumiko , Arinze Ifeanyi J. 
Published: June 2021

The transcription factor Nrf2 (nuclear factor erythroid 2‐related factor 2) induces transcription of a variety of genes by binding to the antioxidant response element(s) (ARE) in target gene promoters. Covalent modification(s) that may impact the biology of Nrf2 after its separation from its inhibitor protein Keap1 are not well‐delineated. Using reporter gene assays and fluorescence imaging in HepG2 cells, we have explored the role of sumoylation in the biological action of Nrf2. In the presence of the SUMO‐specific protease SENP‐1, Nrf2‐induced transcription from ARE‐driven reporter gene constructs was greatly enhanced, suggesting that sumoylation inhibits Nrf2‐induced gene transcription. Treatment with the proteasome inhibitor MG‐132 directed green fluorescent protein‐Nrf2 (GFP‐Nrf2) to promyelocytic leukemia nuclear bodies (PML‐NBs); this treatment also induced co‐localization of GFP‐Nrf2 with red fluorescent protein‐SUMO‐1 (RFP‐SUMO‐1). This localization was abrogated by co‐transfection with an expression plasmid for SENP‐1. We interpret these results to mean that Nrf2 traffics to PML‐NBs in a SUMO‐dependent manner. The results also implicate sumoylation in the transcriptional action of Nrf2.Supported by NIH grants # SC1CA143985 and T32HL007737
Cholecystokinin‐Induced PKC α Activation Mediates the Translocation of RhoA in Mouse Pancreatic Acini
Authors: SABBATINI MARIA EUGENIA , WILLIAMS JOHN A 
Published: June 2021

IntroductionRhoA is a small G protein belonging to the Rho family that cycle between an inactive GDP‐bound and an active GTP‐bound forms. In previous work we showed that cholecystokinin (CCK) induces RhoA activation and translocation from the cytosol to the membrane.AimTo study which intracellular signals are involved in CCK‐induced RhoA translocation in mouse pancreatic acini.ResultsThe PKC stimulator phorbol 12‐myristate 13‐acetate increased the amount of RhoA in the membrane fraction and the PKC inhibitor GF‐203109X blocked CCK‐induced RhoA translocation while agents affecting calcium or cAMP had no effect. In order to test which isoform of PKC mediated the effect of CCK, we infected acini with adenoviruses expressing dominant negative (DN) isoforms of PKC α, δ and ε. Only in DN PKC α‐expressing acini was the effect of CCK on RhoA translocation reduced.ConclusionPKC α, but not calcium or cAMP, is required for CCK‐induced RhoA translocation.
Increased DNA Damage and Decreased DNA Repair Following Poly(ADP‐ribose) Glycohydrolase Inhibition and Chemotherapy
Authors: KOH DAVID W , Zhou Yiran , Feng Xiaoxing , Garcia Katherine D. 
Published: June 2021

The synthesis of poly(ADP‐ribose) (PAR) is an immediate response to DNA damage. We reported previously that the failure to degrade PAR by inhibiting PAR glycohydrolase (PARG) leads to hypersensitivity to DNA damage.OBJECTIVETo investigate the ability of PARG inhibition to increase the effectiveness of DNA‐damaging agents.METHODSUtilizing PARG null trophoblast stem cells, chromatin structure was analyzed by the detection of PAR‐modified nuclear proteins, DNA damage and repair were analyzed by Comet assay after treatment with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), and cell death was measured by FACS.RESULTSPARG null cells displayed high levels of PAR‐modified histones, suggesting chromatin structural change. Low‐dose MNNG led to high levels of DNA damage, which suggests greater DNA access due to PARG inhibition. Further, this DNA damage was not repaired after 12 hr, which suggests unrepairable levels of DNA damage or decreased DNA repair. Finally, treatment of PARG null cells with current chemotherapeutic agents led to increased cell death.CONCLUSIONThe results show that PARG inhibition enhances the efficacy of DNA‐damaging agents by inducing greater amounts of DNA damage and decreasing DNA repair. Therefore, targeting PARG is expected to enhance the effectiveness of DNA‐modifying anti‐cancer agents. Support was provided by ASPET, ACS, and the PhRMA Foundation.
The N‐terminal domain of BLM/Sgs1 displays a strand‐annealing and strand‐exchange activity that is required for in vivo function
Authors: Chen Chi‐Fu , Brill Steven 
Published: June 2021

Mutations in BLM result in Bloom's syndrome, a rare autosomal disease characterized by a variety of symptoms including a predisposition to cancer. BLM is conserved in the budding yeast Saccharomyces cerevisiae as Sgs1. BLM/Sgs1 is a 3′‐5′ DNA helicase with a large N‐terminal domain of unknown function. To determine the role of the N‐terminus, we assayed purified recombinant sub‐domains using biochemical methods. We found that an internal domain of the Sgs1 N‐terminus (amino acids #103–322, named the SA domain) can bind single‐stranded (ss) DNA using gel‐shift assay. Moreover, this ssDNA‐binding activity is conserved in the N‐termini of human and Drosophila BLM. Further studies demonstrate that the SA domain exhibits strand‐annealing and strand‐exchange activities in vitro. To determine the biological function of this domain, we tested the sgs1Δ103–322 allele for a variety of sgs1Δ phenotypes including MMS sensitivity, top3Δ slow‐growth and sgs1Δ slxΔ synthetic‐lethality. The results show that sgs1Δ103–322 complements MMS sensitivity but is defective for top3Δ slow‐growth promotion and sgs1Δslx4Δ and sgs1Δslx5Δ synthetic lethality. While further defining its modular structure, these results identify an essential pro‐recombinogentic function for BLM/Sgs1 and provide a framework to analyze the coordinated activities of Top3, Rmi1 and strand annealing.This work is supported by NIH‐R01 GM071268
Toward the isolation of a novel RNA‐RNA interaction based on an activity of a trans‐acting ribozyme
Authors: Ishikawa Junya , Furuta Hiroyuki , Ikawa Yoshiya 
Published: June 2021

The DSL ribozyme, a class of artificial ligase ribozymes, has a highly modular architecture, which catalyzes template‐directed RNA ligation on a helical substrate that can be either covalently connected (cis‐DSL) or physically separated (trans‐DSL) from the catalytic module. Substrate recognition by the catalytic module is promoted by one or two sets of GNRA/receptor interactions acting as clamps in the cis or trans configurations, respectively.In the field of organic chemistry, modular functional groups are attached into parent compounds to generate new or improved properties. This strategy is well established and a variety of the mudular groups renders the number of compounds infinite. Along with the fact, an abundance of RNA motifs could expand possibilities of functional RNAs. Recent works demonstrated artificially designed RNAs composing naturally occurring structural motifs could be a scaffold to isolate a small RNA unit.In the present study, we are attempting to isolate an artificial loop receptor motif based on the activity of trans‐DSL. For the selection, we have constructed a new derivative of the trans‐DSL by replacing one of its clamp modules with a target loop and a random sequence.This research was supported by Grant‐in‐Aids on Innovative Areas “Emergence in Chemistry” [No. 21111518 to Y.I.] and for the Global COE program, “Science for Future Molecular Systems” [to H.F.] from The MEXT of Japan.
Promoting Dendritic Cell Migration by Airway Epithelial Cells through Interleukin‐17A‐Induced CCL20 Expression
Authors: Hung Li Yin , Huang Fei , Nguyen Tiffany T. , Wu Reen 
Published: June 2021

We previously demonstrated that IL‐17A is a potent inducer of both human β‐defensin (hBD) 2 and CCL20/macrophage inflammatory protein (MIP)‐3α expression in well‐differentiated primary human airway epithelial cell (AEC) cultures. In addition to having a common anti‐microbial activity, both hBD2 and CCL20 are principal chemotactic ligands specific to CCR6‐positive dendritic cells (DC) and T lymphocytes. To further explore the chemotactic significance of IL‐17A in the mouse model, we conducted studies using isolated primary mouse AECs and Cc10‐Il17 transgenic animals. Consistent with human AECs, well‐differentiated mouse AECs also expressed elevated mCcl20 when treated with mIL‐17A. This induction was time‐ and dose‐dependent on the IL‐17A treatment, and the induction was inhibited by isohelenin, a specific inhibitor of NF‐κB activation. Chemotactic assays showed that conditioned media from mIL‐17A‐treated mouse AEC cultures stimulated the migration of CCR6‐positive mouse bone marrow‐derived dendritic cells (BMDC) in vitro; this chemotactic activity was blocked by anti‐CCL20 neutralizing antibody. We found a persistent elevation of mCcl20 message and the presence of CCR6‐positive DCs in bronchoalveolar lavage fluids, accompanied by increased mucin production in the lungs of Cc10‐Il17 transgenic mice, which overexpress IL‐17A specifically in the airway. Blocking the migration of CCR6 expressing cells in vivo lowered mucin production in the airway. These results suggest a functional role of IL‐17A/CCL20 in regulating CCR6‐positive DC migration and mucous cell metaplasia.This research was supported in part by grants from NIH (HL077902, HL077315, and ES00628)
Divergent biochemical roles for cytosolic and chloroplastic pyruvate phosphate dikinase regulatory protein
Authors: Failing Christopher Jon , Chastain Chris J 
Published: June 2021

Pyruvate orthophosphate dikinase regulatory protein (PPDK‐RP) is a unique regulatory protein found in plants. Its properties are highly unusual among all regulatory enzymes. First, it is a bi‐functional enzyme catalyzing both its protein kinase and protein phosphatase on its target protein (PPDK). Second, it uses ADP rather than ATP as a phosphoryl donor. Third, its dephosphorylation mechanism is a Pi‐dependent, pyrophosphate (PPi) forming phosphoryltic cleavage. Bioinformatic analysis of its primary sequence reveals that it is unrelated to any other known class of protein kinase. We describe in this presentation, a PPDK‐RP isoform cloned from Arabidopsis thaliana localized in the cytoplasm that lacks protein phosphatase activity. Our biochemical analysis of this isoform at its native cytsolic pH reveals a phosphoryl substrate preference for GDP and GTP rather than ADP. This difference suggests that this RP is regulated in an unknown yet strikingly different manner than its bi‐functional counterpart.This work was supported by NSF/RUI Grant no.0642190
Characterization of bovine 20a‐HSD gene promoter
Authors: Min Kwan‐Sik , Purevjargal Naidansuren , Kang Myeong‐Hwa 
Published: June 2021

The 20¥á‐hydroxysteroid dehydrogenase (20¥á‐HSD) enzyme catalyzes the NADPH‐dependent reduction of progesterone to 20¥á‐hydroxypregn‐4‐ene‐3‐ one (20¥á‐hydroxyprogesterone), which is biologically inactive. Thus, it is very important enzyme to maintenance of pregnancy in mammlian. We cloned the promoter (−927bp) of bovine 20a‐HSD gene to charaterize the functional role and constructed five deleted mutants into the pGL3‐basic vector. And we also constructed the EGFP gene vector under bovine 20a‐HSD promoter. Bovine 20a‐HSD gene promoter contains TATA box, two putative CCAAT boxes and several transcriptional factor sites (GATA‐1, CRE‐BP, Oct‐1, Ap‐1 and SRY). The promoter activity was remarkably decreased in all deleted vector using the rat corpus luteum cells.But, it was recovery by the co‐transfection of the Sp‐1 factor. The constructed vector (promoter+EGFP) was expressed EGFP signal into the CHO K1 cell at 2 days after transfection.(This work was supported by a grant (20080401034074) from BioGreen 21 Program, Rural Development Administration, Republic of Korea)
Racial differences in correlations between reported dietary carotenoid intakes and plasma carotenoid levels
Authors: Arab Lenore , Cambou Mary Catherine , Craft Neal , Wessling‐Perry Katherine , Jardack Patricia 
Published: June 2021

Because of cost and practicality, a dietary assessment tool that can accurately predict circulating plasma carotenoid levels is of great interest to epidemiologists. However, validity across racial groups needs to be assured. We studied dietary and plasma levels of oxycarotenoid levels (lutein & zeaxanthin, α‐, β‐cryptoxanthin) and hydrocarbons (lycopene, α‐, β‐carotene) among 123 healthy, non‐smoking adult African‐American (AA) and 128 Caucasian (CA) participants from Los Angeles. Age and gender adjusted correlations between diet and plasma differed substantially by race as seen in the Table. Despite similar plasma carotenoid levels, the correlations with dietary intakes were 0.09 to 0.40 lower among AAs. The 6‐day mean intakes from 24‐hr recalls for all carotenoids were lower among the AAs, consistent with similar validation studies. These results suggest that either this dietary assessment tool was less valid among African‐Americans or that other factors influencing circulating carotenoid levels differ between these groups. Supported by NIH R01CA105048.
Pearson's Correlations: Diet and Plasma Carotenoids

Carotenoid
AA N = 123
CA N = 128
Difference

Oxy Carotenoids

Lutein and Zeaxanathin
0.11
0.51
0.40

Total‐Cryptoxanthin
0.29
0.50
0.21

β‐Cryptoxanthin
0.30
0.50
0.20

α‐Cryptoxanthin
0.12
0.31
0.19

Hydrocarbons

Lycopene
0.00
0.09
0.09

β‐Carotene
0.22
0.41
0.19

α‐Carotene
0.18
0.38
0.20
Effects of regular black tea consumption on the antioxidant activity
Authors: Fisunoglu Mehmet , Besler Halit Tanju 
Published: June 2021

Tea consumption is associated with the reduced risk of coronary heart disease.In order to evaluate the effects of black tea consumption on the antioxidant activity, we designed a randomized, controlled study that involves 5 servings (200 ml)/d of black tea for 6 weeks in 16 healthy female volunteers who do not normally consume tea in their life. Subjects were randomly divided into 2 groups (study and control). Plasma levels of total antioxidant activity (TOAC), malondialdehyde (MDA), oxidized LDL (ox‐LDL), 8‐isoprostane (8‐epi PGF2α), and some amino acids susceptible to oxidation, namely histidine, proline, lysine, threonine, leucine, isoleucine, tyrosine, phenylalanine, tryptophan, and valine were measured.TOAC significantly increased in the study group compared to the control group (1.81 ±0.1 vs 1.68 ±0.1). Plasma 8‐epi PGF2α level significantly decreased in the study group compared with the baseline (181.4 ±85.9 vs 164.0 ±94.1) however did not statistically changed between two groups. Although there was a positive trend in the results of oxidative biomarkers (MDA 1.46± 0.14 vs 1.42± 0.50, ox‐LDL 1928.9± 373.9 vs 1892.6± 284.6), we did not find significant difference between two groups. Amino acids susceptible to oxidation, except tryptophan, were significantly increased (p< 0.05) in the study group.Daily 5 servings of black tea consumption significantly increased antioxidant activity.
Regulation of ATP Binding Cassette Transporter A1 (ABCA1) and ABCG1 by Fatty Acids in RAW 264.7 Macrophages
Authors: Ku Chai Siah , Park Young‐Ki , Coleman Sara L , Seo Jeong Min , Lee Ji‐Young 
Published: June 2021

As major transporters for cholesterol efflux in macrophages, ATP‐binding cassette transporter A1 (ABCA1) and ABCG1 play a pivotal role in reverse cholesterol transport, a major athero‐protective property of HDL. We investigated effect of fatty acids (FA) on ABCA1 and ABCG1 expression in RAW264.7 macrophages. Cells were treated with various FA, e.g., 16:0, 18:1, 18:2, 20:5 and 22:6 (100 μM) and T0901317, a liver X receptor (LXR) agonist. Realtime PCR revealed that unsaturated FA (UFA), not saturated FA, reduced basal expression of ABCA1 and ABCG1, which was abolished by T0901317. Trichostatin A, a histone deacetylase (HDAC) inhibitor, increased basal expression of ABC transporters and abrogated UFA‐induced repression. HDAC3 siRNA increased transporter mRNA by 3‐fold. Although repression of ABCA1 and ABCG1 mRNA by UFA was abolished by T0901317, protein levels remained diminished. When cells treated with 16:0 and 18:2 after knockdown of protein kinase C δ (PKCδ), reduction in ABCA1 and ABCG1 protein by 18:2 was further accentuated. Calpeptin or lactacystin, inhibitors for calpain and proteosome inhibitor, could not counteract 18:2‐induced reduction in protein. In conclusion, UFA repress ABCA1 and ABCG1 expression in macrophages by two mechanisms: LXR‐dependent transcriptional repression involving co‐repressor complex with HDAC3; calpain‐ and proteosome‐independent post‐transcriptional inhibition.Grant Funding Source: Faculty Seed Grant from University of Nebraska‐Lincoln Research Council and Layman Award from University of Nebraska Foundation