bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Essential Role of Calmodulin Binding to the Juxtamembranous Segment of the Fourth Submembrane Domain in the Function of the α‐1A Adrenergic Receptor
Authors: Gebert‐Oberle Briana , VerMeer Mark , TRAN QUANG‐KIM 
Published: November 2023

The α1 adrenergic receptor type 1A (α1A‐AR) plays crucial roles in the control of adrenergic functions. Calmodulin (CaM) is the ubiquitous transducer of intracellular Ca2+ signals. Here we show a novel physical and functional interaction between CaM and α1A‐AR. α1A‐AR and CaM coimmunoprecipitate in primary vascular smooth muscle cells and cardiac tissue in non‐stimulated conditions. Novel FRET biosensors revealed direct interaction between CaM and the juxtamembranous segment of the fourth submembrane domain of α1A‐AR (SMD4JM), a domain that encompasses the nuclear localization signal (NLS) of α1A‐AR. Characterization of affinity and Ca2+ sensitivity for the interactions with CaM showed that the NLS itself interacts with CaM, but with significantly lower affinity and Ca2+ sensitivity compared to the SMD4JM, suggesting that the entire SMD4JM is required for full interaction with CaM. Conservative mutations in the non‐NLS segment of SMD4JM that reduced CaM binding affinity of the SMD4JM 3‐fold completely converted α1A‐AR from a membrane targeted receptor to an intracellular receptor. These changes are also associated with substantially reduced α1A‐AR‐mediated ERK1/2 phosphorylation. The results demonstrate an essential role for CaM binding to the SMD4JM in the subcellular localization and function of α1A‐AR and indicate a role for CaM in the control of adrenergic functions.Support or Funding InformationIowa Osteopathic and Educational Research FundsNational Institutes of Health Grant HL‐112184
Signal transduction of lyso‐type phosphatidylethanolamine lipids on intracellular calcium concentration in dopaminergic cells
Authors: Park Soojin , Im Dongsoon 
Published: November 2023

BackgroundMetabolic products of phosphatidylethanolamines, lyso‐type phosphatidylethanolamine lipids, shortly LPEL, are produced by phospholipase A2. LPEL increased intracellular Ca2+ concentration in mammalian cells. G‐protein‐coupled receptor (GPCR) were supposed to be involved in the response. GPCRs for lysophosphatidic acid (LPA) might be responsible for the action in certain cell types but not in others. In the present study, action mode of LPEL on [Ca2+]i in human dopaminergic cells was studied.METHODSHuman dopaminergic cells were cultured at 37°C in a 5% CO2 humidified incubator, and maintained in high glucose RPMI1640, containing 5% (v/v) heat‐inactivated horse serum, 100 units/mL penicillin, 50 μg/mL streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate. Intracellular Ca2+ concentration was measured in the cells that were trypsin‐digested, allowed to sediment, resuspended in Hepes‐buffered medium, and then incubated for 40 min with 5 μM of fura 2‐AM. [Ca2+]i levels were estimated by measuring changes in fura 2 fluorescence using a fluorescence spectrophotometer.RESULTSLPEL induced increase of [Ca2+]i in the dopaminergic cells. Also, LPA induced similar increase of [Ca2+]i in the cells. LPEL‐ and LPA‐induced responses showed homologous and heterologous desensitization. Structurally different antagonists of LPA1/3, Ki16425 and VPC32183 inhibited LPEL‐induced increase of [Ca2+]i. And a specific inhibitor of LPA1, AM‐095, also inhibited the LPEL‐induced Ca2+ response completely in dopaminergic cells. Expression of LPA1 and LPA6 was found in the cells, but not other LPA receptors. Pertussis toxin, s specific inhibitor of Gi/o proteins completely inhibited LPEL‐induced [Ca2+]i increase. And edelfosine, an inhibitor of phospholipase C, and 2‐APB, an inhibitor of IP3 receptor, also completely inhibited LPEL‐induced Ca2+ responses. HA130, an inhibitor of autotaxin/lysophospholipase D, didn't inhibited LPEL‐induced response.DISCUSSIONIn the present study, LPEL‐induced [Ca2+]i increase was found to be mediated via LPA1 in dopaminergic cells. Heterologous desensitization, complete inhibition by AM‐095, LPA1 antagonist, and Gi/o‐coupling character of LPA1 and the PTX‐sensitivity of LPEL‐induced [Ca2+]i increase strongly supported the hypothesis in dopaminergic cells. Also results indicate that LPEL increases [Ca2+]i sequentially via LPA1 receptors, Gi/o proteins, phospholipase C, IP3, Ca2+ rise, and Ca2+ influx in the dopaminergic cells.
Essential Functions of the N‐terminal Domain of Tim17 in Trypanosoma brucei
Authors: Chaudhuri Evan , Weems Ebony , Singha Ujjal K , Chaudhuri Minu 
Published: November 2023

The majority of proteins essential for mitochondrial functions are encoded in the nuclear DNA and subsequently imported from the cytosol by translocases of the mitochondrial outer and inner membranes, termed TOM and TIM, respectively. These translocases are multimeric protein complexes, which are overall conserved from fungi to human. However, mitochondrial protein import machinery in trypanosomatids, a group of ancient and early branching unicellular eukaryotes that causes a fatal human disease African trypanosomiasis, is significantly different. As a part of this machinery, we previously characterized Tim17 from Trypanosoma brucei. TbTim17 is critical for mitochondrial protein import and thus essential for parasite survival. Here we further investigate the function of the divergent N‐terminal domain of this protein. We found that deletion of the first 20 or 30 amino acid residues (Δ20‐ and Δ30‐) of TbTim17 is detrimental for its function. These mutant proteins neither complement the growth defect of TbTim17 RNAi cells nor the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria. However, knock‐in of the full‐length protein (FL‐TbTim17) completely restored these functions in RNAi cells. Further analysis showed that both of these mutants of TbTim17 were properly targeted to mitochondria, however, Δ20‐TbTim17 was not membrane integrated and Δ30‐mutant, though integrated into the mitochondrial membrane was sensitive to protease digestion of the isolated mitochondria, indicating that the N‐terminal region is necessary for proper integration of TbTim17 into the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and mutant mitochondria by Blue‐native gel electrophoresis revealed that none of these mutants were assembled into the TbTim17 protein complex. Co‐immunoprecipitation analysis showed that unlike the wild type TbTim17, the mutant proteins were not associated with the endogenous TbTim17 as well as its interacting partner TbTim62, a novel trypanosome specific Tim. Together, these results show that the N‐terminal domain of TbTim17 is critical for its interaction with TbTim62 and required for the formation of a functional TbTim17 protein complex. To elucidate the assembly of the TIM complex in T. brucei, analysis is under progress to identify essential amino acids residues of this region by site‐directed mutagenesis.Support or Funding InformationNIH Grants 2SC1GM081146‐05, T32HL007727, and G12RR003032
Activation of Human antigen R (HuR) downstream of p38‐MAPK promotes cardiomyocyte hypertrophy through modulation of NFAT activity
Authors: Slone Samuel , Anthony Sarah R , Tranter Michael 
Published: November 2023

The RNA binding protein HuR (Human antigen R) interacts with specific AU‐rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. RNA binding proteins such as HuR are becoming recognized as potential mediators of cardiac physiology and pathology but the role of HuR in cardiac hypertrophy is largely unknown. Activation of Gαq (Gq) proteins downstream of Gq‐coupled GPCRs such as angiotensin receptors (AT1R) or α‐adrenergic receptors (α1‐AR) is a known mediator of pathological hypertrophy, but the responsible signaling pathways downstream of Gq have yet to be fully elucidated.Our results show that HuR cytoplasmic translocation (a marker of HuR activation) is significantly increased in neonatal rat ventricular myocytes (NRVMs) following treatment with phenylephrine or angiotensin II, agonists of two independent Gq‐coupled GPCRs known to induce hypertrophy. It appears that this Gq‐mediated HuR activation is dependent on p38 MAPK, but not canonical Gq‐PKC signaling. Furthermore, we show that HuR activation is necessary for Gq‐mediated hypertrophic growth of NRVMs as siRNA‐mediated knockdown of HuR inhibits hypertrophy as measured by cell size and expression of ANF (atrial natriuretic factor). To decipher the downstream mechanisms by which HuR translocation promotes cardiomyocyte hypertrophy, we assessed the role of HuR in the transcriptional activity of NFAT (nuclear factor of activated T cells), the activation of which is a hallmark of cardiac hypertrophy. Using an NFAT‐luciferase reporter assay, we show that HuR knockdown or inhibition by the use of siRNA and small molecule inhibitors results in acute inhibition of NFAT.In conclusion, our results identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq‐p38 MAPK pathway, and suggest modulation of NFAT activity as a potential mechanism.Support or Funding InformationThis work was partially supported by an AHA Scientist Development Grant to MT (Award #: 16SDG27360004).
RhoA Regulation of Drp1 and Cardiac Mitochondrial Fission
Authors: Brand Cameron S. , Tan‐Sah Valerie P. , Brown Joan Heller , Miyamoto Shigeki 
Published: November 2023

G‐protein coupled receptors can signal downstream through various pathways, including through activation of the small G‐protein RhoA. In cardiomyocytes, RhoA signaling is protective against ischemia/reperfusion injury. We have previously shown that this is mediated through downstream activation of Protein Kinase D (PKD), phosphorylation of PKD targets, and diminished translocation of pro‐apoptotic proteins to the mitochondria (Xiang et al, Sci. Signaling 2013). Mitophagy, a process that removes damaged mitochondria and limits mitochondrial death signaling, has also been suggested to be a cardioprotective response to oxidative stress. Mitochondrial fission is considered to be a preliminary step to clearance of damaged mitochondria via mitophagy. We recently observed that adenoviral expression of constitutively active RhoA in neonatal rat ventricular myocytes (NRVMs) increases RhoA levels at the mitochondria. Adenoviral RhoA expression also results in small, fragmented mitochondria as observed by fluorescent confocal microscopy and electron microscopy, indicative of increased mitochondrial fission. The main protein involved in mitochondrial fission, Drp1, translocates from the cytosol to the mitochondria when activated. We used an adenoviral miniSOG‐tagged Drp1 construct to ask whether expression of active RhoA changes Drp1 levels at the mitochondria. Our results show that mitochondrial miniSOG‐Drp1 is increased within 12 hours of adenoviral expression of constitutively active RhoA. In addition adenoviral RhoA expression increases PKD levels at mitochondria. In summary, we show that RhoA associates with mitochondria and can induce mitochondrial Drp1 and PKD accumulation, as well as mitochondrial fission. The relationship between these mitochondrial signaling events is currently under investigation, with the goal of determining whether RhoA‐dependent Drp1 accumulation and mitochondrial fission contribute to cardioprotection.Support or Funding InformationNIH Grant R37‐HL028143NIH Training Grant T32‐HL007444
“Characterization of mRNA Transcript Expression of IGF‐I/PI3K Pathway Components in Regenerating Skeletal Muscle”
Authors: Geddis Alyssa V , Mary Abdalla N , Leandry Luis A , Carrigan Christopher T , Matheny Ronald W , Hobbs Stuart S 
Published: November 2023

Skeletal muscle regeneration is a complex process involving the coordinated input from multiple intra‐ and extra‐cellular stimuli. Of these processes, actions of the insulin‐like growth factor‐I (IGF‐I) and phosphoinositide 3‐kinase (PI3K) pathways are critical for regeneration to occur. Multiple isoforms of PI3K catalytic and regulatory subunits have been identified. All Class IA PI3K p110 catalytic isoforms (p110alpha, p110beta, and p110delta) are expressed in skeletal muscle, whereas Class IB PI3K catalytic isoform gamma is expressed primarily in immune cells. Given the importance of the IGF‐I/PI3K pathway in skeletal muscle repair, we sought to determine whether gene expression of components of these pathways was sensitive to initial injury intensity. To test this, we injured the left tibialis anterior (TA) muscle of mice by application of a cold (−70° C) probe to induce traumatic injury. The probe was applied for either 5 seconds (5s) or 10 seconds (10s) to induce two different injury intensities, while the uninjured contralateral leg served as an internal control. TA muscles from injured and control legs were harvested three days (3d) or seven days (7d) after the injury for real‐time PCR analysis. Three days following 5s injury, we found increased expression of Igf1 (~4‐fold), Igf1r (~1.3‐fold), Pik3cb (~2‐fold), Pik3cg (~16‐fold), and Pik3r5 (~20.5‐fold) in the injured leg compared to the control leg (P<0.05 for all). Three days following 10s injury, we observed increased expression of Igf2 (~5‐fold), pik3cb (~3‐fold), Pik3cd (~4.5‐fold), pik3cg (~47‐fold), Pik3r1 (~2.6‐fold), Pik3r5 (~52‐fold) (P<0.05 for all). Significant differences between 5s and 10s injuries at 3d were observed for pik3cb, pik3cd, pik3cg, pik3r5 (P<0.05 for all). Seven days after injury, statistical differences were observed only between control leg and 5s injury only for Igf1r (~0.64‐fold; P<0.01); however a number of transcripts were altered following 10s injury [Igf1 (~3.9‐fold); Igf2 (~28‐fold); Pik3cd (~0.71‐fold); Pik3cg (~5.8‐fold); P<0.05 for all)]. These data reveal that during early skeletal muscle regeneration, transcript expressions for some components of the IGF‐I/PI3K pathway are sensitive to initial injury intensity. The increased expression of p110gamma (pik3cg) and its regulatory subunit (Pik3r5) in 3d injured muscle is consistent with injury intensity‐dependent immune cell infiltration.The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.
The roles of peripheral blood natural regulatory T cells and the expression of TNF‐α in aortas and cardiac tissues during the progression of atherosclerosis
Authors: HAO CHUNYAN , DUAN HUBIN , LUO FEITONG , LI SHUZHEN 
Published: November 2023

ObjectsTo explore the immune regulation effect of peripheral blood natural regulatory T cells and the expression of TNF‐α in aortas and cardiac tissues during the progression of atherosclerosis.Methods36 male SD rats (body weight 100±10g) were randomly divided into three groups, healthy control group (A group), early stage atherosclerosis group (B1 group), late stage atherosclerosis group (B2 group), each group have 12 rats. Rat atherosclerosis model can be established by treating rats with celiac injection of vitamin D3 accompanied by high sucrose‐high fat diet in this experiment. The frequency of CD4+CD25+ Foxp3+ regulatory T cell in peripheral blood was measured by fluorescence‐activated cell sorter (FACS) in each group. Aortas and cardiac tissues were observed with HE staining for pathological changes. The expression of TNF‐αin the rat aortic roots and cardiac tissues was detected by immunohistochemical assayResultsThere is no plaque in the rat aortic roots in Group A. There is fibrous plague in the rat aortic roots in Group B1. There is atheromatous plague in the rat aortic roots in Group B2. The expression of TNF‐αprotein in Group B1 and B2 was higher than those in Group A (P<0.05). Peripheral frequency of CD4+CD25+ Foxp3+ regulatory T cell was decreased in Group B1 compared with those in Group A and B2 (P<0.05).ConclusionIn the early stage of atherosclerosis, peripheral frequency of nTreg was decreased, where as the expression of TNF‐αprotein in aortas and cardiac tissues were increased, suggesting an increasing inflammatory response. In the late stage of atherosclerosis, peripheral frequency of nTreg was increased, suggesting an important negative feedback mechanism that results in the attenuation of prolonged or excessive immune responses, which otherwise may cause severe collateral damage.Support or Funding InformationThis study was supported by the Scientific research project of Shanxi provincial health department (No. 201301068), Doctor Start‐up fund of Shanxi Medical University and The first hospital of Shanxi Medical University, Science and technology innovation projects of Colleges and universities of Shanxi Province
Sex and tissue‐dependent expression of transporters and enzymes involved in GHB disposition
Authors: Felmlee Melanie A 
Published: November 2023

PurposeTo investigate tissue‐dependent sex differences in expression of monocarboxylate transporters (MCTs and SMCTs), aldo‐keto reductases (AKRs) and alcohol dehydrogenase iron containing 1 (Adhfe1), key determinants of the disposition of gamma‐hydroxybutyric acid (GHB).MethodsmRNA expression of MCTs (1/4), SMCTs (1/2), AKR1A1, AKR7A2, and Adhfe1 were evaluated by qPCR in male and female Sprague‐Dawley rats (9 weeks of age; N = 3 – 4 per gender) in kidney, liver and intestine.ResultsGender differences were observed for both enzymes and transporters involved in GHB disposition. Hepatic expression of MCT1, MCT4, AKR7A2 and Adhfe1 were significantly lower in females (−1.4 to 2.5 fold). Expression of SMCT1 (+3.5 fold) in the kidney and SMCT1 (+5 fold), SMCT2 (+11 fold) and AKR1A1 (+2 fold) in the jejunum were significantly higher in females. Differences were also observed in expression among tissues. MCT1 had significantly higher expression in the liver (4.5 fold compared to intestine), while MCT4 had significantly higher expression in the intestine (20 – 30 fold compared to liver). SMCT1/2 expression was highest in the kidney and ileum, with SMCT1 expression significantly lower in the jejunum. AKR1A1 and AKR7A2 were expressed in all tissues with AKR7A2 having significantly higher expression in the kidney. Adhfe1 was expressed in liver and kidney, but not in the intestine.ConclusionsThe tissue‐specific sex differences in expression of monocarboxylate transporters, AKRs and Adhfe1 are consistent with the higher plasma concentrations and decreased renal clearance observed in females following supratherapeutic GHB doses. Future studies are needed to determine the mechanisms underlying the observed sex differences in transporter and enzyme abundance. Altered transporter and enzyme expression leading to higher systemic GHB concentrations has the potential for increased risk of GHB‐related toxicity and death in females.Support or Funding InformationUniversity of the Pacific
The Generation Rx Laboratory: An Innovative Pharmacology Education Research Hub
Authors: Kwiek Nicole Cartwright , Rooney Katherine , Downing Molly 
Published: November 2023

The Generation Rx Lab is a multi‐faceted drug education research lab that aims to improve science learning in grades K‐12, educate the public about medication safety principles, and provide a unique teaching opportunity for college students to practice communicating science. In this lab at the nationally renowned Center of Science and Industry (COSI), museum visitors of all ages conduct hands‐on experiments related to the science of drug action. Because drug topics spark young learners’ interest, we hypothesize that teaching science through a pharmacology context provides an effective platform for the general public to learn science, engage in the scientific process, and explore science professions. To test this hypothesis, we created and implemented a variety of educational programs. We share preliminary results from two types of educational programs: hands‐on science experiments and clinical simulations using a human patient simulator. Regardless of program type, our preliminary findings demonstrate that teaching science in the context of drug action increases the general public's understanding of both science and medication safety principles, as well as builds interest in science and health‐related careers. In addition, results from assessing the experiential impact on student lab staff demonstrate that the Generation Rx Lab teaching experience provides our students with a unique opportunity to improve their science communication skills while positively impacting their desire to include a teaching component in their future profession.Support or Funding InformationFunding is provided by the Cardinal Health Foundation and the Ohio State University Office of Research
20‐HETE Increases Large Artery Stiffness and Systolic Blood Pressure in Metabolic Syndrome
Authors: Soler Amanda , Hutcheson Brenda , Hunter Ian , Zhang Frank , Gotlinger Katie , Schwartzman Michal L , Falck John , Rocic Petra 
Published: November 2023

Hypertension is a risk factor that is associated with the metabolic syndrome. Large artery stiffness, which is in part a result of elastin degradation, plays a causal role in the development of isolated systolic hypertension, which is especially common in the elderly. Levels of 20‐hydroxyeicosatetraeonic acid (20‐HETE), a cytochrome (CYP)‐derived arachidonic acid metabolite, are greatly elevated in hypertensive animal models and loosely associated with obesity in humans, but 20‐HETE's role in the regulation of arterial stiffness in the metabolic syndrome has not been investigated. We hypothesized that elevated 20‐HETE in metabolic syndrome increases matrix metalloproteinase 12 (MMP12) activation leading to increased elastin degradation, increased large artery stiffness and increased systolic blood pressure. Our study shows that elastin degradation was increased ~4 fold in large arteries of metabolic syndrome rats (JCR:LA‐cp, JCR) vs. Sprague‐Dawley (SD) control rats. This correlated with increased large artery stiffness (75%±2% JCR vs. SD). 20‐SOLA (2,5,8,11,14,17‐hexaoxanonadecan‐19‐yl 20‐hydroxyicosa‐6(Z),15(Z)‐dienoate) a 20‐HETE antagonist, blocked elastin degradation in JCR rats, concomitant with decreasing MMP12 activation. Importantly, both 20‐SOLA and MMP12 inhibition (pharmacological and MMP12‐shRNA‐Lnv) decreased large artery stiffness in JCR rats (3%±2% JCR+20‐SOLA vs. SD, 18%±4.3% JCR+MMP12 inhibition vs. SD). 20‐SOLA and MMP12 inhibition also decreased systolic but not diastolic blood pressure in JCR rats (113±6 mmHg SD, 159±4 mmHg JCR, 130±4 mmHg JCR+20‐SOLA, 139±5 JCR+MMP12 mmHg inhibition). These data suggest that a portion of the systolic but not diastolic component of hypertension in the metabolic syndrome JCR rats is 20‐HETE‐sensitive and dependent on large artery stiffness/compliance. The source(s) of 20‐HETE and MMP12 responsible for increased elastin degradation and large artery stiffness are likewise unknown. Intra‐abdominal lipectomy (removal of visceral fat=5% body weight) in JCR rats decreased MMP12 activation, elastin degradation and large artery stiffness to levels achieved with 20‐SOLA and observed in SD control rats, suggesting: 1) that this 20‐HETE is largely derived from visceral (intra‐abdominal) adipose tissue, and 2) that MMP12 involved in the regulation of vascular stiffness is also derived primarily from visceral adipose tissue. Future implications of these findings may be important for systolic hypertension management through 20‐HETE and/or MMP12 inhibition.Support or Funding InformationSupported by NIH R01HL093052.