bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydrogen‐Deuterium exchange reveals distinct activation states of PLCβ2 by G‐proteins
Authors: Fisher Isaac , Jenkins Meredith , Tall Greg , Burke John , Smrcka Alan 
Published: June 2021

G‐protein coupled receptors (GPCRs) are an important class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G‐proteins. One major effector of G proteins is phospholipase C (PLC), which hydrolyzes Phosphatidylinositol 4,5 bisphosphate (PIP2) into Diacylglycerol (DAG) and Inositol 1,4,5, trisphosphate (IP3). PLCβ2 and PLCβ3 isoforms are directly activated by binding Gαq, Gβγ or both. The mechanism of activation of PLCβ2 by Gβγ is unknown, and the binding site(s) for Gβγ on PLCβ remain controversial.Here, we use hydrogen‐deuterium exchange mass spectrometry (HDXMS) to understand the conformational dynamics of PLCβ and to answer open questions in the field concerning the mechanism of activation of PLC by Gβγ and Gαq. For these experiments, several complexes were examined with HDXMS: PLCβ2 alone, PLCβ2+ PIP2/Phosphoethanolamine (PE)/Phosphatidylserine (PS) vesicles, or PLCβ2+ PIP2/PE/PS vesicles plus Gβγ or Gαq‐AlF4. Incubation of PLCβ2 with PIP2/PE/PS vesicles strongly protects regions in the C‐terminus, confirming that this region is involved in membrane binding. In the presence of Gαq‐AlF4, PLCβ2 shows protection from deuterium exchange in the helix‐loop‐helix region of the proximal C‐terminal domain (CTD) and the EF‐hand domain on PLCβ2 that exactly correspond to regions shown to interact with Gαq in the Gαq‐PLCβ3 crystal structure. These data validate that our approach accurately reports bona fide protein‐protein and protein‐lipid interactions. Incubation of Gβγ with PLCβ2 does not reveal any regions of strong protection, but rather causes strong increases in deuterium exchange in the of the CTD of PLCβ2, indicating that the CTD of PLC undergoes a large conformational rearrangement upon Gβγ binding. This increase in deuterium exchange of the CTD of PLC is not seen upon Gαq activation. This indicates that there are distinct conformational states of PLCβ induced by Gαq and Gβγ interactions, suggesting that Gαq and Gβγ use different mechanisms for activation of PLCβ. Finally, these data suggest a mechanism for activation of PLC by Gβγ, where Gβγ binding to PLC breaks an autoinhibitory interaction involving the CTD of PLC and either the membrane or another portion of PLCβ2.Support or Funding InformationThis work is supported by NIH R01 GM081772 to AVS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Potential Biomarkers, Mechanisms and Protective Strategies for General Anesthetic‐Induced Neurotoxicity in the Developing Nonhuman Primate
Authors: Slikker William , Han Xianlin , Liu Fang , Zhang Xuan , Gu Qiang , Liu Shuliang , Paterson Tucker A. , Paule Merle G. , Hanig Joseph P. , Wang Cheng 
Published: June 2021

It has been reported that commonly used general anesthetics such as sevoflurane induce neurotoxicity in developing brains. However, there has been limited research evaluating whether and how anesthetic agents affect bio‐lipids, the most abundant components of the brain other than water. Thus, assessing lipid profiles from blood samples may assist in the early detection of general anesthesia‐induced neurotoxic effects.Postnatalday (PND) 5 or 6 monkeys were randomly assigned to control (room‐air; n=4) and sevoflurane‐exposed(n=4) groups. Sevoflurane was delivered using an agent‐specific vaporizer for 9 hours at a clinically‐relevant concentration of 2.5%. Blood samples were collected at 0, 2, 4, 8 and 9 hours during exposure from both the control and sevoflurane‐exposed groups. Lipid extractions and analyses were performed using a mass spectrometer. Four hours after completion of anesthetic exposures front alcortical tissue was collected for histochemical and Western blot analyses. In addition, to evaluate potential protective strategies monkey infants were exposed to sevoflurane in the presence or absence of acetyl‐L‐carnitine(mitochondrial stabilizer), and microPET scanning using [18F]‐FEPPA was carried out to repeatedly visualize and quantify aspects of neurotoxicity.Serumlipidomic analyses demonstrated that the levels of critical lipid components including acylcarnitines, phosphatidylcholines and phosphatidylethanolamines were significantly decreased during prolongedexposure to sevoflurane. In contrast, the amounts of triglyceride and 4‐hydroxynonenal were increased to abnormally high levels in sevoflurane‐exposed monkeys. Consistently, histochemical staining and Western blot analyses of Bax protein revealed increased neuronal apoptotic damage after sevoflurane exposure. Co‐administration of acetyl‐L‐carnitine effectively blocked the neural damage (increased [18F]‐FEPPA signal)‐induced by prolonged sevoflurane exposure.These data suggest that prolonged exposure of neonatal monkeys to a clinically‐relevant concentration of sevoflurane resulted in significant changes in lipidmetabolism and subsequently, neuronal apoptotic damage. Monitoring specificlipid changes may provide insights into the molecular mechanism(s) underlying generalanesthetic‐induced neurotoxicity and serve as sensitive biomarkers for the early detection of anesthetic‐induced neuronal damage. Acetyl‐L‐carnitine may act to preserve cellular membrane integrity and play critical roles in mitochondrial oxidation of long‐chain fatty acids, and thus protect neurons from sevoflurane induced damage.Support or Funding InformationSupported by NCTR/FDAThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluating the effects of HIPK2 Inhibition on Castration‐Resistant Prostate Cancer Cells
Authors: Camacho‐Hernández Elaine Marie , Thomas Phillip , Ledet Russell , Garabedian Michael J. , Logan Susan K. 
Published: June 2021

Prostate cancer is the second leading cause of death in men in the United States. It depends on the signaling by the androgen receptor (AR), which is activated by testosterone. AR signaling promotes normal prostate development as well as cancer. Current treatments for prostate cancer include prostatectomy and androgen deprivation therapy. Although androgen deprivation is effective in halting prostate cancer growth, a group of men are often diagnosed with castration resistant prostate cancer (CRPC), which is refractory to hormone deprivation. Therefore, new targets are needed to stop prostate cancer growth. In this study, we investigated whether Homeodomain‐interacting protein kinase 2 (HIPK2) affects AR signaling and prostate cancer cell growth. To do this, we utilized doxycycline‐induced shRNA‐mediated silencing of HIPK2 expression in 22Rv1 and LNCaP‐95, two cell lines that are refractory to androgen deprivation, and demonstrated substantial knockdown of both HIPK2 mRNA and protein. In addition, we observed significant changes in AR target gene expression. To determine the effects of HIPK2 knockdown on AR subcellular localization, we fractionated control and doxycycline‐treated 22Rv1 and LNCaP‐95 cells. We observed modest changes in chromatin‐bound AR upon HIPK2 depletion, suggesting that HIPK2 influences AR chromatin occupancy and in turn AR‐dependent transcription. We also tested HIPK2 catalytic inhibitors by examining their effects on 22Rv1 and LNCaP‐95 cellular proliferation, and phosphorylation a HIPK2 substrate, SIAH2. We found that HIPK2 inhibitors modestly affected the proliferation and substrate phosphorylation. Thus, HIPK2 can modulate AR activity, highlighting the role of HIPK2 as a new drug target for prostate cancer.Support or Funding InformationNew York University Langone Medical CenterThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Preventive Therapeutic Strategy for Liver Cancer
Authors: Lee Jin , Feng Gen‐Sheng 
Published: June 2021

Liver cancer is the second most common cause of cancer‐related death with difficulties of immune therapeutics. When administered at pre‐cancer stages, the immunostimulatory synthetic double‐stranded RNA (dsRNA), polyinosinic‐polycytidylic acid (pIC) robustly induced tumor remission with no cytotoxic effect in either chemically‐induced or phosphatase and tensin homolog (PTEN) knockout‐induced liver cancer in mice by boosting multiple anti‐tumor immune activities, including natural killer (NK) cells and reprogramming of macrophage polarization. In contrast, a potent anti‐tumor dsRNA, in late stages of PTEN loss‐associated hepatosteatosis markedly increased tumor burden. Injection of dsRNA stimulated macrophage type II and regulatory T cells in PTEN‐knockout mice without affecting lipid accumulation, expression of α‐smooth muscle actin (α‐SMA) and cholangiocyte expansion at late stages. This study provides a new strategy for approaching immune therapeutics for the large population of at‐risk and liver cancer patients at different stages.Support or Funding InformationWe thank Drs. D. Brenner, J Bui, M. Kaplan, M. Karin, T. Kisseleva, B. Schnabl and colleagues in the Feng lab for helpful discussion, critical reading of the manuscript or reagents. This work was supported by R01CA176012 and R01CA188506 to (G.S.F.), and P30DK063491, and JDRF 2‐SRA‐2016‐306‐S‐B (to W.F.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adiponectin regulates T‐cell maturation by modulating the thymic epithelial environment
Authors: Cao Handi , Sun Wai Yan , Zhang Yiwei , Xu Aimin , Wang Yu 
Published: June 2021

ObjectiveAdiponectin is an adipokine possessing potent immunomodulatory activities. The present study investigated T‐cell development and maturation in the thymus tissue of FVB/N‐Tg( PyVT‐MMTV)634Mul/J mice with or without Adipoq allele expressions.MethodsDifferent populations of lymphocytes were analyzed by flow cytometry. Protein expressions were evaluated by immunofluorescence staining and/or Western blotting. Gene expression and mRNA presence were measured by quantitative real time PCR and in situ hybridization respectively.ResultsSignificant reductions of CD3+CD45+ and CD3+CD4+ cells were observed in the blood of FVB/N‐Tg(PyVT‐MMTV)634Mul/J mice lacking adiponectin (AKO), when compared to age‐matched mice with Adipoq allele expression (WT). An increased population of CD45+CD3−CD44high progenitor cells was present in the blood of AKO mice. The CD3+ cells were also significantly reduced in the thymus of AKO mice, where exhibited altered expression and distribution of epithelial markers including cytokeratin 5, cytokeratin 8, p63 and β5t when compared to those of WT mice. Adiponectin mRNA and protein expression were detected in thymus tissue sections and the thymic nurse cell (TNC) complexes of WT mice. The thymus of AKO mice contained significantly reduced amount of TNC complexes, leading to the accumulation of double positive thymocytes. Molecular interactions between adiponectin, CD100 and galectin‐3 might play a role in the engulfment of lymphocytes by TNC complexes.ConclusionThymic adiponectin expression plays a key role in modulating the epithelial environment and T‐cell maturation in thymus.Support or Funding InformationThis work was supported in part by grants from Seeding Funds for basic research of the University of Hong Kong, Research Council Grants (17121714, HKU779712M, HKU780613M) of Hong Kong; And the National Basic Research Program of China (973 program 2015CB553603).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Direct and Indirect Effects of Ephedrine on the Cardiovascular System
Authors: Docherty James R. , Alsufyani Hadeel A. 
Published: June 2021

After many years of use of ephedrine, there is still confusion as to its exact mode of action: direct agonism (Liles et al., 2007) or actions as an indirect sympathomimetic, or both. We have previously shown that cardiovascular actions of the stimulant cathinone are largely indirect by release of noradrenaline (Alsufyani & Docherty, 2015). We have now investigated, using the same techniques, the cardiac and pressor responses to (±)‐ and (−)‐ephedrine in pentobarbitone anaesthetised male wistar rats. The tachycardiac responses to (±)‐ and (−)‐ephedrine were similar, but pressor responses to (−)‐ephedrine (10 mg/kg) were significantly greater than those to (±)‐ephedrine, and for both, the pressor response was followed by a small depressor response. Sympathectomy did not affect the pressor response, but significantly increased the later depressor response to both compounds. Sympathectomy did not significantly affect cardiac or depressor responses to the direct beta‐adrenoceptor agonist isoprenaline. Sympathectomy significantly reduced the tachycardia to (±)‐ephedrine. It is concluded, firstly, that (−)‐ephedrine is more potent than the racemate mixture at producing pressor responses. Secondly, since the depressor response to the direct agonist isprenaline was unaffected, sympathectomy presumably reduced a later pressor component to the response to (±)‐ and (−)‐ephedrine. Hence, the early pressor response to both (±)‐ and (−)‐ephedrine is direct, but the later pressor response is largely indirect. Ephedrine has both direct and indirect actions on blood pressure.Support or Funding InformationSupported by King Abdelaziz UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aspirin Protects Heart Against Ischemia‐reperfusion Injury Via LKB1‐Sestrn2‐AMPK Signaling Cascade
Authors: Lu Shaoxin 
Published: June 2021

IntroductionAMP‐activated protein kinase (AMPK) is a stress signaling enzyme that orchestrates the regulation of energy pathways. Intrinsic AMPK activation protects the heart against ischemic injury, but whether the pharmacologic AMPK stimulation by aspirin mitigates ischemia‐reperfusion (I/R) damage is unknown.HypothesisAspirin as an emerging AMPK agonist could stimulate the cardiac AMPK signaling pathway that attenuates myocardial ischemia‐reperfusion injury.MethodsThe cardioprotective activity of aspirin was evaluated in an in vivo regional I/R (45 min/24 hours) injury model in which the left anterior descending coronary artery (LAD) was occluded and released. The Langendroff perfused heart system was used to approach an ex vivo global ischemia and reperfusion model.ResultsIsolated mouse hearts ex vivo pre‐treated with aspirin had better recovery of left ventricular contractile function (55% vs. 29% of baseline heart rate‐pressure product; p<0.05) and less myocardial necrosis (56% reduction in infarct size; p<0.01) during post‐ischemic reperfusion. Pre‐treatment with aspirin in vivo attenuated myocardial infarction in C57BL/6J mice undergoing left coronary artery occlusion and reperfusion compared to vehicle (36% vs. 18%, p<0.05). Mouse hearts with genetically inactivated AMPK catalytic subunit were not protected by aspirin treatment, indicating the critical role of cardiac AMPK activation by aspirin in cardioprotection against ischemic injury. Moreover, pre‐treatment with aspirin in vivo increased the AMPK downstream phosphorylation and inactivation of eukaryotic elongation factor 2 (eEF2), preserved energy charge during ischemia and delayed the development of ischemic contracture. Aspirin treatment augmented the interaction between AMPK upstream LKB1 and Sestrin2‐AMPK complex, also enhanced activation of AMPK downstream endothelial nitric oxide synthase (eNOS) during ischemia, which partially attenuated myocardial stunning.ConclusionsAMPK is a therapeutic target that can be stimulated by a direct‐acting small molecule in order to prevent injury during I/R. The use of aspirin may represent a novel strategy to protect the heart and other solid organs against ischemia.Support or Funding InformationAmerican Heart AssociationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rho Kinase inhibitors and hypoxia modulate expression of platelet activating factor (PAF) receptor and gene expression in fetal ovine pulmonary vascular smooth muscle cells.
Authors: Ibe Basil O. , Renteria Lissette S. 
Published: June 2021

Hypoxia and PAF augment intracellular calcium flux in fetal ovine pulmonary vascular smooth muscle cells (PVSMC) indicating that high pulmonary vascular tone in fetal pulmonary vessels in utero is consequent on combined effects of hypoxia and PAF receptor (PAFR)‐linked vascular responses. PAFR internalization is said to inactivate PAF effects in PVSMC in vitro. Hypoxia increases PAFR expression, but Rho kinase (ROCK) inhibitors decrease the expression suggesting PAFR internalization may be one pathway to downregulate PAFR‐mediated responses in PVSMC. These findings also suggest that ROCK may act in synergy with PAF to modulate PAFR‐mediated pulmonary vascular responses. We studied effects of PAF, hypoxia and other modulators of pulmonary vascular reactivity in arterial (PA) and venous (PV) smooth muscle cells (SMC) to aid in understanding the signaling pathways of PAFR‐mediated fetal ovine pulmonary vascular reactivity in utero. We hypothesized that ROCK inhibitors attenuate PAFR expression in hypoxia leading to increased expression of the contractile proteins. Cells were cultured in normoxia or hypoxia, with and without 10 nM PAF or ROCK inhibitors (Y27632 and HA 1077, each 10 μM). Proteins were probed for PAFR, and Calponin, MLCK and Desmin, which are contractile protein modulators of PVSMC activity. PAF binding, PAFR protein and gene expression by qRT‐PCR were also determined. In normoxia, PAFR density (Bmax) in PA and PV SMC were 487±37 and 624±17 respectively, which increased by 30–35% in hypoxia, and higher in PV than PA in normoxia and hypoxia. The KD for PAF binding to PASMC was higher than for PVSMC, and then hypoxia decreased the KD of PAFR binding in both cell types. ROCK inhibitors attenuated PAFR binding with comparable effect to cycloheximide, but unlike PAFR inhibitor, CV3988, cycloheximide had no effect on pre‐synthesized proteins. ROCK inhibitors decreased PAFR expression, but increased expression of MLCK2, Calponin and Desmin in PASMC especially in hypoxia, with different effects in PVSMC. Generally, PAFR gene expression studied by qRT‐PCR was higher in PVSMC under all conditions. This study shows that hypoxia in conjunction with ROCK inhibitors modulate PAFR‐mediated signaling in PVSMC in vitro with different effects on cells from PA and PV. Increased expression of the contractive proteins in hypoxia indicates involvement of these proteins in PAF‐induced contractions in PVSMC.Support or Funding InformationLABiomedThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nardostachys jatamansi ameliorates doxorubicin induced cardiotoxicity via down regulation oxidative stress and inflammatory markers in rats
Authors: Madkhali Hassan A. , Ganaie Majid Ahmad , Raish Mohammad , Jan Basit Latief , Ahmad Ajaz 
Published: June 2021

Nardostachys jatamansi (family Valerianaceae) is a perennial herb found in Alpine Himalayas. In Ayurveda system of medicine, rhizomes of Nardostachys jatamansi are used as a bitter tonic, stimulant and antispasmodic and to treat epilepsy, hysteria, and convulsions. In the Unani system of medicine Nardostachys jatamansi has been mentioned as hepatotonic, cardiotonic, diuretic and analgesic. In the present study, we have investigated the protective role of alcoholic extract of N. jatamansi on myocardial injury induced by doxorubicin (15 mg/kg body weight, intraperitoneal single dose). Pretreatment for 5 days with ethanolic extract of N. jatamansi (250 mg/kg body weight and 500 mg/kg body weight orally) showed moderate cardioprotective activity as evidenced by significant decline in the elevated levels of serum marker enzymes and lipid peroxide levels with restoration in myocardial endogenous antioxidants levels. Hyperlipidaemia induced by doxorubicin was also inhibited and inflammation markers TNF‐α, IL‐6, and myeloperoxidase [MPO]), were also assessed. Doxorubicin promoted the upregulation of cardiac cytokines (TNF‐α and IL‐6), and MPO activity were significantly down regulated upon ethanolic extract of N. jatamansi pretreatment. Histopathological changes ranging from edema to degenerative changes in cardiac tissue sections were also restored.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Model‐Based Estimation of Pulmonary Flow Heterogeneity from Observed Oxygen Transport Parameters in Exercise
Authors: Roy Tuhin K. , Secomb Timothy W. 
Published: June 2021

The rate at which oxygen can be taken up into the bloodstream in the lung is a critical determinant of functional capacity, and can be affected by heterogeneity of pulmonary blood flow (leading to maldistribution and poor ventilation‐perfusion matching) as well as alterations in diffusing capacity. Under resting conditions, the high degree of reserve in the healthy lung leads to adequate oxygen uptake even in the face of increased heterogeneity or decreased diffusing capacity; under conditions of exercise, however, oxygen transport and utilization are contingent upon adequate lung function with relatively little reserve. A mathematical model of pulmonary oxygen uptake is used in this study to investigate the impact of pulmonary flow heterogeneity in exercise, which we characterize by the coefficient of variation (CV) of pulmonary capillary blood flow; under conditions of uniform alveolar ventilation, this is theoretically equivalent to impaired ventilation‐perfusion matching. We have previously demonstrated that any level of pulmonary heterogeneity up to a high degree (CV of approximately 3.0) is consistent with the observed level of arterial oxygen tension under resting conditions. We hypothesize that such high levels of heterogeneity would preclude the high oxygen uptake in the lung observed during exercise. In this study, literature data on humans under normoxic and hypoxic conditions was used to estimate the CV of pulmonary capillary blood flow under conditions of moderate and extreme exercise. With the assumption of a normal diffusing capacity, the best fit overall to aggregated data from five studies was found with a CV of 0.61. These results show that a high degree of pulmonary flow heterogeneity does not limit oxygen uptake at rest, but that in exercise much lower levels of heterogeneity are necessary to achieve observed oxygen fluxes. An important mechanism for reducing heterogeneity is flow regulation via hypoxic pulmonary vasoconstriction (HPV), which is activated at the low levels of venous oxygen tension in exercise. The results suggest that HPV plays an essential role in allowing high levels of pulmonary oxygen uptake in exercise.Support or Funding InformationSupported by NIH U01 HL133362This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.