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. To determine the mechanism of activation of PLCβ, we used a purified reconstitution system of PLC activity. In vitro reconstitution experiments show that CTD‐deleted PLCβ2 exhibits increased activity in response to Gβγ. Additionally, the distal CTD of PLCβ inhibits CTD‐deleted PLCβ2 activity in both lipid‐dependent and lipid‐independent reconstitution system. Taken together, these results suggest that Gβγ activates PLCβ by breaking an autoinhibitory interaction of the distal CTD with the core enzyme of PLC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How Do Taste Buds EAT?: Defining the Embryo‐to‐Adult Transition in Mouse Taste Bud Development and Regeneration
Authors: Barlow Linda A , Golden Erin J , Fellin Tim J 
Published: June 2021

Taste buds are collections of heterogeneous taste receptor cells (TRCs) that detect sweet, sour, salt, bitter and umami. TRCs are relatively short‐lived and continually replaced by proliferative progenitor cells situated adjacent to taste buds. In mouse embryos, taste bud precursors are first evident as small clusters of columnar epithelial cells or placodes on the developing tongue surface. Each taste placode comprises 10–20 cells that express both cytokeratin (K) 8 and the secreted protein Sonic Hedgehog (SHH). Previously we showed that SHH+ placode cells differentiate into TRCs in the first postnatal week, but do not give rise to the K14+ progenitor population that supports adult TRC renewal (Thirumangalathu et al., 2009 Development). This raised the questions of how and when TRC turnover commences. Using 3D image analysis, we find that taste placode and taste bud cell number are static during embryonic development and within 1–2 days of birth, respectively; instead EdU incorporation studies and K14 genetic lineage tracing reveal that the progenitor contribution to taste buds begins by postnatal day (P) 2, steadily increasing through P14. Work from our lab and others has shown that SHH is a negative regulator of taste cell fate in embryos but paradoxically SHH promotes TRC differentiation in adults. Thus, we hypothesized that the switch in SHH function coincides with the onset of taste cell renewal. We are currently testing this idea using K14CreER to drive SHH overexpression at specific postnatal time points. In cultured embryonic tongues, excess SHH represses formation of taste placodes, whereas in adult mice, this genetic manipulation induces formation of ectopic taste buds throughout the anterior lingual epithelium (Castillo et al., 2014 Development). Additionally we are using RNAseq analysis to identify candidate regulators of the embryo‐to‐adult transition, and more specifically to define the emerging taste bud progenitor pool in postnatal animals.Support or Funding InformationSupported by R01 DC012383 to LAB and F32 DC015958 to EJGThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
b1‐Adrenergic Receptors in the Golgi Apparatus are Activated by Cell Permeable Agonists and Stimulate PLC‐mediated PI4P Hydrolysis in Cardiac Myocytes
Authors: Wei Wenhui , Nash Craig , Smrcka Alan 
Published: June 2021

Previously, our laboratory identified a phospholipase C (PLC)‐mediated PI4P hydrolysis pathway at the Golgi apparatus that is important for regulation of cardiac hypertrophy, a pathology associated with development of heart failure. cAMP stimulates this pathway via Epac‐mediated activation of Rap1 which directly binds to PLCɛ. We recently found that a cell membrane permeable β‐adrenergic receptor (βAR) agonist, dobutamine (dob), induces Golgi PI4P hydrolysis in neonatal rat ventricular myocytes (NRVMs) while a cell membrane impermeable β‐adrenergic (βAR) agonist, Isoproterenol, does not. We used a YFP‐tagged mini Gs protein2 which is recruited to Gs‐coupled receptors upon their activation, to monitor the membrane location of these activated receptors. Dobutamine induced robust and rapid recruitment of mini Gs to both the plasma membrane, and the perinuclear region corresponding to the Golgi apparatus. In contrast, Isoproterenol, induced a strong recruitment to the plasma membrane but not to the Golgi. Treatment with brefeldin A significantly reversed mini Gs recruitment to the internal membrane induced by dobutamine, supporting the idea that active β1‐ARs are localized to the Golgi membrane. A membrane permeable βAR antagonist, metoprolol, fully reversed dob‐mediated PI4P hydrolysis and mini Gs recruitment to the Golgi membrane while a membrane impermeable antagonist, sotalol had no effects on dob‐mediated PI4P hydrolysis and only partially reversed the mini Gs recruitment to the Golgi membrane. In addition, an Epac inhibitor HJC0726 completely inhibited dob‐mediated PI4P hydrolysis whereas gallein, a Gβγ inhibitor had no effect. Over expression of the RA1 domain of PLCɛ, which competes PLCɛ away from the mAKAPβ scaffold, also inhibited dobutamine stimulation of PI4P hydrolysis. These data indicate that activation of endogenous intracellular β1ARs stimulates production of a local pool of cAMP that activates the perinuclear Epac‐PLCɛ‐PI4P hydrolysis pathway involved in heart failure.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Roles of GPR39 in Regulating Keratinocyte Proliferation and Inflammation
Authors: Satianrapapong Wilasinee , Pongkorpsakol Pawin , Muanprasat Chatchai 
Published: June 2021

Zinc is an important trace element that plays key roles in the human body. Previous reports showed that zinc supplement could relieve dermatitis and improve wound healing process with unknown molecular target. In this study, we investigated the roles of G‐protein coupled receptor 39 (GPR39), a zinc‐sensing receptor, on keratinocyte proliferation and inflammation using a GPR39 agonist TC‐G 1008. Immortalized human keratinocyte cell line (HaCaT cells) was used as an in vitro model. We first verified protein expression and function of GPR39 in HaCaT cells by Western blot and Indo‐1 calcium flux assays. Importantly, at 48 h post‐treatment, Bromodeoxyuridine (BrdU) incorporation assay showed that TC‐G 1008 significantly increased keratinocyte proliferation in a concentration‐dependent manner with a maximal effect being observed at 1 μM. Moreover, TC‐G 1008 enhanced mammalian target of rapamycin (mTOR) phosphorylation in a time‐dependent manner, indicating that GPR39 activation led to keratinocyte proliferation. Next, immunofluorescence staining demonstrated that TC‐G 1008 significantly reduced tumor necrosis factor‐alpha (TNF‐α)‐induced nuclear factor‐kappa B (NF‐κB) nuclear translocation. Interestingly, this effect was abolished by co‐treatment with compound C, AMP‐activated protein kinase (AMPK) inhibitor. These results supported that GPR39 activation promotes wound healing and exerts anti‐inflammatory effect in human keratinocytes, at least in part, by stimulating mTOR and AMPK, respectively.Support or Funding InformationThis work was supported by Thailand Research Fund and Mahidol University (BRG5980008), Faculty of Science, Mahidol University and the Medical Scholars Program (Ph.D.‐M.D. program), Mahidol University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro Evaluation of Excipients as Inhibitors of Human Intestinal P‐glycoprotein
Authors: Bajaj Ruchika , Chong Lisa B , Zou Ling , Tsakalozou Eleftheria , Ni Zhanglin , Giacomini Kathleen M , Kroetz Deanna L 
Published: June 2021

P‐glycoprotein (P‐gp) is one of the major barriers to oral bioavailability and contributes to the interindividual variability observed in intestinal absorption of P‐gp substrates. A well characterized source of variable P‐gp function in the intestine is inhibition by co‐administered drugs. However, the potential interplay between intestinal P‐gp and pharmaceutical excipients used in orally administered drug products has not been systematically studied and is the focus of this investigation. A fluorescence assay using calcein AM (5 mM), which served as a model substrate for P‐gp, was used to assess inhibition of P‐gp transport function by 130 excipients in HEK293 cells stably expressing human P‐gp or transfected with an empty vector (control). Specific inhibition of P‐gp‐mediated calcein AM efflux by excipients was expressed as a ratio of inhibition observed in P‐gp overexpressing cells over control cells. An inhibition screen was performed at an excipient concentration of 200 mM where possible, or at 50 mM where excipient solubility was limiting. Six potential inhibitors (inhibition ratio >1.3) were identified, including five dyes (D&C Red #6, D&C Brown#1, Naphthol blue black, Acid blue 9, Light green CF yellowish) and one suspending agent (Butylparaben). None of these potential inhibitors achieved >30% inhibition of calcein‐AM efflux at concentrations up to 300 mM. Intestinal concentrations of these potential inhibitors estimated from the maximum amount of each excipient allowed per unit dose (http://excipients.ucsf.bkslab.org/) ranged from 0.50–2.3 mM, with the exception of light green CF yellowish (214 mM). While these in vitro findings indicate minimal inhibition of human P‐gp at intestinal excipient concentrations typically expected from marketed drugs, further studies are needed to assess the impact of these excipients on the oral bioavailability of P‐gp substrates.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caveolin scaffolding domain plays an important role in cancer cell migration
Authors: Okerblom Jonathan , Okada Sunaho , Raja Sadaf , Ray Supriyo , Okada Hideshi , Kawamura Itta , Murray Fiona , Murofushi Yoshiteru , Patel Hemal 
Published: June 2021

Caveolin‐1 is the integral membrane protein that plays an important role in the structure and function of terminally differentiated cells. As a structural component of caveolae, the caveolin scaffolding domain (CSD) has been associated with many cellular signaling processes and recent reports have implicated Cav‐1 as a negative regulator in tumor metastasis. We hypothesized that Cav‐1 may be inhibiting cell migration through its CSD. To assess this, HCT‐116 cells (which contain high endogenous Cav1) and HT‐29 cells (which contain low endogenous Cav1) were transduced with a lentiviral construct expressing either Cav‐1 or Cav‐1 without a functional CSD (ΔCSD). HeLa cells were also genetically engineered to stably overexpress Cav‐1(Cav‐1 OE), ΔCSD, or enhanced green fluorescent protein (EGFP) as a comparative control. In all cases, migration was significantly greater in ΔCSD cells compared to Cav‐1 OE and control cells. Phosphorylated STAT‐3 was also decreased in Cav‐1 OE cells compared to control and ΔCSD cells. G2/M phase was significantly increased and G1 was decreased in ΔCSD cells compared to control and CAV1 OE cells. Cdc2 phosphorylation was also increased in ΔCSD cells. To further confirm that CSD affects cell migration, we performed migration assays in Hela cells expressing either a pEGFP tagged CSD or a scrambled sequence. CSD overexpressing cells showed a lower migration rate comparison to control cells. These findings suggest that Cav‐1 CSD affects cell migration, possibly by participating in a G2/M checkpoint. These findings may represent a new approach to the treatment of cancer metastasis.Support or Funding InformationNIH R01 HL091071This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extracts of the medicinal plants Pao Pereira and Rauwolfia vomitoria inhibit ovarian cancer stem cells in vitro
Authors: Chen Qi , Dong Ruochen , Chen Ping 
Published: June 2021

ObjectivesOvarian cancer has an enrichment of stem‐like cancer cells (CSCs) which contribute to the treatment‐resistant tumor's high rate of recurrence and metastasis. Here we investigated two extracts from the medicinal plants Pao Pereira (Pao) and Rauwolfia vomitoria (Rau) each for their activities against ovarian CSCs.MethodsThe Pao and Rau extracts were provided by Natural Source Inc. Cell viability was detected by MTT assay. Ovarian CSC population was examined by tumorsphere formation assays. Ovarian CSC specific cell surface markers were examined by flow cytometry. Western blot and RT‐qPCR was used for protein and mRNA expression.ResultsBoth Pao and Rau inhibited the proliferation of multiple human ovarian cancer cell lines with IC50 values ranging from 210 to 420 μg/mL for 48 hours of treatment, and had limited cytotoxicity to normal ovarian epithelial cells. Pao and Rau had similar potency in inhibiting ovarian CSCs, with IC50s of ~120 μg/mL for 24 hours treatment in reducing CD44+CD117+ sub‐population, and IC50s of ~50 μg/mL for long‐term tumorsphere formation. Nuclear β‐catenin levels were decreased, suggesting suppression of Wnt/β‐catenin signaling pathway.ConclusionsData here showed that Pao and Rau both inhibited ovarian cancer stem cells in preference to the bulk of tumor cells. These data provide the first evidence in the activities of Pao and Rau in inhibiting ovarian cancer stem cells. Further mechanistic studies and in vivo investigation validating these findings are a warrant, giving that inhibition of cancer stem cells holds the promise of comprehensively inhibiting cancer metastasis, drug resistance, and recurrence.Support or Funding InformationThe study was supported by the Beljanski Foundation. The Beljanski Foundation has no influence on the study design, performance of experiments, data collection and analysis.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacologic Inhibition or Genetic Deletion of Soluble Epoxide Hydrolase Improves Survival Following Myocardial Infarction in Aged Mice
Authors: Jamieson Lockhart , Sosnowski Deanna K , Darweshe Ahmed M , Wang Wang , Zhabyeyev Pavel , Edin Matthew , Zeldin Darryl , Kassiri Zamaneh , Oudit Gavin , Seubert John M 
Published: June 2021

ObjectiveMyocardial infarction (MI) accounts for a significant proportion of death and disability in aged individuals. CYP450 metabolism of n−6 PUFA arachidonic acid results in formation of numerous metabolites, called eicosanoids, that exhibit a wide range of cellular effects. Previous studies demonstrate eicosanoids can induce alterations to the mitochondria resulting in preserved cardiac function following ischemic injury. These metabolites are further metabolised by the enzyme soluble epoxide hydrolase (sEH) reducing their endogenous activity. This study investigated post‐ischemic cardiac function in aged mice with either genetic deletion or pharmacologic inhibition of sEH. Furthermore, a primary component of this study was to elucidate potential sex differences between male and female mice undergoing the experimental protocol.MethodsMale and female WT and sEH null mice averaging 15 months old underwent permanent occlusion of the left anterior descending coronary artery (LAD). On the day of surgery, WT mice were given either vehicle (0.1% DMSO) or sEH inhibitor tAUCB (10 mg/ml dissolved in 0.1% DMSO) in drinking water for 28 days ad libitum. Cardiac function was assessed at baseline, 7 days and 28 days post‐MI by echocardiography and electrocardiogram. Protein expression was determined using immunoblotting techniques while mitochondrial enzymatic activities were assessed by spectrophotometry. Mitochondrial respiration in cardiac fibres was measured using a Clark‐type electrode. A full metabolite profile was determined in control and 28‐day post‐MI hearts by LCMS/MS.ResultsPharmacological inhibition or genetic deletion of sEH was associated with fundamental changes in the n−3 and n−6 PUFA metabolite profile in cardiac tissue, correlating with better diastolic function, preserved mitochondrial respiration and overall improved survival at 28‐days post‐MI.ConclusionsThese data suggest that pharmacologic inhibition or genetic deletion of sEH elicits fundamental changes in the aged myocardium following long‐term ischemic injury.Support or Funding InformationThis research is supported by grants from the Canadian Heart and Stroke Foundation and Canadian Institute of Health Research. LJ is supported by a graduate studentship from Alberta Innovates‐Health Solutions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prenatal exposure to Bisphenol‐F followed by chronic estrogen exposure in adulthood increases systolic blood pressure in female rats
Authors: Chuang Yen‐Jun , Jeyaraj Ninitha A. , Jacob Elyssa B. , MohanKumar Puliyur S. , MohanKumar Sheba M. J. 
Published: June 2021

Endocrine disrupting chemicals (EDCs) such as bisphenols and phthalates are produced in high volume in the US and all over the world. They are capable of leaching out of plastics and entering the food chain. They produce a variety of effects on the body, but not all effects are understood. Epidemiological evidence indicates that there is a marked increase in the incidence of hypertension and pre‐hypertension in young children. We hypothesized that exposure to these EDCs during pregnancy can affect brain circuits and cause high blood pressure in offspring. The risk is even greater for female offspring who are subsequently exposed to an environmental estrogen or oral contraceptives in adulthood. To test this, we exposed pregnant Sprague Dawley dams to saline, or 1μg/kg BW of bisphenol‐F (BPF). Two female offspring from each dam were either sham implanted or implanted with estradiol‐17β (E2) pellets that were capable of releasing 20 ng of E2/day for 90 days. Animals were also implanted with a telemeter (Data Sciences International; HD‐S10) in the femoral artery and were allowed to recover. Blood pressure recordings were obtained for once a week for 12 weeks over a 24‐hour time period. In control rats that were treated with E2, there was a modest increase in systolic BP. When prenatal BPF exposure was followed by exposure to E2 in adulthood, it produced a greater increase in systolic BP (p = 0.0031 during the day; p= 0.0081 at night). In contrast, prenatal BPF exposure followed by sham implantation resulted in marked increases in diastolic BP. These results indicate that prenatal exposure to BPF can increase BP in female rats. This risk is augmented when females are subjected to chronic low dose E2 exposure in adulthood. Further studies are needed to understand the underlying mechanisms.Support or Funding InformationSupported by UGA research foundation and start‐up funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DL0108 suppresses the development of pulmonary arterial hypertension in rats through its protective effect on pulmonary arteries
Authors: Yuan Tianyi , Chen Di , Lyu Yang , Fang Lianhua , Du Guanhua 
Published: June 2021

BackgroundPulmonary arterial hypertension (PAH) is a severe disease but in short of an effective drug. DL0108 is a flavonoid discovered by our lab, and is recently found useful in the treatment of PAH. The present study aims to evaluate the potency of DL0108 in PAH rats induced by monocrotaline (MCT).MethodThe PAH rat model was established by subcutaneous injection of 50 mg/kg MCT on day 0. From the second day, 3, 10, or 30 mg/kg DL0108 was respectively administrated (i.p.) daily for 4 weeks in three tested groups, while the model and control group were treated with normal saline. During the experiment, the survival rate and body weight were recorded. At the end of the study, the right ventricular systolic pressure (RVSP) was measured through right cardiac catheterization. The heart ventricle was isolated to calculate the Fulton index (RV/LV+S). The contraction and relaxation of pulmonary artery rings were tested in a classical isolated vessel experiment.ResultOn day 28, the survival rate of the model group was 66.7%. Treatment with 3, 10, or 30 mg/kg DL0108 increased the survival rate to 73.3%, 86.7% and 86.7%, respectively. However, the decrease of body weight induced by PAH was not improved by DL0108. The RVSP of PAH rats in the model group increased from 21.73 to 49.61 mmHg, while the treatment with DL0108 reduced the RVSP to 30.56, 27.42 or 24.9 mmHg, respectively. Meanwhile, DL0108 significantly reduced the right ventricular hypertrophy in a dose‐dependent manner. In the isolated vessel experiment, it was found that 3 and 10 mg/kg DL0108 improved the contraction response of pulmonary artery rings to 10−6 M phenylephrine and the relaxant effect induced by the accumulation of acetylcholine (10−8–10−4 M Ach).ConclusionEarly intervention of PAH rats with DL0108 can delay the progression of the disease and increase the survival rate. The preliminary study indicates that DL0108 protects the function of pulmonary arteries, relieves the remodeling of vessels to control the pressure, and in turn attenuates the injury of heart ventricle. This study contributes to the further development of DL0108 for the treatment of PAH.Support or Funding InformationThis project was supported by grants from the Nation Natural Science Foundation of China (No. 81603101, 81773935, 81573645), Natural Science Foundation of Beijing (No. 7174322) and CAMS Innovation Fund for Medical Sciences (2016‐I2M‐1‐010, 2017‐I2M‐1‐010).
The survival rates of PAH rats treated by DL0108.imageThe survival rates of PAH rats treated by DL0108.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.