bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the Brain Organic Cation Transporter Plasma Membrane Monoamine Transporter (PMAT) by Alternative Splicing
Authors: Wagner David , Duan Haichuan , Wang Joanne 
Published: June 2021

PMAT is a polyspecific organic cation transporter highly expressed at the blood‐cerebrospinal fluid (CSF) barrier and facilitates the removal of monoamine neurotransmitters, organic cation drugs and toxicants from the CSF. We found that human SHSY5Y neuronal cells co‐express the full‐length wild type (wt) PMAT mRNA with two alternatively spliced mRNA species. One spliced variant (PMAT_v1) introduced a premature stop codon before transmembrane domain (TMD) 3 and the other (PMAT_v2) had a frame shift resulting in the deletion of TMD 3‐4. The goal of this study was to determine 1) the function and expression of the spliced variants in normal human tissues; 2) if the spliced variants exhibit a regulatory role on wt PMAT. Variant‐specific PCR indicated the spliced variants were co‐expressed with wt PMAT in all tested human tissues (whole brain, choroid plexus, small intestine and colon) at levels 37‐84% of the wt PMAT. When expressed in HEK293 cells, wt PMAT‐expressing cells showed up to 20‐fold increase in [3H]MPP+ uptake as compared to vector‐transfected cells. In contrast, there was no significant increase in uptake in cells‐expressing PMAT_v1 or PMAT_v2. PMAT_v1 protein was not detected by immunocytochemistry in HEK cells while PMAT_v2 was exclusively expressed in intracellular organelles. Co‐expression of PMAT_v1 or PMAT_v2 with wt PMAT in HEK293 cells did not significantly interfere with the normal activity of wt PMAT. Together our data showed PMAT pre‐mRNA is alternatively spliced in normal human tissues; and the overall PMAT activity in certain human tissues and cells may be regulated by altering the ratio between the functional and the non‐functional mRNA species.This study was supported by NIH Grants R01GM066233 and T32GM07750.
HuR is Activated in Cardiac Hypertrophy and Regulates β1‐AR mRNA
Authors: Kraynik Stephen , Anthony Sarah , Tranter Michael 
Published: June 2021

Heart failure is a worldwide health burden that is often preceded by a long period of increasing hypertrophy, or enlargement, of the heart prior to the loss of cardiac function. This progression of hypertrophy is marked by a decrease in the function and expression of key proteins that regulate cardiac contractility, such as the β1‐adrenergic receptor (β1‐AR). Despite a compensatory increase in sympathetic tone, cardiac contractility and output are substantially impaired, partially through β1‐AR desensitization and decreased receptor expression via mRNA degradation. It is thought that reversing the loss of the β1‐AR mRNA may preserve cardiac function and ameliorate the progression of hypertrophy. Human Antigen‐R (HuR), a known RNA binding protein, has been shown to bind to the 3'‐untranslated region of the β1‐AR mRNA, and we hypothesized that HuR plays a role in the development of cardiac hypertrophy by regulating β1‐AR mRNA stability in cardiomyocytes.In hypertrophic hearts from mice that were subjected to chronic isoproterenol, we observed a shift in HuR subcellular localization from predominantly nuclear to increasingly cytosolic, an indication of HuR activation. Surprisingly, in hypertrophic hearts from mice that had undergone transaortic constriction (TAC), we observed that HuR activation strongly localized to regions of increased fibrosis. Futhermore, HuR translocation in hypertrophic hearts positively correlated with a near 50% decline in β1‐AR mRNA in both models of hypertrophy. Additionally, we show that in vitro knockdown of HuR by siRNA resulted in increased β1‐AR mRNA. In conclusion, these results suggest a novel role for HuR in the development of cardiac fibrosis in hypertrophy, and have demonstrated that HuR may be mediating β1‐AR mRNA degradation.
Clinical Biopotency of Insulin Analogues: Comparative Assessment in Different Species
Authors: Stricker‐Krongrad Alain , Hanks Chris , liu Jason , Bouchard Guy 
Published: June 2021

ObjectiveThe clinical potency of human insulin has classically been evaluated following the U.S. Pharmacopeia (USP) guideline for comparison to an international standard (IS) of activity (IU). However, since insulin analogues cannot be standardized against IS, there is a need for a bioassay to assess clinical specific activity (U). To this extent, we compared the biopotency (U) of different insulin products in different species and used human (recombinant) and pork (natural) insulin as reference standards.Methods: New Zealand White rabbits were fasted and injected subcutaneously (s.c.) at a dose level of 0.5 U/kg. Diabetic Yucatan miniature swine (Sus scrofa) were injected s.c. at dose level of 0.1 U/kg. Normal insulin suppression tests (nIST) were conducted in primates (Macaca fascicularis) receiving a bolus intravenous (i.v.) infusion of glucose. Insulin products were given at dose level of 0.05 U/kg. Glucose levels were recorded using handheld glucometer devices. The blood glucose kinetic (BGPK), the blood glucose area under the curve (BGAUC) and the slope of the blood glucose clearance rate (kG) were used to assess clinical biopotency.Results: The BGPK for short‐acting human and pork insulin were similar in the rabbit assay but their respective BGAUCs were different (ratio of 1.2). Accordingly pork insulin was more potent during nIST in the non‐human primate (slope of ‐0.012 vs. ‐0.010; pork and human, respectively).ConclusionOur data indicate that the clinical biopotency of insulin products can be assessed using BGAUC and kG, but that only the type 1 diabetic miniature swine can discriminate between differences in clinical biopotency for all aspects of BGPK.
Activation of Aryl Hydrocarbon Receptor Dissociates Fatty Liver from Insulin Resistance by Inducing Fibroblast Growth Factor 21
Authors: Lu Peipei , Yan Jiong , Garbacz Wojciech , Xie Wen 
Published: June 2021

Background & AimsThe aryl hydrocarbon receptor (AHR), also known as the dioxin receptor, was originally characterized as a xenobiotic receptor that senses xenotoxicants. Here we investigated the endobiotic and hepatic role of AHR in fatty liver and energy metabolism, and identified the endocrine factor that mediates the metabolic function of AHR. MethodsWild type and liver‐specific constitutively activated human AHR transgenic (TG) mice were used to investigate the role of AHR in fatty liver and energy homeostasis. Adenovirus expressing short hairpin RNA targeting the fibroblast growth factor 21 (FGF21) were used to determine the involvement of FGF21 in the metabolic effect of AHR.ResultsWe showed that despite their severe fatty liver, the TG mice were protected from diet‐induced obesity and type 2 diabetes. We identified the endocrine hormone FGF21 as a mediator for the metabolic benefit of AHR and established FGF21 as a direct transcriptional target of AHR. Interestingly, the transactivation of FGF21 by AHR contributed to both hepatic steatosis and systemic insulin hypersensitivity, both of which were largely abolished upon FGF21 knockdown.ConclusionsThe AHR‐FGF21 endocrine signaling pathway establishes AHR as a pivotal environmental modifier that integrates signals from chemical exposure in the regulation of lipid and energy metabolism.FundingThis work was supported in part by the National Institutes of Health grants DK083952 and HD073070 to Wen Xie.
Role of Toll‐Like Receptor 4 Activation in Mitochondrial Respiration in Mouse Liver and Heart
Authors: Pickering Brian , Sharda Daniel 
Published: June 2021

Ischemia reperfusion (I/R) injury results in activation of Toll‐like receptor 4 (TLR4) in cardiomyocytes, which contributes to tissue loss. Inhibiting TLR4 upon I/R results in improved recovery, as, paradoxically, does pretreatment with lipopolysaccharide (LPS), an activator of TLR4. However, it is unclear how TLR4 affects cardiomyoctes during LPS pretreatment or I/R injury. One clue may come from the recent discovery that TLR4 activation in macrophages enhances production of mitochondrial reactive oxygen species (mROS) due to perturbations in complex I of the electron transport chain (ETC). Therefore, we hypothesized that TLR4 activation in cardiomyocytes would decrease complex I respiration. Furthermore, we hypothesized that TLR4 activation would lead to mitochondrial uncoupling suggestive of mROS. In this study, mitochondria were isolated from hearts and livers of CD1 mice that had been pretreated for 1 hour i.p. with 1mg/kg LPS or vehicle control (PBS), and states 1‐4 of complexes I, II, and IV were analyzed using a Clarke‐type electrode for respiratory affects. Here we report that LPS did not affect complexes I (p=0.38 for state 3), II (p=0.37), and IV (p=0.41) in the heart, or complex II in the liver (p=0.36), indicating that LPS pretreatment does not modify ETC activity. Moreover, mitochondrial coupling displayed similar values for the respiratory control index (RCI) and respiratory control ratio (RCR) suggesting ROS production is unlikely to be affected by LPS pretreatment. Together, these data suggest that any pretreatment affects conferred by LPS at one hour are separate from the ETC, and remain a topic for future investigation.This work was funded by the Elbert Pence and Fanny Boyce Undergraduate Summer Research Grant.
LRRC8A (IClswell) Anion Channels Modulate TNFα‐Dependent Activation of NF‐κB
Authors: Ettinger Nicholas , Choi Hyehun , Nguyen Hong‐Ngan , Lamb Fred 
Published: June 2021

Leucine‐Rich Repeat‐Containing 8 (LRRC8) family members are essential components of IClswell, the evolutionarily‐conserved anion conductance that modulates cytoplasmic volume in response to cell swelling. Our previous work has demonstrated that the 2Cl‐/H+ anti‐porter ClC‐3 is required for TNFα‐induced endosomal superoxide (O2•) production by Nox1, as well as for subsequent activation of IClswell and NF‐κB in both HEK293 and vascular smooth muscle cells (VSMC). We hypothesized that IClswell activation by TNFα is a required step in NF‐κB signaling. Immunostaining of expressed LRRC8A and Nox1 demonstrated co‐localization in cytoplasmic vesicles of HEK293 cells. An NF‐κB‐luciferase reporter construct demonstrated a dose‐dependent decrease in NF‐κB activation by TNFα (10ng/ml) in the presence of the IClswell inhibitors tamoxifen (85±8% at 50μM) or DCPIB (64±6% at 50μM). Targeting LRRC8A expression with siRNA reduced NF‐κB activation by 64±2%. Similar effects of drugs and siRNA were observed in murine aortic VSMCs. N2‐cavitated HEK293 cell lysates were cleared of nuclei and mitochondria (10,000g, 15 min). These lysates made O2• in the presence of NADPH (0.5mM) and lucigenin (5μM). O2• production was inhibited by DPI (75±10%, 10μM), but enhanced by DCPIB (40±17%). These data suggest that endosomal IClswell activity is required for TNFα signaling. We hypothesize that IClswell is present in the endosomal membrane and acts as a pathway by which O2• release mediates local negative feedback to regulate Nox1 activity and O2•‐dependent inflammatory cell signaling.This research was supported in part by an NICHD training grant to the Department of Pediatrics at Vanderbilt University Medical Center (T32 HD068256‐03).
Association Between NADPH Oxidase Activity and NO Bioavailability in Human Blood Arteries of Hyperhomocysteinemic Patients
Authors: KALINOWSKI LESZEK , PLOSKA Agata , SIEKIERZYCKA Anna , DOBRUCKI LAWRENCE , WOJCIECHOWSKI JACEK , WOZNIAK MARCIN , ROGOWSKI JAN , DOBRUCKI IWONA 
Published: June 2021

Nitric oxide (NO) deficiency is a hallmark of the vascular disorders induced by risk factors, including hyperhomocysteinemia (Hhcy). NADPH oxidase and eNOS are sources of reactive oxygen and nitrogen species (NROS). Hhcy can promote NROS stress leading to the endothelial dysfunction. Here we investigated the mechanism of NROS stress in the endothelium of patients with induced Hhcy.Patients qualified for the carotid endarterectomy received 100 mg/kg p.o. methionine or placebo 6 h before surgery. Fragments of isolated carotid arteries were used in the study. NADPH oxidase activity was measured with lucigenin‐based assay. NO, O2‐ and peroxynitrite (ONOO‐) release from endothelial cells (ECs) were measured using electrochemical nanosensors. The expressions of eNOS and NADPH oxidase subunits were analyzed by Western blotting.Hhcy group revealed significant increase of NADPH oxidase activity and the expression of total and membrane fractions of p47phox subunit. eNOS stimulation resulted in lower NO levels and increased production of both O2‐ and ONOO‐ in group with Hhcy. The presence of apocynin improved NO release in ECs with Hhcy. eNOS expression was significantly higher in the Hhcy ECs. The ratio of eNOS dimers to the total eNOS was significantly decreased in patients with Hhcy.In conclusion, the disruption of dimeric structure of overexpressed eNOS contributes to overproduction of O2‐ and induction of eNOS‐dependent NROS stress in patients with Hhcy.
Knockdown of Exocyst Sec10 in Mouse Skeletal Muscle Cells and Analysis of Insulin‐Stimulated GLUT4 Exocytosis
Authors: Lee Amanda , Ines Eden , Lui Vanessa , Fogelgren Ben 
Published: June 2021

Type II diabetes is a metabolic disorder characterized by insulin resistance and high glucose in the blood. The GLUT4 receptor is the major glucose transporter that allows insulin‐stimulated glucose uptake into metabolic tissues such as adipose and skeletal muscle. In cultured adipocyte 3T3‐L1 cells, the eight‐protein exocyst complex has been shown to exocytose GLUT4 vesicles to the plasma membrane when stimulated by insulin. Our lab has extensively studied the activity of the exocyst trafficking complex in renal epithelial cells, and we have successfully generated a conditional knockout mouse for the exocyst subunit Sec10. Previous studies have been limited to cultured adipocytes, but skeletal muscles are responsible for taking in a higher percentage of glucose, and it has been shown that exercise helps maintain glucose levels to prevent the onset of type II diabetes.To evaluate whether the exocyst also plays a role in GLUT4 regulation in skeletal muscle cells, we used shRNA to generate stable Sec10 knockdowns in C2C12 mouse skeletal muscle cells. After confirming the knockdown, we measured glucose uptake using two different methods. First, we measured the amount of glucose in the media of Sec10 knockdown and control cells over the course of 2 weeks. Second, we compared insulin‐stimulated glucose uptake using a fluorescent glucose analog, 2‐NBDG. Based on preliminary findings, knockdown of Sec10 in mouse skeletal muscle cells results in decreased glucose uptake. Ongoing work with skeletal muscle‐specific Sec10 knockout with our new mouse model will help with better characterization of the role of the exocyst in insulin signaling and GLUT4 exocytosis that may reveal new therapeutic targets for human type II diabetes and pre‐diabetic insulin resistance.
P2X7R Antagonism Improves Renal Medullary Blood Flow and Oxygenation in Angiotensin II‐dependent Hypertension
Authors: Bailey Matthew , Menzies Robert , Mullins John 
Published: June 2021

Kidney disease is a complex disorder with a genetic contribution. We have shown that the Fischer (F344) rat is more susceptible to hypertensive renal injury than the Lewis rat. Interrogation of microarray identified P2X7R as a candidate gene for injury susceptibility in F344 rats. Renal P2X7R were expressed in the endothelium of the preglomerular arteries and in the afferent arteriole, vasa recta and renal tubules. Infusion of angiotensin II for a 14‐day period did not increase the abundance of P2X7R in whole kidney homogenates but induced expression of the receptor in the vascular smooth muscle of the renal, interlobar and arcuate arteries, the site of vascular injury. P2X7R were also expressed in the glomerulus of hypertensive rats, in which increased numbers of macrophages were identified. The specific P2X7R antagonist AZ11657312, administered IV to angiotensin II treated rats, caused a dose‐dependent reduction in arterial blood pressure and increased blood flow in the renal artery and renal medulla, measured by Doppler flow and flux respectively. BOLD‐MRI also identified a transient decrease in renal R2* signal following P2X7R antagonism, consistent with improved oxygenation of the renal medulla. These data support the hypothesis that P2X7R activation exerts adverse vascular effects within the kidney. Acute antagonism of this receptor lowers blood pressure and increases renal perfusion/oxygenation and may offer a multi‐layered approach to treat kidney disease.We thank the British Heart foundation for funding and AstraZeneca for AZ11657312.
MD2 may be a new target for the treatment of obesity‐induced cardiac injury
Authors: Qian Yuanyuan , Zhong Peng , Wang Yi , Liang Guang 
Published: June 2021

BackgroundObesity is strongly associated with cardiovascular diseases. Accumulating evidences have demonstrated a vital role of TLR4 signaling in the pathogenesis of obesity‐induced complications. Myeloid differentiation 2 (MD2), an obligate partner for TLR4, was required for LPS‐associated inflammation and TLR4 signaling activation. However, whether MD2 is also involved in the obesity‐associated inflammation is still unknown. The aim of this study is to investigate the involvement of MD2 in the pathogenesis of obesity‐induced cardiac diseases.MethodsCardiac H9C2 cells exposed to free fatty acid were pretreated L6H9, a specific MD2 inhibitor or MD2 silencing siRNA. Rats and APOE‐/‐ mice were given high fat diet for eight weeks and administrated with L6H9 by daily gavage.ResultsIn H9C2 cells, FFA was found to induce inflammation, oxidative stress, ER stress, mitochondrial dysfunction, and ultimately cardiac hypertrophy, fibrosis, and apoptosis. Interestingly, all these alterations were significantly inhibited by pharmacological inhibition and genetic silence of MD2. In vivo, two obese models markedly induced cardiac inflammation and oxidative stress and ultimately lead to cardiac remodeling including cardiac disorganization, hypertrophy, apoptosis and fibrosis. All these molecular and pathological alterations were remarkably attenuated by the administration with MD2 inhibitors.ConclusionMD2 is involved in the pathogenesis of obesity‐associated cardiac remodeling via mediating FFA‐induced inflammation, and MD2 can be a therapeutic target in treating obesity‐induced complications.
S‐glutathionylation of Epithelial Sodium Channels in Neonatal Mouse Lung Regulates Alveolar Fluid Clearance in vivo
Authors: Trac Phi , Helms My 
Published: June 2021

Hyperoxia causes oxidative stress and leads to a positive shift in the GSH/GSSG redox potential (Eh) in the lung. We hypothesize that an increase in the concentration of GSSG can post‐translationally modify lung epithelial sodium channels (ENaC) via S‐glutathionylation. We measured the Eh value of bronchoalveolar fluid (BAL) from postnatal day (PN) 10 C57Bl6 mice housed under room air (CTR; 21% O2) or hyperoxia (H85; 85% O2) and found that chronic H85 exposure elevates GSSG concentrations. In support of our hypothesis, single channel patch clamp analysis showed that GSSG (0.4 mM) decreases ENaC activity in alveolar type 2 cells (from 0.39 + 0.06 to 0.13 + 0.05). Real‐time X‐ray imaging illustrated that H85 and GSSG (0.4 mM) treatment significantly impairs the rate of lung fluid clearance compared to PN 10 controls; thus verifying the translational significance of single channel measurements. Predictive software analysis showed that a, b, and g‐ENaC subunits have conserved Cys thiols that can be directly S‐glutathionylated by GSSG. ENaC subunit pull‐down with biotinylated glutathione ethyl esters (BioGEE) confirms post‐translational modification of ENaC via S‐glutathionylation under hyperoxia‐induced oxidative stress. In conclusion, our data indicated that S‐glutathionylation of ENaC Cys thiols attenuates net salt and water reabsorption in the lungs. A better understanding of this novel regulatory pathway could lead to improved therapeutics for newborn infants under respiratory distress reliant on oxygen therapy.Acknowledgements: We acknowledge support from the APS Short‐Term Research Education Program to Increase Diversity in Health‐Related Research Fellowship.
Molecular Dynamics Modeling of Sodium And Water Transport through a Claudin Pore
Authors: Yu Alan , Laghaei Rozita , Rosenthal Rita , Coalson Rob 
Published: June 2021

BackgroundClaudin‐2 is a paracellular channel. Na and water flow through it driven by an osmotic or Na concentration gradient, suggesting that Na and water pass through a common pore and that their fluxes are frictionally coupled. We investigated the mechanism by molecular dynamics simulations.MethodsThe claudin pore was modeled either as a double cone or a cylindrical nanotube with narrowest diameter of 6.5 Å and 6 negatively charged sites in the middle of the pore (each ‐0.2e). A hydrostatic pressure gradient was applied via an additional constant force in the channel axis direction to a layer of water molecules parallel to the membrane interface. Simulations were performed using the CHARMM27 force field and the TIP3P water model, with full electrostatics was included.ResultsWith an osmotic gradient, water molecules oriented within the pore in 2 water wires and exhibited directional flow with Pf= 4.7 x 10‐13 cm3/s and a water:Na flux ratio of 233. Increasing the magnitude of the negative charge increased permeability only when the charges were smeared evenly along the pore surface. Application of a Na concentration gradient, also drove water flow with water:Na flux of 14.6. Increasing the pore diameter to 7Å also increased water flow significantly by allowing 3 water wires to form.ConclusionsOur findings confirm that water can permeate through claudin‐2 via a water wire mechanism, demonstrate that water permeability is dependent on the magnitude and distribution of intrapore charges and on the pore diameter, and provide a molecular explanation for the frictional coupling of Na and water fluxes within the pore.
Claudin‐2 Mediates Sodium and Water Transport through a Common Pore
Authors: Rosenthal Rita , Günzel Dorothee , Schulzke Joerg , Fromm Michael , Yu Alan 
Published: June 2021

BackgroundThe tight junction protein Claudin‐2 is mostly expressed in leaky epithelia like proximal kidney tubule and small intestine. Claudin‐2 protein abundance is increased in inflammatory bowel diseases like Crohn's disease, ulcerative colitis, and celiac disease. Former studies of our group revealed that claudin‐2 forms paracellular channels for small cations like sodium and potassium and also paracellular channels for water. Aim of this study was to analyze whether the diffusive transport of sodium and water occurs through a common pore of the claudin‐2 channel.MethodsWild‐type claudin‐2 and claudin‐2 mutants with substituted amino acids of the first extracellular loop were expressed in the tight epithelial cell line MDCK I, using a TetOff inducible system. Sodium and water permeability were measured in modified Ussing chambers.ResultsNeutralization of the negatively charged cation interaction site in the pore with the mutation D65N caused a reduction of both, sodium and water permeability. Steric blocking of the claudin‐2 pore by thiol‐reactive reagents was performed after substitution of the pore‐lining amino acids, isoleucine‐66 and serine‐68 with cysteine. Thiol‐reactive reagents caused a decrease in both, ion conductance and water permeability in these claudin‐2 cysteine mutants whereas wild‐type claudin‐2 was not affected. Plotting all data yielded a strong correlation of claudin‐2‐mediated sodium and water permeability.ConclusionClaudin‐2‐mediated sodium and water transport are loosely coupled and share a common pore lined by the amino acids of the first extracellular loop of claudin‐2.