bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phenotypic Differences Between Tissues and Sex Observed in Mice with Human Gout Causing ABCG2 Variant
Authors: Hoque Kazi Mirajul , Lewis Raychel M. , Woodward Owen M. 
Published: June 2021

Hyperuricemia is a risk factor for gout and other complex human disorders. We have established in humans that ABCG2 codes a high‐capacity uric acid (UA) efflux transporter, that when dysfunctional causes hyperuricemia and significantly increases the risk for gout. Recent work has demonstrated ABCG2 mediated secretion is important for intestinal excretion of UA, so to better understand the intestinal contribution of ABCG2 dysfunction to hyperuricemia and gout, we created a new model where a Q140K (human Q141K) point mutation in the Abcg2 gene was made using CRISPR Cas9 gene editing techniques on a C57BL/6 mouse background. We employed both western blot and confocal microscopy to generate profiles of ABCG2 in both sexes and multiple tissues and assessed the differences of luminal UA excretion using mouse intestinal closed loop method. We observed in the wild type mice ABCG2 expression varies along the GI tract with the highest levels in the jejunum and ileum, and with low levels in the colon of both male and female mice. The localization of the wild type ABCG2 protein was exclusive to the brush border of villus cells and luminal membrane of the crypt cells. In contrast, one or two copies of the Q140K Abcg2 allele resulted in significant decreases (53% and 88% respectively) in total intestinal ABCG2 expression and apical membrane staining in villus cells. In female mice, the Q140K ABCG2 mutation resulted in little difference of ABCG2 expression, similar to the mostly unaltered expression of ABCG2 in the renal cortex of both male and female Q140K ABCG2 mice. Concomitant with the protein abundance defect, Q140K ABCG2 male mice had higher serum UA (3.31 ± 0.37 mg/dl) than WT mice (2.13 ± 0.14 mg/dl) and significantly reduced (−40%) acute UA flux in an intestinal loop assay. The reduction in the UA flux in the Q140K loop model was comparable to the reduction observed when the WT intestinal loop was treated with the ABCG2 inhibitor FTC (30%), suggesting the Q140K mutation approximates a complete loss of ABCG2 function in the small intestines. The sex dependence of the pathology of the Q140K mutation suggested that ABCG2 and UA secretion could be under hormonal control. In intestinal loop experiments we found FBS and more specifically angiotensin II modulated UA secretion. We conclude that (i) The Q141K ABCG2 mutation (Q140K in mouse) results in severe loss of ABCG2 mediated UA secretion in the small intestine (ii) sex determines the pathology and abundance of the mutant ABCG2 protein along the intestine and (iii) Small intestines may be the site of hormonal regulation of both ABCG2 and UA secretion.Support or Funding InformationNational Institute of Health RO1 grant (DK114091‐01) & American Hear Association grant: 14SDG18060004This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tiliacora triandra (Colebr.) Diels Ameliorates Hepatic Steatosis by Inhibition of HMGR and Down‐regulation of FAT/CD36
Authors: Pasachan Tipthida , Ontawong Atcharaporn , Soodvilai Sunhapas , Duangjai Acharaporn , Pongchaidecha Anchalee , Amornlerdpison Doungporn , Srimaroeng Chutima 
Published: June 2021

Background & AimsTiliacora triandra (Colebr.) Diels (TT) is a native plant of Southeast Asia widely used in northeastern Thai cuisines, and is known in Thai language as Yanang. High concentration of polyphenolic compounds found in TT aqueous extract (TTE) include epicatechin (EC), quercetin (QC), cyanidine (CD), and gallic acid (GA), respectively. TTE has shown various beneficial pharmacological effects such as anti‐inflammatory, anti‐cancer, anti‐aging, anti‐oxidant, anti‐hypercholesterolemia, and inhibition of intestinal cholesterol absorption. However, the effects of TTE on cholesterol synthesis and hepatic steatosis are limited. This study aimed to investigate the direct effects of TTE on cholesterol synthesis and hepatic lipid accumulation in hepatocellular carcinoma (HepG2) cells.MethodsHepG2 cells at the cell density of 5×105 cells/well were treated with TTE, EC, and QC in a molar ratio for 24 hr. The cytotoxic effect of TTE was evaluated using MTT assay. Hepatic lipid accumulation was quantified by Nile red staining and detected by flow cytometry. The mRNA expression of genes involved in cholesterol synthesis, HMGR, and the fatty acid transporter, FAT/CD36, was also evaluated using quantitative real‐time PCR.ResultsTTE significantly reduced hepatic lipid accumulation up to the dose of 100 μg/ml similarly to that of EC and QC without any cytotoxic effect. This effect also corresponded to the reduction in HMGR and FAT/CD36 gene expressions, resulting in a significant decreased in de novo hepatic cholesterol synthesis and free fatty acid accumulation.ConclusionThese findings suggest that TTE exhibits lipid‐lowering action by decreasing both hepatic cholesterol synthesis and lipid accumulation, leading to improved non‐alcoholic fatty liver. Thus, TTE could potentially be developed as nutraceutical product for prevention of progressive liver diseases in obesity, diabetes, or metabolic syndrome.Support or Funding InformationThis study is supported by the Faculty of Medicine Endowment fund (093/2560), Chiang Mai University, Research and Researcher for Industry (RRI) by Thailand Research Fund (TRF), (PHDI0009 to CS) and the NSTDA Chair Professor grant (the Fourth Grant) of the Crown Property Bureau Foundation and the National Science and Technology Development Agency to Professor Dr. Vatcharin Rukachaisirikul.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced expression of colonic non‐heme iron transporters divalent metal transporter‐1 (DMT‐1) and ferroportin (FPN) in patients with ulcerative colitis
Authors: Minor Emily , Kupec Justin , Nickerson Andrew , Rottgen Trey , Rajendran Vazhaikkurichi 
Published: June 2021

Iron is an essential micronutrient that is involved in many different physiological processes, such as redox reactions of the electron transport chain, oxygen transport, and DNA synthesis. Additionally, iron is an important limiting factor for microbial growth. The tight regulation of whole body and cellular iron concentration is part of the innate immune system whereby the body can restrict the amount of available iron to help prevent the growth of pathogenic bacteria. This is crucial because microbial dysbiosis is detrimental to health and is thought to play a causative role in the development of ulcerative colitis (UC), a chronic inflammatory bowel disease (IBD) associated with bloody, mucous‐laden stools. Patients with ulcerative colitis (UC) frequently suffer from iron deficiency and may be prescribed oral iron supplementation, but there is fine line between trying to correct the iron deficiency and providing excess iron that can exacerbate disease activity and alter the microbial community. Most iron absorption occurs in the duodenum, but the colon is also known to contain non‐heme iron transporters. The purpose of this study was to characterize the location of iron transporters in normal human colon and determine whether their expression varied in patients with UC. RNA and protein were isolated from colonic biopsy specimens obtained during previously scheduled colonoscopies. We found a trend of decreasing mRNA and protein abundance from the cecum to the distal colon of DMT‐1 and FPN. Patients with ulcerative colitis had increased expression of colonic iron transporters, with the highest expression of those transporters found in patients with active disease activity. The upregulation of iron transporters in the colonic epithelium of patients with UC may have a two‐fold benefit: increased colonic iron absorption would help alleviate the iron deficiency frequently seen in patients with UC and it would limit the amount of available iron in the colonic lumen, thus reducing the growth of pathogenic bacteria.Support or Funding InformationR01: 10019896.1.1006856RThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impairment of renal organic anion transport mediated by organic anion transporter 1 (Oat1) and 3 (Oat3) in acute myocardial ischemic and reperfusion injury in rats
Authors: Srimaroeng Chutima , Sirijariyawat Kungsadal , Ontawong Atcharaporn , Palee Siripong , Thummasorn Savitree , Maneechot Chayodom , Boonphang Oranit , Chatsudthipong Varanuj , Chattipakorn Nipon 
Published: June 2021

Acute kidney injury (AKI) is frequently associated with acute myocardial infarction (AMI). Kidney plays a crucial role in the clearance of uremic toxins, produced from organ dysfunctions, mainly through organic anion transporters 1 (Oat1) and 3 (Oat3). This study aims to examine the effects of acute myocardial ischemia and ischemic/reperfusion (I/R) on renal transporter functions, and identify the mechanisms involved in the impairment of these transporters. Myocardial ischemia and I/R were induced in rats by either 30‐minute left anterior descending coronary artery occlusion or 30‐minute occlusion followed by 120‐minute of reperfusion, respectively. The renal hemodynamic parameters and renal transporter functions were subsequently determined along with renal biochemical markers. Results showed that myocardial ischemia led to reduction in renal blood flow, perfusion pressure, and kidney function by approximately 40%, while the reperfusion phase partially recovered renal hemodynamic parameters. Moreover, myocardial ischemia and I/R progressively induced renal oxidative stresses and mitochondria dysfunction, all of which directly deteriorated renal Oat1 and Oat3 functions. Thus, this study indicates that myocardial ischemia and I/R conditions leads to a progressively declined renal transport function, which could potentially worsen AKI. Renal Oat1 and Oat3 transporters, therefore, might be potential therapeutic targets for reversing such injury.Support or Funding InformationThis research was supported by the Thailand Research Fund (RSA5980009 to CS), Faculty of Medicine Endowment Fund, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of a new splice variant of large‐conductance Ca2+‐activated K+ (BK) channel α subunit from human chondrocyte
Authors: Suzuki Yoshiaki , Ohya Susumu , Yamamura Hisao , Giles Wayne R. , Imaizumi Yuji 
Published: June 2021

Changes in intracellular calcium concentration ([Ca2+]i) modulate chondrogenesis, proliferation, cell death and baseline anabolic and catabolic activities in chondrocytes. Under pathophysiological conditions, like osteoarthritis (OA), histamine release from resident mast cells enhances the production of pro‐inflammatory mediators and the activities of matrix degrading enzymes. Specific patterns of [Ca2+]i changes are considered as essential components initiating and/or progressing the disease. In an established model cell‐line of the human chondrocyte, OUMS‐27 cells, large‐conductance Ca2+‐activated K+ (BK) channels are involved in the enhancement of histamine‐induced Ca2+ influx by the activation of Ca2+ release‐activated Ca2+ (CRAC) channels consisting of Orai1‐Orai2 heteromers and STIM1 via the modulation of membrane potential. We have identified a novel BKα splice variant (BKαΔe2) in OUMS‐27 cells. Although BKαΔe2 lacks exon2 that codes the intracellular S0–S1 linker (Glu127‐Leu180), significant expression was detected in several human tissues including chondrocytes of OA patients and in mouse tissues as well. BKαΔe2 channels are not expressed on plasma membrane (PM), but can traffic to PM after forming hetero‐tetramer units with wild‐type BKα (BKαWT). These hetero‐tetramers of BKα have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo‐tetramers in a stoichiometry‐dependent manner. BKαΔe2 knockdown in OUMS‐27 chondrocytes increased BK current density, and augmented the responsiveness to histamine assayed as cyclooxygenase‐2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as OA.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant Numbers, 26293021 and 15H01408 to Y.I., 26860059, 16H06215 and 16K15127 to Y.S., 16K08278 to H.Y. This work was also supported by a Grant‐in‐Aid from Takeda Science Foundation (to Y.S.) and Salt Science Research Foundation [Grant 1637] (to Y.S.). W.G. holds an Alberta Innovates‐Health Solutions Scientist Award, and CIHR funding.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Role for the Na‐K‐2Cl Cotransporter in Mitochondrial Respiration
Authors: Omer Salma , Delpire Eric 
Published: June 2021

A patient recruited by the undiagnosed disease program (UDP) at the NIH suffers from multi organ failure and deficits in energy homeostasis. The multi organ failure consists of complete gastrointestinal and bladder dysfunction, as well as thyroid, parathyroid, adrenal, and pancreatic deficiencies. The deficits in energy homeostasis were evidenced in lab tests conducted on the patient's muscle and liver biopsies, where there was an abnormal increase in glycogen content and mitochondrial DNA copy number. Whole exome sequencing showed that the patient expresses a de novo mutation in an allele of the Slc12a2 gene encoding the Na‐K‐2Cl cotransporter‐1 (NKCC1). Previous studies in the laboratory demonstrated that the 11 base pair deletion causing a truncation of the carboxyl‐terminus, leads to a non‐functional transporter. We have also demonstrated that the UDP patient's fibroblasts displayed reduced total NKCC1‐mediated K+ influx. Additionally, trafficking studies in transfected HEK293 cells expressing the NKCC1 truncation mutation have revealed that this mutated protein dimerizes with wild‐type (WT) NKCC1 and is trafficked to the plasma membrane along with WT NKCC1.To address a possible role of the cotransporter in metabolism, I utilized fibroblasts isolated from the patient and from healthy controls and performed mitochondrial respiration stress tests. I showed that mitochondrial respiration and ATP production was significantly higher in the patient's fibroblasts. Because the cells from the patient might carry additional mutations, I transfected MDCK cells with EGFP‐tagged wild‐type or mutant transporter, sorted the cells, and tested them. Cells expressing the mutant cotransporter also demonstrated higher mitochondrial respiration, compared to cells expressing wild‐type EGFP‐NKCC1.To further confirm the role of NKCC1 in mitochondrial respiration, I subjected control MDCK cells to bumetanide, a NKCC1 specific inhibitor. MDCK cells treated with 20 mM bumetanide for 20 min also demonstrated an increase in mitochondrial respiration. To further assess whether this effect in mitochondrial respiration is NKCC1 specific, I subjected bumetanide to two stable lines of MDCK cells (LK‐A3 and LK‐C1) that carry a distinct mutation in NKCC1, resulting in a non‐functional transporter. There was no change in mitochondrial respiration levels in these cells, which indicated that the rise in mitochondrial respiration levels in wild‐type cell is NKCC1 specific. These data reveal a novel role for NKCC1 in mitochondrial respiration. Future work will address the mechanism by which NKCC1 activity affects mitochondrial function.Support or Funding InformationSupported by NIH grants R21GM118944 and RO1DK093501This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Orai1‐mediated store‐operated Ca2+ entry by a novel compound RP4010 for anti‐proliferative activity against esophagus squamous cell carcinoma
Authors: Chang Yan , Cui Chaochu , Zhang Xiaoli , Penmetsa Kumar , Viswanadha Srikant , Fu Liwu , Pan Zui 
Published: June 2021

Targeting Orai1‐mediated store‐operated Ca2+ entry by a novel compound RP4010 for anti‐proliferative activity against esophagus squamous cell carcinomaThe University of Texas System STARs AwardEsophageal cancer is the 6th leading cause of cancer mortality worldwide with poor prognosis and 5‐year survival rate as low as 18%. It is an unmet need to develop more effective chemotherapeutic drugs for esophageal cancer patients. We previously reported that the expression of Orai1, a Ca2+ channel, was elevated in esophageal squamous cell carcinoma (ESCC), a major form of esophageal cancer, and the high expression is associated with poor survival rate in patients. Orai1‐mediated store‐operated calcium entry (SOCE) has been demonstrated to contribute to cancer cell proliferation, migration and invasion in ESCC and other cancers. Here we evaluated the anti‐cancer effect of a novel Orai1 channel inhibitor, named RP4010, in cultured ESCC cells and xenograft animals.RP4010 is a novel, oral inhibitor of Orai1 channel currently in Phase I/IB clinical development. Compared to commonly used Orai1 channel blockers (such as BTP‐2), RP4010 is more potent to block Orai1‐mediated SOCE and cell proliferation in ESCC cells. The IC50 of RP4010 in cell viability analysis were calculated as 1.2, 1.4, 1.4 and 5.5 μM for KYSE‐30, KYSE‐70, KYSE‐150 and KYSE‐790, respectively while IC50 for BTP‐2 was 17.7 – 17.8 μM for these cells. Treatment of RP4010 (10 μM) inhibited intracellular Ca2+ oscillations, halted Ca2+‐dependent nuclear translocation of NF‐κB, and resulted in cell cycles arrested at G0/G1 phase. Interestingly, RP4010 did not induce significant cell death even at rather high concentrations. In vivo study revealed that the mice can tolerate daily I.P. injection of 20 mg/kg RP4010 without body weight loss or noticeable adverse effects up to 4 weeks. At this dose, RP4010 could significantly reduce tumor growth compared to vehicle treated control group.In summary, these data demonstrated that R4010 is a novel potent inhibitor for Orai1‐meidated SOCE. It has potential as a new kind of targeted compound blocking Orai1‐mediated intracellular Ca2+ signaling in ESCC treatment.Support or Funding InformationNational Cancer Institute and The University of Texas System STARs AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pannexin channels regulate cell‐cell calcium mobilization and motility during epithelial wound repair
Authors: Gonzales Selina Grace , Lee Yoonjoo , Trinkaus‐Randall Vickery 
Published: June 2021

The cornea is a transparent avascular tissue that transmits light to the retina and covers the lens and iris. According to the World Health Organization, blindness due to injury and disease in the cornea is the fourth leading cause of preventable blindness. Previous research demonstrates that epithelial cells release nucleotides that activate purinergic receptors P2X7 and P2Y2, which induces cell‐cell calcium mobilization. This calcium mobilization is reported to potentially play a role in collective cell migration and wound healing. To better understand this specific mobilization, we studied pannexin, a channel forming glycoprotein. ATP is able to move through the Pannexin channel and binds with P2X7. We want to investigate the mechanism of the calcium signal propagation and whether pannexins are involved. We hypothesize that pannexin channels mediate cell‐cell calcium mobilization and regulates actin activity in response to injury or agonist stimulation. Experiments were performed using 10Panx, an inhibitory peptide for pannexin channels, and scramble Panx, a control peptide. Human Corneal Limbal Epithelial (HCLE) cells were cultured to confluence on glass bottom dishes. Twenty‐four hours prior to experimentation, growth supplements were removed. Cells were preincubated with either 100uM 10Panx or 100uM scramble Panx to inhibit pannexin channels. In order to visualize the change in calcium, cells were pre‐loaded with 5 μM Fluo‐3AM, a fluorescent dye, at a final concentration of 1% (v/v) DMSO and 0.02% (w/v) pluronic acid for 20 minutes at 37°C and 5% CO2. Images were collected every 3 seconds on a Zeiss Axiovert LSM 880 confocal microscope for a period of 45 minutes after either agonist stimulation or injury. The agonist used for stimulation was BzATP, a synthetic nucleotide that selectively activates P2X7 and UTP, an agonist for P2Y2. Baseline and post‐wound frames were captured. Analysis was performed using FIJI/ImageJ and MATLAB programs. We found that 10PanX correlated with dampened calcium mobilization after injury. In uninjured cultures the decrease was only detected in response to BzATP and not to UTP. Actin activity was also inhibited by 10PanX after injury. MATLAB analysis using heatmap indicates that 10PanX reduces correlation of cells during calcium mobilization (Scr BzATP mean: 0.030 10PanXBzATP mean:0.079). Our data indicates that P2X7‐mediated Pannexin channels play a role in the injury response. This provides greater insight as to the level of involvement of pannexin in calcium signal propagation in response to injury in HCLE cells.Support or Funding InformationNIH 5R25HL118693‐05, NIH RO1 EY06000, NIH R21 EY024392, The Mass Lions Foundation New England Corneal Transplant FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.