bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting hepatic thioredoxin reductase by the acetaminophen metabolite N‐acetyl‐p‐benzoquinone (844.15)
Authors: Jan Yi‐Hua , Dragomir Ana‐Christina , Gardner Carol , Heck Diane , Laskin Debra , Laskin Jeffrey
Published: June 2021

Acetaminophen is biotransformed by cytochrome P450's in hepatocytes to an electrophilic metabolite, N‐acetyl‐p‐benzoquinone imine (NAPQI). This reactive species is known to bind to glutathione and a variety of hepatic proteins, an important downstream event mediating acetaminophen hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a critical cellular antioxidant which contains a selenocysteine in its C‐terminal redox center, an accessible target for electrophilic modification. In the present studies, we characterized the reaction of NAPQI with TrxR. Acetaminophen treatment of mice (300 mg/kg, i.p.) caused a marked time‐dependent inhibition of the activities of both cytosolic TrxR1 and mitochondrial TrxR2. Using purified rat liver TrxR1, we found that inactivation of the enzyme was correlated with APAP metabolism by P450s. This suggested that enzyme inhibition was a result of the formation of reactive metabolites of acetaminophen. To examine the mechanism underlying the inhibitory actions of acetaminophen, the effects of NAPQI on purified TrxR were characterized. We found that NAPQI‐mediated enzyme inhibition required reduced TrxR. A recombinant mutant enzyme, in which selenocysteine was replaced with cysteine was only weakly inhibited by NAPQI indicating that selenocysteine residues in the redox motifs of the enzyme are critical for enzyme inactivation. This was supported by our findings that alkylation of TrxR with biotin‐conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. Moreover, LC‐MS/MS analysis confirmed that NAPQI selectively modified cysteine 59, cysteine 497 and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. Collectively, these data demonstrate that reactive intermediates of acetaminophen can target TrxR demonstrating a novel mechanism by which this analgesic induces oxidative stress and hepatotoxicity.

Grant Funding Source: Supported by AR055073, ES004738, CA132624, and ES005022.
The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen (APAP) toxicity in mouse hepatocytes (844.16)
Authors: Banerjee Sudip , Melnyk Stepan , Letzig Lynda , James Laura , Hinson Jack
Published: June 2021

3‐Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of APAP correlates with toxicity. Nitration is by peroxynitrite (ONOO‐), a reactive nitrogen species formed from nitric oxide (NO) and superoxide (O2‐). We postulated that APAP toxicity is mediated by peroxynitrite. Since iNOS knockout mice are equally susceptible to APAP toxicity as wildtype mice we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10µM) (N‐[(4S)‐4‐amino‐5‐[(2‐aminoethyl) amino] pentyl]‐N’‐nitroguanidine tris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from B6C3F1 mouse were incubated with/without APAP (1mM). Every 0.5h 1million cells were removed and assayed spectrofluorometrically for NO and superoxide production using diaminofluorescein (DAF) and Mitosox, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO and acetaminophen‐cysteine (APAP‐Cys) adducts were measured by HPLC‐EC. APAP significantly increased cytotoxicity at 1.5‐3.0h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or covalent binding which indicated that NANT did not inhibit metabolism. APAP significantly increased NO and superoxide at 0.5 and 1.0h, respectively. The increases in both NO and superoxide were blocked by NANT. APAP significantly increased 3NT at 1.5‐3.0h and the increase was blocked by NANT. The data support the hypothesis that APAP hepatotoxicity is mediated by reactive nitrogen formed by nNOS.

Grant Funding Source: NIH RO1 DK079008
Nicotinic acetylcholine receptors: expression demonstrates functional diversity in parasitic nematodes (844.4)
Authors: Buxton Samuel , Charvet Claude , Neveu Cedric , Robertson Alan , Martin Richard
Published: June 2021

Nicotinic acetylcholine receptors mediate fast neurotransmission in both vertebrates and invertebrates. Selective targeting of parasite nAChR by antiparasitic drugs requires functional and/or structural differences between the receptors of the host and the parasite. Anti‐parasitic drugs like levamisole, pyrantel, tribendimidine, and derquantel (cholinergic anthelmintics) selectively target the nAChRs of nematodes. Unfortunately, regular use of anthelmintics has resulted in increasing reports of resistance in livestock parasites. Our aim has been to understand the molecular pharmacology of the nAChRs in parasitic nematodes, to inform development of better anthelmintics. We have cloned homologues of the nAChR subunit genes unc‐29, unc‐38, unc‐63 and acr‐8 from Oesophagostomum dentatum, a pig GI parasite. By employing the Xenopus laevis oocyte expression system, we demonstrated that four, pharmacologically different receptor subtypes can be reconstituted with various combinations of the four subunits. Single‐channel properties of one of the reconstituted receptor subtypes matched with one of the four subtypes recorded in vivo from O. dentatum, demonstrating the physiological relevance of the reconstituted receptor subtypes. Comparing these results with reconstitution results from other nematodes demonstrates the diverse, plastic nature of nAChRs in parasitic nematodes.

Grant Funding Source: Supported by NIH R01 AI047194‐12, R21AI092185‐01 and Chateaubriand Fellowship
Calpain mediates PDGF‐induced Akt phosphorylation via TGFβ1 in pulmonary artery smooth muscle cells (847.1)
Authors: Abeyrathna Prasanna , Su Yunchao
Published: June 2021

Calpain cleaves and activates transform growth factor β1 (TGFβ1) and mediates platelet‐derived growth factor (PDGF)‐induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs). Akt is downstream of PDGF receptor and participates in the collagen synthesis and cell proliferationof PASMCs. In this study, we tested whether calpain mediates PDGF‐induced Akt activation. PASMCs were incubated with PDGF in the presence and absence of Akt inhibitor triciribine, calpain inhibitor MDL23170, and TGFβ receptor inhibitor SB431542, as well as siRNA against calpain‐1 or ‐2. Calpain activity, the protein levels of active TGFβ1, phospho‐Akt (S473) and collagen‐I, and cell proliferation were measured. We found that incubation of PASMCs with PDGF resulted in increases in calpain activity, active TGFβ1, phospho‐Akt (S473), collagen‐I, and cell proliferation. Triciribine reduced PDGF‐induced increases in collagen‐I and cell proliferation. MDL23170 and siRNA against calpain‐2 other than calpain‐1 prevented PDGF‐induced increases in phospho‐Akt (S473), collagen‐I, and cell proliferation. Moreover, SB431542 blocked PDGF‐induced increases in phospho‐Akt (S473), collagen‐I, and cell proliferation. These results indicate that PDGF‐induced phosphorylation of Akt is attributed to TGFβ1 activation caused by calpain‐2.
Consumption of diets high in fat and/or sucrose enhances the locomotor stimulatory effects of cocaine in male and female C57Bl/6J mice (848.7)
Authors: Collins Gregory , Chen Edward , Rush Elise , Chris Tcshumi , Koek Wouter , France Charles
Published: June 2021

Drug abuse and obesity are serious public health problems. Dopamine systems play a central role in mediating the reinforcing effects of drugs and food, and the prolonged use of drugs or consumption of palatable food is known to alter the function and/or sensitivity of many neurotransmitter systems, including dopamine systems. The current studies evaluated the impact of consuming a high‐fat diet and/or a 10% sucrose solution on the sensitivity of adult mice (8 males and 8 females per condition) to the locomotor effects of cocaine (3.2‐32.0 mg/kg) across four weekly tests. The acute locomotor stimulatory effects of cocaine were enhanced in both male and female mice that consumed diets high in fat and/or sucrose, relative to mice that consumed standard chow and water. Although these diet‐induced enhancements generally persisted with repeated cocaine testing, sensitization to the locomotor effects of cocaine developed more rapidly in female mice drinking sucrose (and consuming either standard or high‐fat chow), relative to sex‐matched mice consuming standard chow and water; mice from all other feeding conditions developed sensitization at a rate comparable to their standard chow and water counterparts. The results of these studies provide clear evidence that consuming diets high in fat and/or sugar enhances cocaine effects in ways that might increase vulnerability to abuse cocaine.

Grant Funding Source: Supported by K05 DA017918 and a Zannoni (SURF) Fellowship through ASPET
Nanog regulation of endothelial asymmetric cell division (851.20)
Authors: Baruah Jugajyoti , Wary Kishore
Published: June 2021

Objective: To test whether inhibition of Glycogen Synthase Kinase‐3 (GSK‐3) enzyme or GSK‐3α/β‐ knockdown, induces Asymmetric Cell Division (ACD) in endothelial cells (ECs). Accordingly, we test the hypothesis that a subset of mature ECs retain the ability to divide unequally, a process called asymmetric cell division (ACD) and this event requires the activation of canonical Wnt signaling pathway.

Methods and Results: Stimulation of cultured ECs with 6‐bromoindirubin‐3‐oxime (BIO) or shRNA mediated GSK‐3α/β‐knockdown stabilized β‐catenin and increased expression of NANOG, thereby increased proliferation as well as migration of these cells in culture. Accordingly, BIO stimulation and 5‐bromo‐2'‐deoxyuridine (BrdU) pulse labeling of ECs followed by microscopy showed increased ACD in a subset of BrdU‐positive cells. The immunostaining of BrdU positive ECs (stalk‐cells) showed expression of NOTCH‐1 (NICD) and DLL4 (tip‐cells). Immunohistochemistry and biochemical analyses indicated altered expression of NOTCH and NUMB in asymmetrically dividing cells. Western blot analyses confirmed increased expression of NOTCH‐1, DLL4, NUMB and JAM‐A, protein differentially expressed during ACD, in response to BIO stimulation. In a converse experiment, NANOG‐knockdown carried‐out in ECs after receiving BIO showed decreased ACD, while control knockdown had no effect. Interestingly, re‐expression of VEGFR‐2/Flk1 cDNA into NANOG‐depleted ECs partially restored the ability of these cells to divide asymmetrically.

Conclusion: Thus, our data indicate that the activation of canonical Wnt signaling induces ACD in a subset of ECs, an event that may partly be responsible for mediating EC dedifferentiation and regeneration.

Grant Funding Source: Supported by American Heart Association Grant to KKW (12GRNT12070159).
Measurement of Evans blue dye concentration in central and peripheral tissue following administration in vitro and in vivo: dose‐ and replicate‐dependent effects on absorbance, fluorescence, and extraction by trichloroacetic acid (851.3)
Authors: Wang Hwai‐Lee , Lai Ted
Published: June 2021

The Evans blue dye (EBD) is the most commonly used inert tracer for tracking endogenous plasma protein extravasation into the brain. Even though several methods have been developed for measuring EBD concentration extravasated into central and peripheral tissues, the accuracy and precision of these methods remains poorly defined. First, we measured the absorbance and fluorescence of EBD solutions prepared in 96‐well plates using standard photo‐spectrometer. EBD absorbance increased in a dose‐dependent manner, and logarithm‐transformation revealed a sigmoidal curve with Hill coefficient of 2.086. The steepest slope of the curve lied between 5 µg/ml to 500 µg/ml, and hence this concentration range was the region at which measurements would be most precise. On the other hand, EBD fluorescence also increased in a concentration‐dependent manner, but with region of precision from 0.2 µg/ml to at least 5 µg/ml. Nevertheless, substantial photo‐bleaching of EBD fluorescence was found with increasing concentrations. Thus, our results recommends the use of fluorescent measurements to define EBD concentrations between 0.2 µg/ml to 5 µg/ml, and the use of absorbance measurements to define EBD concentrations between 5µg/ml to 500 µg/ml. Second, we determined the efficiency at which EBD can be retrieved from plasma proteins using the trichloroacetic acid (TCA) extraction technique. The degree of extraction was substantial even with 1:1 plasma‐to‐TCA ratio, and reached maximum with 1:2 ratio where increased ratios failed to further increase EBD extraction. Notably, contrary to earlier reports, we failed to completely extract EBD from plasma sample even with substantial amount of TCA. Third, we determined the sample‐to‐TCA ratio required to maximally extract EBD from central and peripheral tissue in rats receiving intravenous EBD. Taken together, the data presented here will be of great value in guiding future research using EBD as a tool to study central and peripheral vascular permeability.

Grant Funding Source: Supported by the National Science Council of Taiwan
Differential role for STIM1 in the regulation of vascular function (851.7)
Authors: Kassan Modar , Zhang Wei , Ait Aissa Karima , Stolwijk Judith , Feske Stefan , Trebak Mohamed , Matrougui Khalid
Published: June 2021

Background: The aim of this study was to determine the role of stromal interacting molecule‐1 (STIM1) in the regulation of vascular function using tissue‐specific STIM1 knockout mice either in endothelial cells or in smooth muscle cells.

Methods & results: We used male C57/BL6 homozygous and heterozygous mice lacking STIM1 specifically in either smooth muscle cells (STIM1SMC‐/‐, STIM1SMC‐/+) or in endothelial cells (STIM1EC‐/‐, STIM1EC‐/+). Systolic blood pressure and glucose levels were similar in all mice, but body weight was significantly reduced in STIM1EC‐/‐ and STIM1SMC‐/‐ mice. The contraction of resistance and conductance arteries in response to phenylephrine was significantly reduced in STIM1SMC‐/‐ mice only. However, contraction to thromboxane and potassium chloride was similar in all groups. The endothelium‐dependent relaxation was significantly impaired in STIMEC‐/+ and drastically reduced in STIM1EC‐/‐ mice while the endothelium‐independent vasorelaxation was similar among all groups. Acute down regulation of STIM1 in resistance arteries reduced endothelium‐dependent relaxation to acetylcholine and the contractile response to phenylephrine, while the contractile response to thromboxane was not affected. NADPH oxidase activity was only increased in STIMEC‐/+ and STIMEC‐/‐ mice. Calcium Ca2+ entry in microvascular endothelial cells stimulated with thrombin and histamine, two vasoactive compounds that cause NO production, had the pharmacological features of store‐operated Ca2+ entry (SOCE) and was dependent on STIM1 expression.

Conclusions: STIM1 plays opposing roles in vascular smooth muscle vs. endothelial cells in the regulation of vascular reactivity. We conclude that STIM1 is a critical factor in the basic mechanisms of vascular endothelium‐dependent relaxation and the contractility in response to sympathetic activity but not in the intrinsic contractility of smooth muscle.
A novel estimate of endothelial cell autophagic flux is associated with greater vascular endothelial function and reduced oxidative stress in healthy middle‐aged/older adults (855.3)
Authors: van Boekel Anna , Santos‐Parker Jessica , LaRocca Thomas , Seals Douglas , Kaplon Rachelle
Published: June 2021

Using only indirect, static markers of the dynamic cellular quality control system, autophagy, we have reported impaired autophagy in biopsied vascular endothelial cells (EC) of humans with aging that is related to endothelial dysfunction, as indicated by reduced endothelium‐dependent dilation (EDD), and oxidative stress. Here, we estimated dynamic autophagic flux in EC from 10 healthy adults aged 51‐73 by calculating the within‐subject difference in the expression of microtubule‐associated protein light chain 3 (LC3 ratio) between ECs incubated in the absence vs. presence of chloroquine (50 μM 1.5 hrs) to inhibit autophagy. EDD was assessed by: 1) brachial artery flow‐mediated dilation (FMD); and 2) forearm blood flow (FBF) to incremental brachial artery infusions of acetylcholine. Oxidative stress‐suppression of EDD was determined by ∆FBF to co‐infusion of vitamin C (vitC). The LC3 ratio was positively related to FMD (r=0.76, P<0.05) and FBF (r=0.56, P<0.05), but not endothelium‐independent dilation to NO donors (both P>0.05). The LC3 ratio also was inversely related to ∆FBF with vitC (r=‐0.57, P<0.05). These findings provide novel evidence that greater EC autophagic flux is associated with improved endothelial function linked to reduced oxidative stress in healthy middle‐aged/older adults.

Grant Funding Source: NIH AG044031 TR001082 AG013038
Regulation of the epithelial sodium channel by specific palmitoyltransferases (DHHC) (860.6)
Authors: Mukherjee Anindit , Poland Paul , Wang Zhijian , Kleyman Thomas , Hughey Rebecca
Published: June 2021

The epithelial sodium channel (ENaC) has a key role in the maintenance of extracellular fluid volume and control of blood pressure. ENaC is a member of the ENaC/degenerin family and is assembled with three structurally similar subunits (alpha, beta and gamma). We have shown that palmitoylation of specific cytoplasmic Cys residues in the beta and gamma subunits activate mouse ENaC by increasing channel open probability. In assessing channels that lack palmitoylation sites on the beta and/or gamma subunit, our data indicate that gamma subunit palmitoylation has a dominant role in activating ENaC. There is a family of 23 palmitolytransferases (referred to as DHHCs) in mice that catalyze the transfer of palmitate to cytoplasmic and transmembrane Cys in proteins. To directly assess the physiological significance of ENaC palmitoylation, we co‐expressed ENaC individually with all 23 palmitoyltransferases to identify specific palmitoyltransferases that can activate ENaC in Xenopus oocytes. We observed that five DHHCs significantly increased (1.8‐2.5 fold p < 0.05 ‐ 0.001) wild type channel activity. Co‐expression of these activating DHHCs with ENaC lacking Cys for either beta or gamma subunit palmitoylation revealed one DHHC with specificity for the gamma subunit. Using RT‐PCR we found that all five of the activating DHHC are present in mpkCCDc14 cells that express endogenous ENaC providing a model system to study regulation of ENaC palmitoylation. Results of our studies provide new insights regarding a regulatory pathway that has a large effect on the modulation of channel open probability.

Grant Funding Source: supported by NIH 5R01DK065161‐09
Regulation of SERCA2a by chronic ethanol: role of Akt (864.8)
Authors: Jeffress Miara , Umoh Nsini , Haddad Georges
Published: June 2021

Alcoholic cardiomyopathy is one of the leading causes of non‐ischemic dilated cardiomyopathy accounting for 36% of cases. Almost 50% of alcoholics develop this disease. Previously our lab has demonstrated the up‐regulation of intracellular Ca2+ (IC) in dilated cardiomyopathy by Akt. Here we investigate the role of Akt in regulating Ca2+ cycling proteins and contractility in chronic ethanol exposed cardiocytes. Parallel measures of IC and sarcomere/cellular contractions were assessed (Ionoptix) with the expression level (western blot) of Ca2+ handling proteins (sarcoplasmic reticulum calcium ATPase(SERCA2a), phospholamban(PLN), Na+‐Ca2+ exchanger (NCX) and L‐type Ca2+ channels(LTCC)) from control, low (LA: 0.02%), and high (HA: 0.45%) alcoholic rat hearts (4 months on Lieber DiCarli liquid diet), with and without Ad.EGFP and Ad.Akt transfection. Our results show an inotropically damaging increase in IC with HA but not with LA. In parallel, NCX protein expression is diminished in HA whereas that of LTCCs is increased. In addition, SERCA2a levels are increased in LA and decreased in HA. Upon Ad.Akt transfection SERCA2a levels increased in HA. Protein levels of phosphorylated PLN also increased in LA and decreased in HA hearts. This suggests that chronic ethanol elevates IC to harmful levels through cytoplasmic trapping via NCX which is exacerbated by reduced SERCA2a through PLN in an Akt‐dependent manner.

Grant Funding Source: Supported by NIH/NIAAA/1R15AAA019816‐01A1 and 2G12 RR003048
Ebselen prevents the carotid body chemosensory potentiation and reverses the hypertension induced by intermittent hypoxia (873.1)
Authors: Moya Esteban , Arias Paulina , Carlos Varela , Oyarce Maria , Iturriaga Rodrigo
Published: June 2021

Oxidative stress is associated with hypertension and enhanced carotid body (CB) chemosensory responses induced by chronic intermittent hypoxia (CIH), but the underlying mechanism is not fully understood. We tested if ONOO‐ is involved in the CB enhanced chemosensivity. Accordingly, we studied the effects of Ebselen, a specific ONOO‐ scavenger, on 3‐nitrotirosine immunoreactivity (3‐NT‐ir) in the CB, the chemosensory discharge and arterial blood pressure (BP) in rats exposed to CIH. Male Sprague‐Dawley rats were exposed to CIH (5% O2, 12 times/h, 8 h/day), BP was measured with telemetry and Ebselen was administrated using subcutaneous osmotic pumps (10 mg/Kg day). At the end of the experiments, CB chemosensory discharge was recording, and rats were perfused with parafolmaldehyde 4% for 3‐NT‐ir. CIH increased 3‐NT‐ir in the CB, enhanced the chemosensory responses to hypoxia, elevated BP during normoxia and potentiated the BP responses to hypoxia. Ebselen prevented the CIH‐induced CB chemosensory potentiation, and reversed the elevated BP and the potentiated pressor responses to hypoxia. Our results showed that 3‐NT formation caused by CIH, contributes to the CB chemosensory potentiation, and that ONOO‐ is involved in the CIH‐induced hypertension.

Grant Funding Source: Supported by FONDECYT 1100405
Cytokine‐mediated regulation of catecholamine biosynthesis in adrenal chromaffin cells (876.5)
Authors: Byrne Collin , Khurana Sandhya , Peltsch Heather , Kumar Aseem , Tai T.C.
Published: June 2021

Catecholamines, neurohormones with extensive functions in the sympathetic regulation of blood pressure, have a well‐established role in cardiovascular disease and hypertension. Epidemiological data has shown that, in addition to elevations in the catecholamine adrenaline, hypertensive patients demonstrate altered plasma cytokine profiles, suggesting that hypertension entails a complex interplay of the neuroendocrine and immune systems. From preliminary work, we have found that spontaneously hypertensive rats (SHR) exhibit higher plasma levels of the cytokine CCL2, and decreased levels of interleukin (IL)‐1β and interferon (IFN)‐δ, compared to their normotensive counterparts. To delineate the relationship between cytokine‐mediated signalling and catecholamine synthesis, we tested a panel of cytokines for their ability to affect the expression of tyrosine hydroxylase (TH), dopamine beta hydroxylase (DBH) and phenylethanolamine N‐methyltransferase (PNMT), key enzymes for catecholamine biosynthesis. Adrenal medulla derived PC12 cells were treated with the cytokines IL‐1β, IL‐2, IL‐6, IL‐10, tumor necrosis factor‐α, IFN‐α, transforming growth factor‐β1, or CCL2, and transcriptional changes were analyzed using RT‐PCR. Alterations in the transcripts of TH, DBH and PNMT upon cytokine treatments were observed illustrating cytokine‐mediated regulation of catecholamine biosynthesis.

Grant Funding Source: Supported by NSERC and CIHR
Inhibition of thiazide‐sensitive sodium chloride cotransporter phosphorylation provokes a global compensatory response (892.17)
Authors: Grimm P , Wall Susan , Delpire Eric , Wade James , Welling Paul
Published: June 2021

We hypothesize an adaptive transport process preserves salt balance when activation of the thiazide‐sensitive sodium‐chloride cotransporter (NCC) is impaired, as in Gitelman Syndrome. Here, we describe such a compensatory mechanisms taking place in SPAK kinase null mice, which have a Gitelman‐like phenotype owing to the loss of NCC phospho‐activation. Global transcriptional profiling by microarray, together with real‐time PCR, western blot analysis, immunolocalization and metabolic assays were statistically quantified to reveal that an intercalated cell (IC) reprogramming pathway synergizes with a α‐keto‐glutarate (α‐KG) paracrine signaling system to augment sodium chloride reabsorption in distal nephron. Acid secreting α‐IC are replaced with chloride reabsorbing pendrin positive IC (PPIC). Coordinate transcriptional activation of a multi‐gene transport system in PPIC, involving solute carriers (Slc26a; Slc4a8; Slc4a9), carbonic anhydrase 2 and 15, and V‐type H+‐ATPase subunits, creates a new electroneutral salt reabsorption pathway. In the proximal tubule, glutamate and α‐KG carriers (Slc3a1, Slc13a2, Slc38a3) increase while α‐KG metabolizing enzymes (Odc1 and Got2) decrease, causing [α‐KG] to increase in the tubular fluid. The GPCR (Oxgr1) for α‐KG increases on the PPIC apical surface, allowing paracrine delivery of α‐KG to stimulate the transport pathway. This study identifies the induction of an integrated compensatory NaCl reabsorption mechanism in PPIC, which may provide new molecular targets for diuretics.

Grant Funding Source: Supported by RO1DK093501
Transcriptional regulation of renal organic anion transporter 1 by B‐cell CLL/lymphoma 6 (892.33)
Authors: Henjakovic Maja , Wegner Waja , Burckhardt Birgitta , Burckhardt Gerhard
Published: June 2021

The human organic anion transporters 1 (OAT1) is crucial for the excretion of organic anions in renal proximal tubular cells and was classified as clinically relevant transporter in the kidneys. Our previous study indicated that renal male‐predominant expression of rat Oat1 and Oat3 appears to be regulated by transcription factor B‐cell CLL/lymphoma 6 (BCL6). The aim of this study was to characterize the effect of BCL6 on human OAT1 promoter.

Luciferase assays were carried out on Opossum kidney (OK) cells transiently transfected with promoter constructs of OAT1 and expression vectors for BCL6. Transcription factor (TF) activation profiling plate array was used for monitoring the activation of multiple transcription factors in nuclear extracts of pcDNA3‐BCL6 and pcDNA3 transfected OK cells. The protein expression of several transcription factors was investigated using fluorescence microscopy and Western blot.

BCL6 enhanced the promoter activity of OAT1, independently of predicted BCL6‐binding sites. Promoter sequence ‐63 to +1 bp of OAT1, which contained HNF1a binding sites, is the minimal regions required for BCL6 dependent activation. BCL6 enhanced the protein expression of HNF1α, a known OAT1 activator. HNF1α dependent activation of OAT1 promoter was increased by BCL6.

BCL6 constitutes a promising candidate gene for the regulation of human OAT1 transcription, possibly via activation of HNF1α.
Identification of an accessory protein necessary for the activation of the Na+/glucose cotransporter 2 (892.37)
Authors: El Tarazi Abdulah , Coady Michael , Bissonnette Pierre , Bichet Daniel , Lapointe Jean‐Yves
Published: June 2021

The Na+/glucose cotransporter SGLT2, which accounts for over 90% of renal glucose reabsorption, has become a major pharmaceutical target for type 2 diabetes treatment. Unfortunately, functional studies on SGLT2 have been hindered due to its lack of activity when expressed heterologously. Using an expression cloning strategy to identify a required accessory protein for SGLT2 function, we have identified MAP17 (membrane associated protein of 17 kDa) which is specifically expressed in the kidney proximal tubule, along with SGLT2. Using electrophysiology (oocytes) and radioactive uptakes (oocytes/OK cells), we demonstrate that MAP17 increases the activity of SGLT2 in both oocytes (150 fold) and OK cells (15 fold). Furthermore, Western blot and immunofluorescence studies using FLAG‐SGLT2 show that the cell surface density for SGLT2 is independent of MAP17 expression, which suggests that MAP17 stimulates the activity of SGLT2, but not its targeting. Finally, functional assays of disease‐causing mutations to SGLT2 (familial renal glucosuria) expressed in OK cells confirm these mutations to be non‐functional, even in the presence of MAP17. In conclusion, we have identified a cofactor responsible for the activation of SGLT2 which enables its characterization in heterologous systems.
Role of mitochondrial fission and fusion in cardiomyocyte contractility (893.10)
Authors: Givvimani Srikanth , Pushpakumar Sathnur , Metreveli Naira , Narayanan Nithya , Kundu Sourav , Veeranki Sudhakar , Chaturvedi Pankaj , Tyagi Suresh
Published: June 2021

Mitochondria constitute 30% of myocardial cell volume and are continuously engaged in two dynamic processes called fusion and fission which are regulated by Drp‐1(Dynamin related protein) and mitofusin protein‐2 (Mfn‐2) respectively. Previously, we showed that inhibition of Drp‐1 ameliorates cardiovascular dysfunction following pressure overload in an aortic banding model and myocardial infarction studies. As dynamic organelles, mitochondria are capable of changing their morphology in response to stress conditions. However, whether such changes can alter their function and in turn the cellular function is not known. Further, a direct role of mitochondrial fission and fusion on cardiomyocyte contractility has not yet been studied. In the present study, we hypothesized that modulating mitochondrial fission and fusion processes will alter cardiomyocyte contractility. To verify this, we used freshly isolated ventricular myocytes from wild type mouse hearts and transfected them with siRNA for DRP‐1 and Mfn‐2. Myocyte contractility studies were performed by IonOptix using a myopacer. Intracellular calcium and potassium measurements were done using flow cytometry. Immunocytochemistry was done to evaluate live cell mitochondria and its membrane potential sensitivity. Our results suggested that inhibition of mitofusion‐2 by siRNA significantly decreased the myocyte contractility suggesting the importance of fusion dynamics of mitochondria.

Grant Funding Source: HL108621, HL 074185
Voltage‐gated K+ channel, Kv3.3 is involved in hemin‐induced K562 differentiation (893.13)
Authors: Song Min Seok , Choi Seon Young , Ryu Pan Dong , Lee So Yeong
Published: June 2021

Voltage‐gated K+ (Kv) channels are well known to be involved in cell proliferation. However, even though cell proliferation is closely related to cell differentiation, the relationship between Kv channels and cell differentiation remains poorly investigated. This study demonstrates that Kv3.3 is involved in K562 cell erythroid differentiation. Down‐regulation of Kv3.3 using siRNA‐Kv3.3 increased hemin‐induced K562 erythroid differentiation through decreased activation of signal molecules such as p38, cAMP response element‐binding protein, and c‐fos. Down‐regulation of Kv3.3 also enhanced cell adhesion by increasing integrin β3 and this effect was amplified when the cells were cultured with fibronectin. The Kv channels, or at least Kv3.3, appear to be associated with cell differentiation; therefore, understanding the mechanisms of Kv channel regulation of cell differentiation would provide important information regarding vital cellular processes.
Analysis of OSR1 kinase‐peptide binding substrate interaction through computational protein docking software (893.37)
Authors: Austin Thomas , Nannemann David , Meiler Jens , Delpire Eric
Published: June 2021

The oxidative‐stress‐responsive kinase 1 (OSR1) and the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na+‐K+‐2Cl‐ cotransporters (NKCC1‐2) and Na+‐Cl‐ cotransporter (NCC). Both kinases have a conserved carboxyl‐terminal (CCT) domain, which recognizes a unique peptide (Arg‐Phe‐Xaa‐Val) motif present in OSR1‐ and SPAK‐activating kinases [with‐no‐lysine kinase 1 (WNK1) and WNK4] as well as their substrates (NKCC1, NKCC2, and NCC). In this study, various modalities of the Rosetta Software Suite including flexible peptide docking and protein design were utilized to predict several constructed hexapeptides that would favorably bind to the CCT domain after individual residue mutation or whole hexapeptide design. These constructs are all derived from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide (Gly‐Arg‐Phe‐Gln‐Val‐Thr) derived from WNK4 (2v3s). Among the favorable point mutations include an arginine to histidine substitution at position 2 and a phenylalanine to tyrosine substitution at position 3 of the hexapeptide. In addition, the designed hexapeptides FRFEVT (Phe‐Arg‐Phe‐Glu‐Val‐Thr) and TRFDVT (Thr‐Arg‐Phe‐Asp‐Val‐Thr) were both predicted to bind to the CCT domain. The results of these computations were confirmed through the use of yeast two‐hybrid screening and frequencies of computationally derived motifs were calculated in known proteins sequence by searching the NCBI mouse protein database. Our study show that the Rosetta Molecular Sotware Suite can very precisely model the binding of the GRFQVT peptide to the CCT/PF2 domain of OSR1 and our in silico mutagenesis study shows that the nature of the residues required for binding might be more flexible than previously recognized.

Grant Funding Source: RO1 DK0935010, RO1 GM074771
RyR1 mutation associated with malignant hyperthermia facilitates catecholaminergic stress‐included arrhythmia via mitochondrial injury and oxidative stress (893.8)
Authors: O‐Uchi Jin , Porter George , Kang Sung Hyun , Boncompagni Simona , Sokolova Niina , Gross Polina , Jhun Bong Sook , Beutner Gisela , Brookes Paul , Blaxall Burns , Dirksen Robert , Protasi Feliciano , Pan Shi , Sheu Shey‐Shing
Published: June 2021

Mutations of the type 1 isoform of the ryanodine receptor (RyR1) form leaky channels and exhibit inherited skeletal muscle disorders including malignant hyperthermia (MH). Cardiac arrhythmias and sudden cardiac death in MH patients are also frequently observed during anesthesia or even under the conscious condition, but the mechanism is still unknown. We hypothesize that chronic mitochondrial Ca2+ overload through leaky mutant RyR1 expressed at cardiac mitochondria incudes mitochondrial injury and oxidative stress, which leads to cardiac arrhythmia. We used the knock‐in mice carrying a RyR1 MH mutation Y522S (YS) and found that YS‐RyR1 was expressed at mitochondria but not at SR in heart. Heterozygous YS hearts showed disrupted mitochondrial morphology as well as compromised mitochondrial functions with high cellular oxidative state. In vivo measurements of cardiac functions showed that basal function of YS heart is maintained at the similar level of WT, but YS heart are insensitive to Isoproterenol (Iso)‐induced positive inotropy and chronotropy in vivo due to its higher basal levels of catecholamine signaling. Moreover, only YS heart frequently developed multiple ventricular extrasystoles in response to Iso bolus under ex vivo Langendorff perfusion. In summary, chronic mitochondrial damage by Ca2+ overload via leaky mutant RyR1 induces cellular oxidation, which facilitates catecholaminergic stress‐triggered arrhythmia.
Cardiac α1‐adrenergic signaling accelerates mitochondrial Ca2+ uptake, ROS generation and cell death signaling through Pyk2‐dependent phosphorylation of the mitochondrial Ca2+ uniporter (893.9)
Authors: O‐Uchi Jin , Jhun Bong Sook , Xu Shangcheng , Hurst Stephen Hurst , Raffaello Anna , Liu Xiaoyun , Yi Bing , Gross Polina , Ainbinder Alina , Kettlewell Sarah , Smith Godfrey , Dirksen Robert , Wang Wang , Rizzuto Rosario , Sheu Shey‐Shing
Published: June 2021

Mitochondrial Ca2+ homeostasis controls the balance of cell survival and death. The molecular identity of the mitochondrial Ca2+ uniporter (MCU) has been just reported and basal tyrosine phosphorylation of MCU was shown in mass spectroscopy of human tissues. However, the physiological and pathophysiological relevance of MCU post‐translational modifications are unknown. Here we reported that α1‐adrenoceptor (α1‐AR) signaling induces mitochondrial Ca2+ overload and reactive oxygen species (ROS) generation and activates cell death signaling through MCU tyrosine phosphorylation in cardiomyocytes. α1‐AR signaling activates mitochondria‐localized proline‐rich tyrosine kinase 2 (Pyk2) and accelerates mitochondrial Ca2+ uptake via Pyk2‐dependent MCU phosphorylation which enhances tetrameric MCU channel pore formation. Moreover, mitochondrial Ca2+‐overload thorough this signaling stimulates mitochondrial superoxide generation, permeability transition pore (mPTP) activity, and initiates apoptotic cell death in adult cardiomyocytes. Thus, α1‐AR‐Pyk2‐MCU signaling may become novel potent therapeutic targets for preventing mitochondrial Ca2+ overload, oxidative stress and cardiac injury under persistent adrenergic stimulation such as during heart failure.
Effect of nicotine exposure on cellular and molecular parameters of vitamin B1 (thiamin) uptake by pancreatic acinar cells (896.3)
Authors: Srinivasan Padmanabhan , Subramanian Veedamali , Kapadia Sameer , Said Hamid
Published: June 2021

Pancreatic acinar cells take in thiamin by a specific carrier‐mediated process that involve both THTR‐1 and THTR‐2 (encoded by SLC19A2 and SLC19A3, respectively). In this study we examined the effect of chronic exposure of mouse pancreatic acinar in vivo (wild‐type and transgenic mice carrying the SLC19A2 and SLC19A3 promoters) and human pancreatic acinar cells in vitro to the cigarette smoke component nicotine on physiological and molecular parameters of the thiamin uptake process. Chronic exposure of mice to nicotine (0.77 mM nicotine in drinking water for 4 wks) leads to a significant decrease in thiamin uptake, expression of THTR‐1 and ‐2 protein, mRNA levels and activity of the SLC19A2 and SLC19A3 promoters expressed in transgenic mice. A decrease in the level of expression of protein and mRNA of the thiamin‐metabolizing enzymes TPKase and thiamin pyrophosphatase (TPPase) was observed. Similarly chronic exposure of human pancreatic acinar cells to nicotine (0.5 µM, 48 h) leads to a significant inhibition in thiamin uptake, as well as in the level of expression of THTR‐1 and ‐2 protein and mRNA. These results demonstrate, for the first time, that chronic exposure of pancreatic acinar cells to nicotine negatively impacts the physiological and molecular parameters of thiamin uptake and that this effect is exerted, at least in part, at the level of transcription of the SLC19A2 and SLC19A3 genes.

Grant Funding Source: Supported by NIH grants AA 018071 and DK‐56061
IMP1 loss in intestinal epithelial cells promotes altered Paneth cell morphology and autophagy defects (899.2)
Authors: Lundsmith Emma , Hamilton Kathryn , Noubissi Felicite , Hahn Christopher , Spiegelman Vladimir , Rustgi Anil
Published: June 2021

Background Insulin‐like growth factor‐2 mRNA‐binding protein 1 (IMP1) is an oncofetal protein that exhibits high expression during embryonic development and lower expression in adult tissues. Mice with global Imp1‐/‐ gene deletion exhibit perinatal lethality due to altered gut morphology, suggesting a potential role in stem cell maintenance. We hypothesize that IMP1 contributes to intestinal homeostasis through modulation of the stem cell niche, specifically through a novel role in Paneth cells.

Methods Using immunohistochemistry (IHC), immunofluorescense (IF), and immunoblotting, we evaluated mice with intestinal epithelial‐specific loss of Imp1 (VillinCre;Imp1floxed mice) for Paneth cell morphology (lysozyme), and alterations in markers of autophagy (LC3 and p62).

Results VillinCre;Imp1floxed mice exhibit Paneth cell mislocalization and altered morphology. Furthermore, VillinCre;Imp1floxed mice exhibit increased LC3+ puncta and decreased p62 expression, representing up‐regulation in autophagic flux.

Conclusions Our data suggest that IMP1 may mediate intestinal homeostasis through a novel role in Paneth cells. The intersection of Paneth cell function and autophagy pathway components is an emerging area of interest in patients with inflammatory bowel diseases. As such, Villin‐Cre;Imp1fl/fl mice provide a compelling model for elucidating this relationship.

Grant Funding Source: Supported By HHMI Medical Fellowship (ETL), NIH grants (F32 DK093207‐01, RO1 DK056645‐12, UO1 DK0855
Kinetics and expression of copper reductases in hepatocyte and embryonic fibroblast cell lines (900.2)
Authors: Gaite Michaella , Nguyen Alex , Su Jason , Kidane Theodros , Linder Maria
Published: June 2021

We have demonstrated in prior studies that in mammals, Cu is delivered directly to the cells of various organs by interaction with the plasma proteins that carry it. Since Cu(II) is the main form bound to these proteins, and Cu uptake transporters transfer Cu(I), cell surface reduction would appear to be required and might be rate‐limiting. To begin to explore these concepts, we measured the kinetics of reduction and expression of potential Cu(II)/Fe(III) reductases in HepG2 cells and mouse embryonic fibroblasts that do and do not express Ctr1. Cu(II) reduction rates were measured with whole cells by following formation of Cu(I)‐BCS. Vmax was 3x higher in the hepatocytes vs. fibroblasts (~1 vs. 0.36 nmol/min/mg cell protein); Kms were also higher (~8 vs. 12 uM). Non‐competitive inhibition by Fe(III) was observed. At physiological (5 uM) Cu concentrations, reduction rates exceeded those for uptake. Kinetic plots suggested single reductases were involved. Quantitative Real Time PCR (relative to 18S rRNA) showed HepG2 cells to primarily express Steap 3 (and traces of Steap 2 and dCytb). The fibroblasts expressed Steap 4 almost exclusively. Although we will have to confirm and localize the reductases, our findings suggest that at least in the cell types examined, single but different reductases mediate uptake of Cu from its plasma carrier proteins, and reduction may not be rate‐limiting for uptake.
Role of glycine N‐methyltransferase in experimental ulcerative colitis (902.13)
Authors: Lee Kuan‐I , Chou Wen‐Yueh , Zhao Jing‐Feng , Chen Yi‐Ming , Lee Tzong‐Shyuan
Published: June 2021

Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with unclear etiology and mechanism(s). Glycine N‐methyltransferase (GNMT) plays a central role in inflammatory diseases such as hepatitis and atherosclerosis. However, little is known about the impact of GNMT and the involved mechanism in the pathogenesis of IBD. In current study, we investigated the role of GNMT in colitis with the mouse model of dextran sulfate sodium (DSS)‐induced colitis. GNMT was expressed in the epithelium of the colon under normal conditions, and with DSS treatment, its expression was predominant in infiltrated leukocytes of lesions. Mice with genetic deletion of GNMT (GNMT‐/‐) showed increased susceptibility to DSS induction of colitis, as revealed by the progression of colitis. Additionally, severe colonic inflammation, including increased crypt loss, leukocyte infiltration and hemorrhage, was greater with DSS treatment in GNMT‐/‐ than wild‐type mice. Furthermore, the expression of adhesion molecule and inflammatory mediators in the colon was significantly higher with DSS treatment in GNMT‐/‐ than wild‐type mice. Moreover, loss of GNMT decreased cell apoptosis in colitis lesions with DSS treatment. Collectively, our findings suggest that GNMT may be a crucial molecule in the pathogenesis of DSS‐induced colitis. This finding may provide new information for a potential therapeutic target in treating IBD.
Variations in the incidence of sacral hiatus in macerated human sacred: gender differences (915.4)
Authors: Garcia Arthur , da Silva Jairo , Fazan Valeria , Thomazini Jose Antonio
Published: June 2021

The sacral hiatus is a wide opening that arises through the absence of fusion blades S5 in the posterior midline, 5 cm above the distal end of the sacrum, which is used for administration of corticosteroids and other applications in the field of anesthesiology (Ganen, Hamaji, Carneiro, 2004). There are anatomical variations in the formation of the sacral hiatus and its lack can create difficulties in medical procedures described (NAJMAN et al, 2011). The aim of this work is to identify variations in the sacral hiatus in human cadavers Brazilian age, ethnicity and origin is not known. We evaluated 110 post‐exhumation sacral hiatus, 57 male and 53 female classifying them according to forms already known, “U”, “V” inverted bell shape, irregular, bifid, elongated and agenesis dorsal wall of the sacral canal (NAGAR, 2004). In males, 14% of the identified gaps in the shape of inverted “U”, 22.8% in the form of inverted “V”, 5.2% bell‐shaped, jagged 14%, 12.3% as bifida, 24 , 6% elongated and 7% agenesis. In females 24.5% identified as inverted “U”, 24.5% in the form of inverted “V”, 3.8% in bell shape, irregular 7.5%, 11.3% bifidus, 22 6% elongated and 5.7% agenesis. We emphasize that there are a variety of ways with the sacral hiatus significant percentage differences between genders, especially in inverted “U” and irregular, being more frequent in men and in women elongated shape the inverted “V”. It is evident that a morphometric evaluation will be needed to obtain data of clinical importance, applicable in anesthesiology. Other measures are being carried out to prove these alterations.
CAVE ő a proposed ontogeny ontology (917.1)
Authors: Cork R.
Published: June 2021

The Virtual Human Embryo (VHE, virtualhumanembryo.lsuhsc.edu) is a digital atlas that has become a canonical reference for the microscopic anatomy of human embryos. We propose to extend this concept to a variety of other species, with a project called CAVE (Comparative Atlas of Vertebrate Embryology). CAVE will include model species used for developmental biology research, as well as evolutionary significant species that can provide insights into the field of evolutionary developmental biology (evo‐devo). To allow researchers to search and compare these databases we are also proposing to construct a developmental anatomy ontology. This ontology will not only provide a space‐filling description of the anatomy at different time points during development but will also put the developmental anatomy into a temporal context that will enable the processes of development to be described. Thus, anatomical structures will be related to each other using relationships such as is_a and part_of, terms such as first_appears_at and disappears_at will describe the anatomical structures on a temporal axis, and terms such as develops_from and develops_into will be used to describe temporal relationships between anatomical structures. The ontology will be annotated with section images from the VHE and CAVE databases so that specific anatomical structures can be quickly located in the databases and compared across species.