bluuebunny/crossref_metadata_2025 huggingface dataset:
Autophagy‐enhancing therapy reduces oxidative stress and restores vascular endothelial function in old mice
Authors: LaRocca Thomas J , Henson Grant D , Sindler Amy L , Seals Douglas R
Published: June 2021

Aging results in vascular endothelial dysfunction caused by oxidative stress and reduced nitric oxide (NO) bioavailability. Autophagy (process of cell recycling of damaged biomolecules) is a key mediator of oxidative stress. We assessed ex vivo acetylcholine (ACh)‐induced endothelium‐dependent dilation (EDD) in adult C57/Bl6 mice: young (Y: 5 mo, n=5), old (O: 27 mo, n=5) and old treated with the autophagy enhancer trehalose (OT: 27 mo, n=5). Aging was associated with a ~40% reduction (P<0.05) in aortic expression of the autophagy markers beclin‐1 and LAMP‐2a, and impaired carotid artery EDD (Max EDD: Y: 96 ± 2%, O: 73 ± 3%, P<0.01) mediated by reduced NO bioavailability (ACh + L‐NAME). Trehalose (20 g/L drinking water, 4 weeks) increased aortic beclin‐1 and LAMP‐2a and restored EDD (OT: 95 ± 2%). TEMPOL, a superoxide scavenger, improved EDD in O (P<0.05), but not Y or OT. Consistent with this, aortic superoxide production (electron paramagnetic resonance) was ~150% greater in O vs. Y (P<0.01), but was similar to Y in OT. Compared with O mice, OT had ~30% greater expression of endothelial NO synthase (eNOS) and ~80% higher levels of SIRT1 and AMPK, key modulators of autophagy and eNOS activity (all P<0.05). Impaired autophagy may contribute to vascular endothelial dysfunction with age. Autophagy‐enhancing therapy restores endothelial function in old mice by reducing oxidative stress and increasing NO bioavailability.
Circulating matrix metalloproteinases levels and their inhibitors in clinical and experimental hypertension
Authors: Fontana Vanessa , Silva Pâmela Souza , Belo Vanessa Almeida , Antonio Raquel Carros , Ceron Carla Speroni , Biagi Celso , Gerlach Raquel Fernanda , Tanus‐Santos José Eduardo
Published: June 2021

The aim of this study was to evaluate circulating levels of metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of MMP (TIMP) and alpha‐2‐macroglobulin (A2M), in human and experimental hypertension. MMP‐2, MMP‐8, MMP‐9, TIMP‐1, TIMP‐2 and A2M plasma levels were determined in 38 untreated hypertensive patients and 33 normotensive subjects. We also assessed plasma levels of MMP‐9 and MMP‐2 and A2M in Spontaneously Hypertensive Rats (SHR) and in Wistar Kyoto (WKY) rats. We found no significant differences in MMP‐2, MMP ‐8, TIMP‐1, TIMP‐2 and A2M levels between the groups. However, hypertensives patients had higher levels of MMP‐9 (p≤0.05) than normotensives. In addition, MMP‐9/A2M and MMP‐8/A2M ratio are significantly higher in hypertension group. Similarly to human hypertension, no change in MMP‐2 and A2M plasma levels was observed in SHR compared to WKY. Also, significantly higher MMP‐9 levels and MMP‐9/A2M ratio were found in hypertensive group. Briefly, our findings show abnormal net plasma MMP‐9 activity in untreated hypertensive patients and in experimental hypertension, reinforcing the relevance of MMPs in pathophysiology of hypertension.

Financial Support: CNPq, FAPESP and CAPES
Effect of dimethylsulfoxide on hepatic HbNO formation in septic shocked rats
Authors: Chavez Francisco Javier Mendez , Rosales Daniel Ramirez , Ulloa Rafael Zamorano , Villanueva Cleva
Published: June 2021

It is known that dimethylsulfoxide (DMSO) scavenges superoxide. Oxidative stress as well as nitric oxide (NO) have a role on the pathophysiology of septic shock. Since NO and superoxide form peroxynitrite, the goal of the study was to evaluate the effect of DMSO on NO availability in septic shock. Septic shock was produced in male Wistar (230–250 g) rats through lipopolysaccharide (LPS) administration (8mg/kg) in the absence or presence of DMSO treatment (1 ul/g, subcutaneous, one hour before LPS). 3.5 h after LPS or isotonic saline solution (control) all the animals received a NO spin trapping. 30 min later animals were sacrificed by anesthesia overdose (pentobarbital) and a liver biopsy was frozen on liquid nitrogen. The adduct hemoglobin‐NO (HbNO) was detected through electron paramagnetic resonance (EPR) in 100 mg of liver. HbNO increased significantly in the samples of shocked rats. However the higher values of HbNO were found in samples isolated from septic rats treated with DMSO. It is concluded that DMSO increased NO availability in the liver of rats subjected to septic shock. The biological significance of DMSO effect remains to be established
Autophagy‐enhancing treatment reverses age‐associated large elastic artery stiffening and modulates arterial superoxide production, inflammation and collagen I
Authors: Henson Grant D , LaRocca Thomas J , Sindler Amy L , Fleenor Bradley S , Seals Douglas R
Published: June 2021

Aging leads to stiffening of large elastic arteries associated with increased superoxide (O2‐) production, inflammation and collagen protein. Impairments in autophagy, a process that recycles damaged cellular components and influences oxidative stress and inflammation, may contribute to age‐related arterial stiffening. We tested the hypothesis that treatment with the natural disaccharide trehalose, an autophagy inducer, would reduce aortic pulse wave velocity (aPWV), a clinically important measure of arterial stiffness, and decrease superoxide production, inflammation and collagen I in old (O) male C57/BL6 mice. Autophagy was impaired in aorta from O (28 mo, n=6) vs. young (Y: 5 mo, n=6) mice as indicated by a ~40% reduction (p<0.05) in beclin‐1 and LAMP‐2a, and these markers were restored by trehalose treatment (4 weeks, 20 g/L drinking water) in O mice. aPWV was higher in O vs. Y mice (471 ± 23 vs. 405 ± 25 cm/s, p<0.05), and trehalose reduced aPWV in O mice (pre: 478 ± 32 vs. post: 417 ± 18 cm/s, p<0.05). Trehalose treatment reversed age‐associated increases in aortic O2‐ production and inflammatory cytokines (tumor necrosis factor α and interleukin‐1), and reduced aortic collagen I by ~50%. Our results suggest that autophagy may be a promising therapeutic target for treating age‐associated large elastic artery stiffening, possibly by reducing O2‐/inflammation modulation of collagen.
Synthesis and Functional Analysis of a Structure‐Switching Aptamer with Homogeneous 3′‐ends
Authors: Baltis Ariel , Morse Daniel Paul
Published: June 2021

A structure‐switching signaling aptamer that could detect the antibiotic tobramycin was previously produced by a novel in vitro selection strategy. To facilitate the analysis of the mechanism by which this RNA detects tobramycin, the aptamer was synthesized in a manner that ensures that the 3′ ends of the RNA are homogeneous. This was done by incorporating a self‐cleaving ribozyme at the 3′ end. The resulting RNA will be subjected to enzyme and lead ion‐catalyzed hydrolysis to confirm that the ends are homogenous and to analyze the nature of the tobramycin‐induced conformational change.
Calcium Homeostasis in Vascular Smooth Muscle Cells is altered in Type 2 Diabetes by Bcl‐2 Protein Modulation of InsP3R Calcium Release Channels
Authors: Velmurugan Gopal Viswanathan , White Carl
Published: June 2021

The current study examines the extent to which antiapoptotic Bcl‐2 proteins Bcl‐2 and Bcl‐XL contribute to aortic vascular smooth muscle cell (VSMC) Ca2+ dysregulation and contractility in the db/db mouse model of type 2 diabetes. Expression levels of Bcl‐XL were found to be higher in tissues isolated from diabetic (db/db) mice compared to non‐diabetic (db/m) controls. In single cells, inositol 1,4,5‐trisphosphate receptor (InsP3R)‐dependent [Ca2+]i signaling was enhanced in db/db VSMCs. This was attributed to alterations in the intrinsic properties of the InsP3R because there were no differences between db/db and db/m in the steady state [Ca2+]SR or InsP3R expression levels. Moreover, in permeabilized cells the rate of InsP3R‐dependent SR Ca2+ release was increased in db/db compared to db/m, and attenuated only in db/db cells by the Bcl‐2 protein inhibitor ABT‐737. Experiments in which [Ca2+]i and constriction were recorded simultaneously in cannulated intact arteries, application of ABT‐737 similarly attenuated enhanced agonist‐induced [Ca2+]i signaling only in intact db/db vessels. In contrast, ABT‐737 had no effect on agonist‐evoked contractility in either db/db or db/m vessels. In conclusion, elevated Bcl‐XL in diabetic VSMCs increases InsP3R excitability, the resulting dysregulated [Ca2+]i signaling, however, does not correlate with increased vessel reactivity.
Impaired vascular responses to nitrite in hypertensive rat aortae
Authors: Machha Ajay , Murugan Dharmani Devi , Mustafa Mohd Rais , Schechter Alan N
Published: June 2021

Recent data demonstrate that nitrite is a physiological vasodilator, suggesting that impaired vascular responses to nitrite may be a causative factor for hypertension. However, little is known about the vasodilator effects of nitrite in hypertension. We investigated the relaxant effects of sodium nitrite in spontaneously hypertensive rat (SHR) aortae and the responses were compared to those of age‐matched normotensive Wistar Kyoto (WKY) rat aortae. Sodium nitrite elicited relaxation in WKY and SHR aortae, but with a lesser potency in SHR. In particular, sodium nitrite at concentrations that reflect plasma level of endogenous nitrite (< 1 μmol/L) elicited relaxation in WKY aortae but not in SHR aortae. Free radical scavenging with ascorbic acid normalized responses to sodium nitrite in SHR but had no effect in WKY. Nitric oxide synthase inhibition with Nω‐L‐arginine methyl ester abolished responses to lower concentrations, but not to higher concentrations (≥100 μmol/L), of sodium nitrite in WKY but had no effect in SHR rings. Cyclooxygenase inhibition with indomethacin marginally potentiated responses to sodium nitrite in WKY but markedly increased in SHR. These findings suggest that the relaxation responses to nitrite are impaired in SHR aortae, possibly coupled to increased production of free radicals. It is possible that this abnormality participates in the pathogenesis of hypertension.
Tissue‐specific metabolic regulations of respiration and ROS production of the heart, brain and spinal cord mitochondria
Authors: Panov Alexander , Dikalov Sergey , Dambinova Svetlana
Published: June 2021

In vivo mitochondria oxidize several metabolites simultaneously. Therefore, changes in metabolic condition associated with age (menopause in women) or disease (diabetes) may have distinct effect on mitochondria from different organs. We tested this hypothesis by analysis of heart (HM), brain (BM) and spinal cord (SCM) mitochondria isolated from young Sprague Dawley rats using Percoll purification step. Pyruvate, glutamate, malate, succinate, palmitoyl‐carnitine and their mixtures were used as substrates. Clark electrode and Amplex Red method were used to measure respiration and ROS generation. HM oxidized palmitoyl‐carnitine or succinate alone very poorly in State 3, and respiration was inhibited upon uncoupling. Combination of palmitoyl‐carnitine with any other mitochondrial substrate increased the rates of oxidative phosphorylation and uncoupled respiration by 40 to 70%, State 4 respiration by 64%, and ROS production increased 3–4 folds. BM and SCM did not oxidize palmitoyl‐carnitine. However, when palmitoyl‐carnitine was mixed with glutamate, pyruvate or succinate, the rates of ROS generation increased 4‐fold as compared with glutamate + malate.

Conclusions

Rat HM, BM and SCM show distinct substrate preferences for maximal rates of oxidative phosphorylation and ROS production. Thus changes in metabolic conditions or food supplements such as carnitine may affect oxidative stress in a tissue‐specific manner. Research was supported by RFFI grant N 09‐04‐01376.
Pitavastatin treatment normalizes VEGF signaling and cardiac function in STZ‐induced diabetic rat hearts
Authors: Jesmin Subrina , Mowa Chishimba , Zaedi Sohel , Sultana Sayeeda Nusrat , Yamaguchi Naoto
Published: June 2021

Abnormal alterations in cardiac expression of vascular endothelial growth factor (VEGF) as well as its receptors and impairment in the development of coronary collaterals have recently been reported in diabetic subjects. However, the effectiveness of pharmacological intervention on these diabetic defects remains unsettled. Here, we studied the effects of statin (pitavastatin) on cardiac VEGF signaling pathways and cardiac function in Sprague‐Dawley rats 5 wk after induction of type I diabetes with streptozotocin (65 mg/kg ip) in comparison with age‐matched control rats. After streptozotocin (1 wk), some diabetic rats were treated with pitavastatin (1 mg/day) for 4 wk. VEGF, its receptors, and its angiogenic signaling molecules [phosphorylated Akt and endothelial nitric‐oxide synthase (eNOS)] were analyzed by Western blot, ELISA, real‐time PCR, and immunohistochemistry, and cardiac function was evaluated by echocardiography. Coronary capillary morphology was assessed by lectin and enzymatic double staining. We found significant decreases in cardiac expression of VEGF, its receptors, phosphorylation of Akt and eNOS, and coronary capillary density in diabetic rats compared with controls. Treatment of diabetic rats with pitavastatin reversed these alterations to control levels and ameliorated impairment of cardiac function. The present study is the first to show the potential usefulness of a statin in the treatment of cardiac dysfunction in type I diabetes at the molecular level.
Aqueous extract of Ginger (Zingiber officinale) prevents metalaxyl fungicide induced hepatotoxicity in albino mice
Authors: Lamfon Hawazen Ahmad , sakr saber
Published: June 2021

The present work aims to study the hazardous effect of metalaxyl, an acylalanine fungicide, on the liver of albino mice and the possible role played by the aqueous extract of Ginger (Zingiber officinale) in attenuating the hepatotoxicity of metalaxyl. Animals were divided into 4 groups. Group 1: given metalaxyl at a dose level of 1/10 LD50 for 4 weeks, Group 2: given metalaxyl and ginger, Group 3: given ginger and Group 4: served as controls. Histopathological and biochemical observations of the liver function parameters and oxidative enzymes were carried out. Metalaxyl treatment induced many histological changes in the liver including congestion of blood vessels, cytoplasmic vacuolization of the hepatocytes, necrosis and fatty degeneration. Metalaxyl caused marked elevation in serum ALT and AST. It also caused an increase in malondialdehyde and depletion of the activity of the antoxidant enzymes, catalase and superoxide dismutase in the liver. Treating animals with metalaxyl and ginger extract led to an improvement in both the histological and biochemical alterations induced by metalaxyl. Moreover, ginger reduced the level of malondialdehyde and increased the activity of antioxidant enzymes, SOD and CAT. This preventive effect of ginger is attributed to its antioxidant and free radicals scavenging properties. The present work is supported by Umm Al‐Qura University and Menoufia University.
Regulation of Renin Gene Expression by Oxidative Stress
Authors: Itani Hana Ahmad , Liu Xuebo , Sarsour Ehab H. , Goswami Prabhat C. , Born Ella , Keen Henry L. , Sigmund Curt D.
Published: June 2021

Increased arterial pressure, angiotensin II(AII), and cytokines each result in feedback inhibition of renin gene expression. Because AII and cytokines can stimulate reactive oxygen species (ROS) production, we tested the hypothesis that oxidative stress may be a mediator of this inhibition. Treatment of renin‐expressing As4.1 cells with the potent cytokine tumor necrosis factor‐á (TNFá) caused an increase in the steady‐state levels of cellular ROS, which was reversed by the antioxidant NAC. Exogenous H2O2 (HP) caused a dose‐ and time‐dependent decrease in the level of endogenous renin mRNA and decreased the transcriptional activity of a 4.1‐kb renin promoter fused to luciferase, which was maximal when the renin enhancer was present. The TNFá–induced decrease in renin mRNA was partially reversed by either NAC or panepoxydone, a nuclear factor êB (NFêB) inhibitor. Interestingly, HP did not induce NFêB in As4.1 cells, and panepoxydone had no effect on the downregulation of renin mRNA by HP. The transcriptional activity of a cAMP response element‐luciferase construct was decreased by both TNFá and HP. These data suggest that cellular ROS can negatively regulate renin gene expression via an NFêB‐independent mechanism involving the renin enhancer and inhibiting cAMP response element–mediated transcription. TNFá decreases renin expression through both NFêB‐dependent and NFêB‐independent mechanisms.
Hypoxia inducible factor 1Î±, hypoxanthine‐guanine phosphoribolsyl transferase and NADPH oxidase 4 from northern elephant seals: cDNA characterization and tissue expression
Authors: Organis Jose Guadalupe Sonanez , Medina Jose Pablo Vazquez , Aguilar Andy , Forman Henry , Crocker Daniel , Ortiz Rudy
Published: June 2021

Northern elephant seals (Mirounga angustirostris) are naturally adapted to prolonged periods (1–3 months) of food deprivation (fasting) and tissue ischemia. Fasting elephant seal pups do not exhibit signs of oxidative damage or inflammation despite increases in the renin‐angiotensin system, NADPH oxidase activity and the superoxide‐generating enzyme, NADPH oxidase 4 (Nox4). Inducible factor 1α (HIF‐1α) and hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) may be activated in response to fasting‐ and hypoxia‐induced increases in ROS. We report the molecular characterization and tissue expression of Nox4, HIF‐1α and HGPRT from fasting northern elephant seal pups. The full‐length sequence for HGPRT is 761 bp long and codes for 218 amino acids, which is similar to other mammalian homologs. The partial sequences of Nox4 and HIF‐1α are 1131 bp and 745 bp long respectively and correspond to approximately 65% and 35% of the full sequence of mammalian homologs, respectively. HGPRT, Nox4 and HIF‐1α sequences are similar to vertebrate homologs and contain conserved functional and regulatory domains. HGPRT, Nox4 and HIF‐1α expression was detected in both muscle and adipose tissue, with higher expression in muscle than in adipose. The present study represents a critical first step towards advancing our understanding of the molecular mechanisms of oxidant regulation in response to prolonged fasting and apnea‐induced ischemia.
Short‐term, high‐intensity interval training (HIT) alters ATP pathway fluxes during maximal muscle contractions in humans
Authors: Larsen Ryan Godsk , Maynard Logan , Kent‐Braun Jane
Published: June 2021

Phosphorus magnetic resonance spectroscopy (MRS) was used to examine the effects of 2 weeks of cycling training on bioenergetics of vastus lateralis in vivo. Eight healthy, untrained men (27 ± 4 yr) performed 6 sessions of HIT (4–6 bouts of 30‐s all‐out cycling). At baseline, and after the 1st and the 6th training sessions, rates of ATP synthesis from net PCr breakdown (ATPCK), glycolysis (ATPGLY) and oxidative phosphorylation (ATPOX) were measured during a 24‐s maximal voluntary isometric contraction (MVIC) in a 4T magnet. Training increased peak whole‐body oxygen uptake (36 ± 4 to 39 ± 5ml·kg−1·min−1, p=0.01) and exercise capacity (225 ± 32 to 239 ± 36 W, p=0.04) on the cycle ergometer, with no effects on total ATP production or force‐time integral during the MVIC. While ATP production through the 3 pathways remained unchanged after the first training session, 6 training sessions increased the relative contribution of ATPOX (31 ± 2 vs. 39 ± 2% of total ATP turnover, p<0.001), and lowered the relative contribution from ATPCK (49 ± 2 vs. 44 ± 1%, p=0.004) and ATPGLY (20 ± 2 vs. 17 ± 1%, p=0.03). Thus, 6 sessions of HIT alters muscle energetics, such that maximal contractions are performed with increased support from oxidative ATP synthesis and relatively less contribution from anaerobic ATP production, which may contribute to improved exercise capacity.

Support: NIH K02 AG023582
Endothelial progenitor cell responses to hypoxia: Time‐course dependent changes in Akt signalling and gene expression
Authors: Brazil Derek Patrick , Ali Imran Hussein , McKenzie Rebecca A. , O' Brien Amy C. , Carroll Elizabeth C. , Neill Christina O' , Medina Reinhold J. , Doherty Michelle O' , Stitt Alan W. , Hookham Michelle B.
Published: June 2021

The aim of this work is to examine endothelial progenitor cells (EPCs) and their potential role as a cell based autologous therapy to revasularise the ischaemic tissue of the diabetic retina. Our group identified a distinctive human blood derived EPC with high proliferative capacity known as outgrowth endothelial cells (OECs). Characterisation of these cells highlighted their endothelial‐like, as opposed to inflammatory cell phenotype. To mimic low O2 concentrations in the diabetic retina, we have exposed OECs to 1% O2 for varying times. Induction of HIF1α was detected at 5h and maintained up to 48h at 1% O2 (in contrast to HeLa cells). Changes in Akt and p70 S6K phosphorylation were evident after 30 min exposure to hypoxia. Chronic hypoxia activated the AMPK→TORC2→p70S6K pathway leading to inhibition of p70S6K phosphorylation. Global gene array analysis using Illumina bead chip platform identified distinct cohorts of early response (8h) and late (24, 48h) response genes in OECs to hypoxia. Genes such as ADM, ANGPTL4, JMJD1A and the recently documented hypoxia inducible gene ANKRD37 were elevated in our array. A number of novel hypoxia genes including several involved in the BMP/Smad signalling pahway were also elevated. These groups of genes will then form the basis of comparison between control and diabetic OECs, to identify differences in diabetic cells that may explain their poor vaso‐reparative potential.
Protein Targeting and Degradation Pathways are Coupled for Elimination of Mislocalized Proteins
Authors: Hessa Tara , Sharma Ajay , Mariappan Malaiyalam , Eshleman Heather , Gutierrez Erik , Hegde Ramanujan
Published: June 2021

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis. The mechanisms of mislocalized protein (MLP) degradation are unknown.

Here, we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome‐associating chaperone. Bag6 complex capture depends on unprocessed or non‐inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 clients is transferred to TRC40 for membrane insertion, while the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex impairs efficient ubiquitination selectively of MLPs. Thus, by its presence on ribosomes synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling permits fast‐tracking of MLPs for degradation without futile engagement of cytosolic folding machinery.
Systemic enhancement of blood flow as a potential therapeutic strategy to counteract high altitude related health problems
Authors: Radiloff Daniel R. , Zhao Yulin , Shan Siqing , Irwin David , Hamilton Karyn , Schroeder Thies
Published: June 2021

Introduction

Acute high altitude exposure leads to hypoxemia resulting in decreased exercise capacity. We hypothesize a pharmaceutical strategy to counteract tissue hypoxia by increasing cardiac output, and decreasing pulmonary vascular resistance resulting in increased oxygen transport to tissue.

Methods

Rats were habituated on motorized running wheels. Time to exhaust in a wheel was tested after i.p. injection of cardiotonic drug/vasodilator under hypobaric hypoxia using the Duke institutional hyperbaric facility. Exhaust was determined by behavioral endpoints. Data was analyzed using log rank test. Hemodynamic experiments were done on anesthetized animals under inspired hypoxia. Cardiovascular parameters were measured using pulse oximetry, fluid‐filled catheters, and oxygen needle probes.

Results

Combinational treatment with a cardiac stimulant and a vasodilator showed significant enhancement of exercise capacity under hypobaric hypoxia, but single compounds alone did not. Hemodynamic experiments confirmed enhanced cardiac activity after drug injection without increase of blood oxygenation levels. When given together, the drug combination appears to increase tissue oxygenation under hypoxia.

Conclusions

Modulation of cardiac activity and pulmonary vascular resistance appears a viable option to facilitate rapid acclimation to high altitude.
Glucocorticoid receptor signalling contributes to muscle atrophy caused by acute inflammation
Authors: Adams Olivia Joan , Haegens Astrid , Hermans Rob , Kelders Marco , Snepvangers Frank , Schols Annemie , Langen Ramon
Published: June 2021

Skeletal muscle atrophy accompanies acute inflammatory conditions including ARDS, COPD exacerbations and sepsis, and closely correlates with increased mortality. Increased circulating glucocorticoid levels have been linked to muscle atrophy. Nevertheless, the contribution of glucocorticoid receptor (GR) signalling to muscle atrophy caused by acute inflammation remains to be elucidated. To investigate this, acute pulmonary inflammation was induced by intra‐tracheal lipopolysaccharide (IT‐LPS) installation, in muscle‐specific GR knockout (MLC‐Cre+/− GR‐LoxP) and control (GR‐LoxP) mice. Circulating corticosterone levels were increased within 24h in response to IT‐LPS. Increased expression of mRNA transcripts encoding glutamine synthetase and phosphoenolpyruvate carboxykinase, as well as Atrogin1 and MuRF1, was detected in skeletal muscle of control mice following IT‐LPS, suggestive of GR‐ and atrophy signalling. Importantly, the reduction in body and muscle weight following IT‐LPS observed in GR‐LoxP control mice, was strongly attenuated in muscle‐specific GR knockout mice. These data implicate an important role for muscle GR‐signaling in muscle atrophy following acute (pulmonary) inflammation.

This study was supported by the Dutch Top Institute Pharma (project # T1–201).
Alterations in intracellular free Ca2+ handling in mouse models for Duchenne muscular dystrophy
Authors: Mazala Davi Augusto , English Samuel Andrew , Chen Dapeng , Chin Eva R
Published: June 2021

Duchenne muscular dystrophy (DMD) is a progressive degenerative muscle disease that affects 1 out of 3500 boys. It is known that the mouse model most frequently used in DMD research, the mdx mice, upregulates utrophin (utr), a protein with similar roles as dystrophin. Thus, the mdx/utr−/− double mutant (DM) is an alternative model that may be a more appropriate model for studying DMD disease mechanisms. One proposed mechanism behind the disease progression is related to poor intracellular calcium ([Ca2+]i) handling. The purpose of the current study was to evaluate the [Ca2+]i levels in response to repeated tetanic stimulation in intact single muscle fibres from the DM mouse model in comparison to fibres from mdx mice. Single muscle fibres were obtained from the flexor digitorum brevis by collagenase digestion and loaded with Fura‐2 AM for [Ca2+]i assessment at a range of stimulation frequencies (10, 30, 50, 70, 100, 120 and 150Hz). There were no differences for peak Fura‐2 ratio at any of the stimulation frequencies but the resting Fura‐2 ratio was reduced in the DM compared to the mdx mice (0.361±0.029 vs 0.381±0.019; p<0.05). The diameter and length of muscle fibres from DM were smaller compared to the mdx group (p<0.05). Furthermore, 65% of the fibres from DM animals failed to maintain peak Ca2+ during a tetanus, while 43% of the mdx fibres failed. These data suggest that alterations in the Ca2+ handling properties may be due to plasma or t‐tubule membrane conductance abnormalities and/or impaired Ca2+ release or storage by the sarcoplasmic reticulum. This project has been funded by Department of Kinesiology Graduate Research Initiative Project (GRIP).
The influence of the cardiopulmonary baroreflex on the arterial baroreflex resetting during dynamic exercise in elderly humans
Authors: Volianitis Stefanos , Yoshiga Chie C , Vogelsang Thomas , Secher Niels H
Published: June 2021

The cardiopulmonary baroreceptors influence the resetting of the arterial baroreflex during exercise. We evaluated the effect of aging on the interaction between the cardiopulmonary and the arterial baroreflexes during exercise by determining the carotid baroreflex function (CBR) in 12 older (71 ± 2 yr; mean ± SD) and 12 young (24 ± 1 yr) healthy normotensive individuals during seated and supine light cycling [supine heart rate (HR), ~100 beats min−1; mean arterial pressure (MAP, 94 ± 2 mmHg)]. HR and MAP responses to brief 5 s pulses of neck pressure and suction (NP/NS), ranging from +40 to −80 mmHg, were used to calculate the maximal gain of the carotid‐HR (Gmax‐HR) and carotid‐MAP (Gmax‐MAP) baroreflex function curves as measure of CBR responsiveness. During seated vs. supine cycling there was a larger increase in Gmax‐HR in the young compared with the old subjects (0.09 ± 0.02 vs. −0.03 ± 0.01 beats min−1 mmHg−1, respectively, P<0.05), while there was no difference in the increase in Gmax‐MAP, (0.24 ± 0.04 vs. 0.17 ± 0.03 mmHg−1 mmHg−1, young vs. older, respectively). These data demonstrate that aging attenuates the influence of the cardiopulmonary baroreflex on the cardio‐vagal responsiveness of the carotid baroreflex, while vasomotor responsiveness is maintained during exercise. This study was supported by The Danish Heart Foundation (03‐1‐3‐69A‐22074).
Temporal profile of cardiac and splanchnic sympathetic tone in AngII‐salt rats
Authors: Kuroki Marcos T , Guzman Pilar A , Fink Gregory D , Osborn John W
Published: June 2021

We recently reported that sympathetic nerve activity (SNA) to the kidneys and hindlimb do not contribute to AngII‐salt hypertension (HTN) despite elevated whole body sympathetic tone. We tested the hypothesis that AngII‐salt HTN is associated with enhanced sympathetic tone to the heart and splanchnic vascular bed. Mean arterial pressure (MAP), heart rate (HR) and superior mesenteric artery blood flow (SMABF) were measured in rats fed a high (2%) NaCl diet during 4 days of control and 14 days of AngII infusion (150ng/kg/min, sc). Changes in sympathetic tone on MAP, HR, and mesenteric vascular resistance (MVR; SMABF/MAP) were assessed during control, and days 3, 7, and 13 of AngII by ganglionic blockade (hexamethonium; 20mg/kg, iv). MAP (140±4 mmHg) and depressor response to hexamethonium (−86±5) on Day 13 of AngII was significantly elevated compared to control (97±1 and −41±1, respectively). HR and MVR were 402±7 BPM and 9.7±0.8 mmHg·min/mL during control. By day 13 of AngII, HR was 397±5 and MVR was 14±1. The change in HR after ganglionic blockade was −37±10 during control, +20±24 on Day 3, −50±10 on Day 7 and −60±10 on Day 13 of AngII. The decrease in MVR after ganglionic blockade was similar during control (−2.6±0.3) and Day 3 (−3±1), but enhanced on Day 7 (−7±1) and Day 13 (−5.8±0.9) of AngII. We conclude that increased sympathetic tone on MAP in AngII‐sat HTN is associated with enhanced sympathetic tone on HR and MVR (HL076312).
Obese predicts increase in blood pressure associated with enhanced oxidative stress and inflammatory markers in young adults
Authors: Yoshida Michiko , Mori Takefumi , Takahashi Kuniko , Hirose Takuo , Kawamata Akihiro , Yoneki Yoshimi , Ito Hiroko , Ogawa Susumu , Hida Wataru , Ito Sadayoshi
Published: June 2021

The present study was designed to determine whether obese could predict future hypertension and associated with oxidative stress and inflammation in young adults.

1511 young adults, averaged 18±0.8 years old were enrolled. Subjects were divided into two groups according to BMI (above and below 25). Systolic blood pressure (SBP) was compared after two years. Urinary thiobarbituric acid reactive substances (U‐TBARS, as a marker of oxidative stress, n=1794) and monocyte chemotactic protein‐1 (U‐MCP‐1, as a marker of inflamation, n=794) excretion was analyzed in 103 young adults (obese students BMI avaraged 32.6±2.2, n=51) and non‐obese students BMI averaged 20.4±0.9, n=52).

SBP was significantly increased in subjects BMI above 25 (136.7±18.4 mmHg) compared with subjects below 25 (123.7±15.8 mmHg, P<0.05). Percentage of subjects whose SBP increased after two years was higher in BMI above 25 compared with below 25. Significant increase (P<0.05) in SBP (129.3±14.8 mmHg), U‐TBARS (42.9±18.6μmol/day) and U‐MCP‐1 (35.1±15.9 pg/day) was observed in obese group compared with non‐obese group (148.6±14.5, 64.5±15.5 and 44.4±15.8, respectively).

The results in the present study indicate that obese predicts increase in blood pressure after two years in young adults. Oxidative stress and inflammation were increased in obese young adults which is associated with increase in blood pressure.
The impact of Kosovo industrial pollution in activity of some enzymes in hemolymph and hepatopancreas of snail (Helix aspersa)
Authors: Gashi Agim , Bajraktari Ismet , Bislimi Kemajl , Ferizi Rrahman , Sahiti Hazbije , Mazreku Ilir , Halili Fetah
Published: June 2021

The activity of following enzymes: AST ‐ Asparat amino‐transferase, ALT ‐ Alamin amino‐transferase, ALP ‐ Alkaline phosphatase and LDH ‐ Lactat‐ dehydrogenase has been examined in hemolymph and hepatopancreas of Helix aspersa (N=75). Snails were sampled in several localities in Kosovo contaminated with different physical and chemical pollutants. The snails of control group were taken from Gjilan region, far away from polluted area.

The results of preliminary research show that enzyme activity depends on the area where snails were sampled. Compared to the control group snails, in hemolymph and hepatopancreas belonging to snails from contaminated localities the activity of ALP and AST was significantly lower while the activity of ALT and LDH higher. Significant decline in activity of ALP (P<0.01) and AST (P<0.01) is registered in hemolymph of snails taken from Mitrovica, whereas on the other hand significant increase has been noted in activity of ALT (P<0.001) in snails taken from Obiliq and of LDH in snails taken from Mitrovica (P<0.001) and Skenderaj (P<0.01). Opposite trend was noted in the activity of these enzymes in hepatopancreas of snails from different contaminated localities, especially in snails taken from Hani i Elezit and Mitrovica. This research shows that garden snail (Helix aspersa) can be used as bioindicator of complex environmental pollution effects.
Assembly and Pre‐steady‐state Kinetic Analysis of the Hepatitis C Virus RNA Polymerase Elongation Complex
Authors: Jin Zhinan , Leveque Vincent , Ma Han , Johnson Kenneth A. , Klumpp Klaus
Published: June 2021

The synthesis of RNA molecules by HCV RNA polymerase (NS5b) proceeds through two major phases: de novo initiation and elongation. To date, mechanistic studies of HCV polymerase at its elongation phase have been limited by the apparent difficulty to obtain preparations of the polymerase/primer/template elongation complex. The goal of this study was to assemble a functional HCV polymerase elongation complex and characterize the kinetics of nucleotide incorporation catalyzed by the polymerase in its elongation mode. NS5b(Δ21) was used in this study. After initiation from a GG primer and a 20‐nucleotide RNA template, the majority of NS5b(Δ21) was trapped into an elongation complex containing NS5b(Δ21) bound to the 20‐mer template and 9‐mer primer (P9/T20). Active site titration at various protein concentrations showed that more than 80% NS5b was trapped in the elongation complex, which was different from previous reports on HCV polymerase suggesting that only a small proportion of NS5b protein was active. The NS5b(Δ21)/P9/T20 complex was characterized using pre‐steady‐state kinetic methods. Challenging the elongation complex with enzyme trap (heparin or RNA) for up to 21 hr caused little dissociation of the complex, suggesting an extremely slow dissociation rate of the elongation complex. The kinetics of incorporation of CTP opposite GTP and misincorporation of A, U, G opposite GTP catalyzed by NS5b(Δ21) in the elongation complex were measured. The HCV elongation complex will be a valuable tool to study the mechanism of action of NS5b inhibitors.
Expression of Cyclic GMP‐inhibited Phosphodiesterases 3A and 3B (PDE3A and PDE3B) in Human and Mice Cardiac Myocytes
Authors: Khan Zoha , Manganiello Vincent
Published: June 2021

Biochemical and confocal microscopy approaches were used to investigate the expression of two cyclic GMP‐inhibited cyclic nucleotide phosphodiesterase 3 genes (PDE3A and PDE3B) in isolated murine cardiac myocytes and in cultured human cardiac myocytes. Western immunoblotting experiments were done with anti‐PDE3B and anti‐PDE3A antibodies, which allowed detection of both PDE3A and PDE3B proteins. PDE3A was expressed as a 130 kDa protein in total cardiomyocyte lysates of mice, while several PDE3A isoforms (70–130 kDa) were detected in cultured human cardiac myocytes. PDE3B was expressed as a 135 kDa protein in total mice and human myocyte lysates. Expression of PDE3B was increased in PDE3A‐KO mice myocytes. Immuno‐histochemical staining in mice cardiomyocytes showed that PDE3A co‐localizes to sarcomere Z‐bands with desmin and SERCA2 (Ca+2 ATPase).
Interaction of p67phox with Prdx6 regulates PLA2 activity
Authors: Krishnaiah Saikumari Yegnisettipalli , Dodia Chandra , Chatterjee Shampa , Feinstein Sheldon I , Fisher Aron B
Published: June 2021

Phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6) is necessary to activate NADPH oxidase (NOX2). P67phox is an essential component of the NOX2 enzyme complex that produces superoxide ions in response to agonist stimulation. It has been previously reported that p67phox interacts with Prdx6 in PMN. We further investigated the interaction of these two proteins and its physiological role. In vitro assays used recombinant proteins and phosphoPrdx6 that was generated by incubation of Prdx6 with Erk2 plus ATP. His‐tagged Prdx6 pulled down p67phox and his‐tagged p67phox pulled‐down Prdx6 and phosphorylated Prdx6. A monoclonal anti‐p67phox Ab immunoprecipitated Prdx6 and phosphoPrdx6, while a polyclonal anti‐Prdx6 Ab immunoprecipitated p67phox. Phosphorylation of Prdx6 increased its apparent binding to p67phox. Prdx6 and phosphoPrdx6 also were co‐immunoprecipitated from lysates of angiotensin II‐stimulated mouse pulmonary microvascular endothelial cells by anti‐ p67phox antibody. PLA2 activity, measured at pH 7 in Ca2+‐free buffer was 50 nmol/min/mg for Prdx6 and 1303 nmol/min/mg for phosphoPrdx6, but only 14–17 nmol/min/mg in the presence of p67phox. Our data indicate that p67phox binds to Prdx6 and inhibits its PLA2 activity and thus could function to terminate the PLA2 ‐mediated NOX2 activation signal.
The indolocarbazole, Gö6976, is a competitive inhibitor of guanylyl cyclase‐A and ‐B
Authors: Robinson Jerid W , Lou Xiaoying , Potter Lincoln R
Published: June 2021

The inhibitory mechanism of Gö6976 on the enzymatic activity of guanylyl cyclase‐A and ‐B was examined to better understand the regulation of these enzymes. Atrial natriuretic peptide (ANP) and B‐type natriuretic peptide (BNP) activate guanylyl cyclase‐A (GC‐A) and C‐type natriuretic peptide (CNP) activates guanylyl cyclase‐B (GC‐B). In addition, ATP activates both receptors by reducing the KM for GTP. Gö6976 blocked cellular CNP‐dependent cGMP elevations in 293T‐GC‐B cells. The t1/2 for Gö6976 inhibition was 7 seconds and IC50 was 380 nM. Cellular exposure to Gö6976 reduced basal and hormone‐dependent, but not detergent‐dependent, GC‐A and ‐B activity in a reversible manner. A constitutively phosphorylated form of GC‐B was inhibited as well. Gö6976 increased the EC50 for CNP 4.5 fold, but increasing the CNP concentration did not overcome the inhibition. Kinetic analyses indicate that Gö6976 is competitive with substrate. These data demonstrate that Gö6976 potently, rapidly and reversibly inhibits GC‐A and ‐B via a competitive process that does not require intact cell architecture or known phosphorylation sites. This is the first report of an intracellular inhibitor of a membrane GC and provides the foundation for the development of small molecule inhibitors of receptor guanylyl cyclases. This work is supported by NIAMS Grant T32AR050938 to JR.
The role of Desnutrin/ATGL in promoting a brown‐adipose‐phenotype and regulation via AMPK
Authors: Abbott Marcia J , Ahmadian Maryam , Sul Hei Sook
Published: June 2021

Desnutrin/ATGL has been identified as the major triacylglycerol (TAG) lipase in adipose tissue and is induced during fasting. To further establish the role of desnutrin in adipocyte lipolysis we generated adipose‐specific knock‐out (ASKO) of desnutrin in mice. ASKO of desnutrin resulted in an obese phenotype with increased insulin sensitivity due to a decrease in circulating fatty acids (FA). The brown adipose tissue (BAT) in the desnutrin ASKO mice displayed a white adipose tissue (WAT)‐like phenotype with increased TAG, lower FA oxidation and less PPARα binding to its promoter. Desnutrin ASKO mice were cold intolerant and had lower expression of UCP‐1 in BAT. We also determined that the S406 residue of desnutrin is an important regulation site and is able to be phosphorylated by AMPK to increase its hydrolase activity. Immunoprecipitates from desnutrin overexpressed HEK 293 cells, treated with AICAR, had an increase in phosphorylation of desnutrin at S406. Mutation of S406 to alanine decreased lipase activity and increased TAG content in livers of adenovirus infected mice. These results suggest that desnutrin activity, in adipose tissue, may be regulated through AMPK to provide FA ligands for PPARα and promotes a BAT phenotype in WAT.
Natural alcohol exposure: Is ethanol the main substrate for alcohol dehydrogenases in animals?
Authors: Julián‐Sánchez Adriana , Hernández‐Tobías Aída , Piña Enrique , Riveros‐Rosas Héctor
Published: June 2021

In animals, three non‐homologous alcohol dehydrogenase (ADH) protein families are reported: Zn‐ADHs are zinc‐containing enzymes; type II ADHs lack metal cofactor, and Fe‐ADHs are iron‐activated enzymes. The presence of these ADHs in animals has been assumed to be a consequence of chronic ethanol exposure. The main natural source of ethanol is fermentation of fruit sugars by yeast. Therefore, if substrate availability must precede enzyme selection, then it can be expected that the enzymogenesis of ADHs began after the appearance of fleshy fruits. In this work, this possibility is discussed.

Phylogenetic analyses reveal that type II ADHs suffered several duplications, all of these restricted to flies. Induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow flies to exploit niches with high ethanol content.

In contrast, Zn‐ADHs and Fe‐ADHs in animals appeared before land plants, independently of ethanol availability. Because these enzymes are not induced by ethanol and possess a high activity with non‐ethanol endogenous substrates, it can be concluded that their participation in ethanol metabolism can be considered as incidental, and not adaptive.

Supported by DGAPA‐UNAM grant IN208510.
Calcium supplements and the risk of myocardial infarction
Authors: Wallace Taylor C , Heaney Robert , Kopecky Stephen L , Maki Kevin C , Hathcock John , MacKay Douglas
Published: June 2021

This poster presents an expert consensus position on the current controversy regarding calcium supplement use and risk of myocardial infarction. In November 2011, The CRN Foundation with the support of the National Osteoporosis Foundation and National Bone Health Alliance convened an expert panel of scientists from the nutrition and medical communities to review the total body of evidence and specifically studies relating calcium supplements to an increased risk of myocardial infarction. Because of the many data limitations present in the supporting studies relating calcium supplements to an increased risk of myocardial infarction and contrast to the large majority of the literature accompanied with recent reanalyses of the data, no change in prescribing practice is currently recommended. Prescribers should consider the potential benefits of calcium supplements for prevention of osteoporotic fractures based on an individual's personal dietary intake. Positive outcomes of calcium supplementation such as prevention of obesity and colorectal cancers may also carry weight in support of calcium supplements when individual dietary intakes are inadequate to meet IOM recommendations.
Co‐Transcriptional Splicing and Dynamic Rearrangements in the Spliceosome
Authors: Johnson Tracy L , Pradhan Suman , Merkhofer Evan , Soule Erik
Published: June 2021

Recent studies have shown that pre‐mRNA splicing occurs co‐transcriptionally. The next key challenge is to identify specific factors that coordinate pre‐mRNA splicing with transcription, particularly within the context of a chromatin template. We have previously shown that Gcn5, which encodes the HAT activity of the co‐activator SAGA complex, mediates co‐transcriptional recruitment of the U2 snRNP to pre‐mRNA. As further evidence of a role for SAGA in mediating ATP‐dependent rearrangement of the U2 snRNP, we have identified interactions between the DExH/D box protein Prp5, which hydrolyzes ATP to remodel the U2 snRNP and facilitate branchpoint recognition, and components of SAGA. A lethal mutation in PRP5 that eliminates ATP‐binding is suppressed when components of the SAGA complex are deleted, thereby for the first time linking SAGA with a catalytic activity that drives spliceosomal rearrangements. These results have led us to further analyze how histone acetylation contributes to U2 snRNP recruitment. We find that chromatin remodeling complexes that associate with acetylated histones physically and functionally interact with the U2 snRNP and contribute to spliceosome assembly. These data support a model whereby co‐transcriptional spliceosome assembly is intimately coordinated with histone acetylation and chromatin remodeling. Support: NSF CAREER MCB0448010 and NIGMS GM085474; GM085764.