bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of Branched‐Chain Amino Acid Supplementation on Exercise Performance and Recovery in Highly Endurance‐Trained Athletes
Authors: Jafari Hanieeh , Ross J. Barrett , Emhoff Chi‐An W. 
Published: November 2023

Branched‐chain amino acid (BCAA) supplementation has recently entered the spotlight as a potentially key nutritional component to skeletal muscle recovery during periods of high training volume in endurance athletes. The purpose of this study was to examine the effects of BCAA supplementation on anaerobic and aerobic performance, substrate utilization during constant‐load exercise to exhaustion, and plasma creatine kinase and lactate dehydrogenase levels during post‐exercise recovery. Nine well‐trained cyclists (5 male, 4 female) underwent a morning session and an afternoon session of exercise testing under BCAA or Placebo conditions. Morning tests included two 15‐second Wingates to determine peak and mean anaerobic power output, followed by an incremental test to exhaustion to determine peak oxygen consumption (VO2peak). Afternoon tests included two 15‐second Wingates, followed by a constant‐load exercise to exhaustion at 75% VO2peak, during which only water was provided. During the latter test, pulmonary gas exchange, blood lactate and blood glucose were periodically measured to monitor substrate utilization. Pre‐ and post‐exercise blood tests were conducted to quantify plasma creatine kinase and lactate dehydrogenase levels as biomarkers for muscle damage. Post‐exercise ratings of perceived soreness were also collected. Subjects were then instructed to continue training for at least 2000 MET‐min/week for the next four weeks while supplementing with either a BCAA (5 g amino acids + 56 g carbohydrates) or Placebo (56 g carbohydrates) powdered drink during each workout. At the completion of the four weeks, subjects repeated two 15‐second Wingates and an incremental test to exhaustion to determine any differences in anaerobic and aerobic power due to BCAA or Placebo supplementation. There were no differences in VO2peak pre‐ and post‐training between BCAA and placebo conditions. Peak anaerobic power was slightly improved when athletes supplemented their training with the BCAA drink. The total energy expenditure achieved during the constant‐load exercise test was not different between conditions. Post‐exercise plasma creatine kinase levels were significantly reduced with BCAA compared with Placebo, while there were no differences in plasma lactate dehydrogenase levels. Post‐exercise leg muscle soreness was slightly lower with BCAA supplementation, although not significant. In conclusion, BCAA supplementation with endurance training may augment adaptations in muscle anaerobic power and post‐exercise recovery in well‐trained athletes.Support or Funding InformationThis research was supported by the Saint Mary's College Faculty Development Fund and a gift from GU Energy Labs, Berkeley, CA.
NSAIDs Inhibit Cell Migration by Disrupting the Cellular Adhesion Signaling Cascade
Authors: Silver Kristopher , Lillich James 
Published: November 2023

Treatment with non‐steroidal anti‐inflammatory drugs (NSAIDs) is associated with a wide range of negative side effects including gastrointestinal toxicity. Various mechanisms have been proposed to account for these effects, including promotion of ulceration and erosion by inhibition of cell migration and wound healing. Our lab has previously demonstrated that NSAIDs inhibit expression and activity of calpains resulting in stunted and uncoordinated cell migration, but the downstream effects of suppression of calpain activity/expression are currently unknown. Accordingly, we used a combination of immunoblotting and immunofluorescence in both migrating and non‐migrating IEC‐6 cells to identify total and phosphorylated forms of adhesion signaling proteins, such as vinculin, PAX, FAK, and talin, which are affected by NSAID treatment. Whereas vinculin was unaffected by NSAID treatment in either resting or migrating cells, there was significance suppression of expression of phosphorylated PAX and talin in both cell populations following treatment with indomethacin or NS‐398. Additionally, there was also a significant decrease in expression of phosphorylated FAK with indomethacin, but a significant increase in phosphorylated FAK with NS‐398 treatment. These data suggest that not only do NSAIDs affect calpain activity, as previously reported, but NSAIDs also significantly disrupt the adhesion signaling cascade required for coordinated cell migration. Further, our results also provide a mechanism to explain the apparent retention of rear cellular adhesions observed in NSAID‐treated migrating cells and provide a broad foundation for understanding the precise molecular mechanisms through which NSAIDs disrupt cell migration and wound healing.Support or Funding InformationThis work was supported by a grant from the National Institutes of Diabetes and Digestive and Kidney Disease (1R15DK091791)
Oxygen Kinetics and Muscle Twitch Tension in the Rat Extensor Digitorum Longus Muscle After Creatine Kinase Inhibition
Authors: Batten Spencer M , Dobard Sarah E , Buell Joshua H , Cammack Scott A , Sexton William L 
Published: November 2023

Creatine kinase (CK) catalyzes the reversible reaction in which high‐energy phosphates are shuttled between phosphocreatine and ADP. CK plays a role in the initial production and maintenance of muscle force during contractions before the onset of glycolytic and oxidative metabolism. The onset of oxidative metabolism leads to changes in interstitial PO2 (PiO2) reflecting the dynamic balance between oxygen delivery (QO2) and oxygen consumption (VO2). The purpose of this study was to investigate the role of CK in QO2 to VO2 matching as reflected in changes in PiO2 at the onset of contractions in the rat extensor digitorum longus muscle (EDL). Female Sprague‐Dawley rats (n=9, 291 ± 5 g) were anesthetized (sodium pentobarbital, 50 mg/kg ip). The trachea was cannulated to maintain a patent airway, and the carotid artery was cannulated to monitor mean arterial pressure and heart rate. The EDL was isolated and connected to a force transducer coupled with a muscle tension analyzer. In each EDL, PiO2 (measured by phosphorescence quenching) was determined at rest and during a 150 s contraction bout (1 Hz, 6 V) where PiO2 and twitch tension were simultaneously measured during the transition to steady‐state contractions. After a 10 min recovery period the rats received an intravenous bolus of IA (20 mg/kg in saline). After a 15 min equilibration period, the same contraction protocol was repeated. Thus, each rat served as its own control. This protocol was validated in preliminary experiments wherein PiO2 and twitch tension data were not different during a repeated stimulation. Mean arterial pressure (120 ± 5, 128 ± 4 mmHg) and heart rate (363 ± 19, 349 ± 9 bpm) were similar before and after IA. There was no difference in resting PiO2 between the control stim and post‐IA (28 ± 3, 29 ± 2 mmHg). At the onset off contractions, PiO2 fell more rapidly after IA as reflected by a faster time constant (16 ± 2, 12 ± 1 s; P<0.05) and mean response time (20 ± 2, 16 ± 2 s; P<0.05). After IA treatment twitch tension (N/g) and the rate of tension development (dT/dt) were both lower (P<0.05) throughout the 150 s contraction bout. However, muscle fatigue was similar between both contraction bouts (3 ± 1, 3 ± 1%; P>0.05). The results of this study reveal that inhibition of creatine kinase with IA in fast‐twitch rat skeletal muscle depressed developed twitch tension and dT/dt. IA treatment also sped the fall in PiO2 reflecting a dramatic imbalance between QO2 and VO2 during the transition to steady state contractions, which may have contributed to the decline in contractile performance.Support or Funding InformationSupported by a grant from the Graduate Program Committee, KCOM – ATSU.
Crystal Structure of Human Tetraspanin CD81 Reveals a Conserved Intramembrane Binding Cavity
Authors: Zimmerman Brandon , McMillan Brian , Seegar Tom , Kruse Andrew , Blacklow Stephen 
Published: November 2023

Tetraspanins comprise a diverse family of four‐pass transmembrane proteins that play critical roles in cell activation, adhesion, migration and proliferation. CD81/Tspan28, the most widely studied member of the family, forms a complex with CD19, CD21 and CD225 to regulate B cell receptor function, and also acts as a receptor or co‐receptor for pathogens such as Hepatitis C virus and Plasmodium species. Despite the fundamental importance of tetraspanin proteins in a broad range of physiologic and pathophysiologic processes, structural information about a full‐length tetraspanin has remained elusive. Here we present the first crystal structure of a full‐length tetraspanin, human CD81. The transmembrane segments of CD81 pack as two largely separated pairs of helices, not as the four‐helix bundle predicted previously. The structure is capped by the large extracellular loop 2 (EC2) at the outer membrane leaflet, and the two pairs of helices converge at the inner leaflet to form a shape resembling an inverted teepee. The transmembrane region contains a large pocket suggestive of a ligand‐binding site, offering a potential mechanism for binding lipids or other molecules. In support of this idea, we observe electron density consistent with a bound cholesterol molecule in the pocket, and we detect specific binding of cholesterol to this site in vitro. The discovery of the hydrophobic pocket in the core of the transmembrane domain presents a novel therapeutic avenue for targeting CD81 and other tetraspanins in pathogenic infections or malignancies.Support or Funding InformationBrandon Zimmerman is supported by a CIHR postdoctoral fellowship. NIH grant NCI 5ROI CA092433 to SCB and DP5 OD021345 to ACK.
Novel gene expression changes dependent on 2 mitochondrial enzymes in a rescued model of Parkinson's disease
Authors: Theisen Christopher Shane , Kogel William , Tiege Angela , Kaston Essa , Perry Kelley 
Published: November 2023

Parkinson's disease (PD) is a progressive disorder of the central nervous system for which there is no cure. The pathogenesis of PD involves insufficient formation of dopamine as a result of dopaminergic cell death in the midbrain. The cause of demise of these cells is poorly understood, but mitochondrial dysfunction has been implicated as a central pathway in the neurodegenerative process. We have observed cellular protection in a model of PD by increasing the levels of 2 important mitochondrial enzymes, NAMPT and SIRT3. Our current model of Parkinson's disease employs the use of immortalized dopamine progenitor neurons and zebrafish larvae. In this study, we examined the effects of overexpressing Nampt in dopaminergic neurons as well as neurons stably expressing lentiviral mediated shRNA against Nampt and the essential mitochondrial sirtuin gene, Sirt3. After establishing stable populations of dopaminergic neurons with either increased or decreased levels NAMPT or SIRT3 we have identified various genes that may be involved in the pathways of neuroprotection in dopaminergic neurons susceptible to cell death in the progression of PD. We compared the expression levels of major genes involved in neurotoxicity. A sub cohort of genes has been identified as over expressers when NAMPT and SIRT3 have reduced expression. There are 15 genes that have significant changes in gene expression consistent with our hypothesis. 7 genes have dramatic changes reciprocal to NAMPT over vs under expression. Our results indicate that NAMPT and SIRT3 alter similar pathways. These mitochondrial enzymes are also being examined in a zebrafish model of PD that shows to have rescued motor function when the genes are overexpressed.Support or Funding InformationKCUMB Division of Research
Spatial expression of Receptor for Advanced Glycation End‐Products (RAGE) in diverse tissue and organ systems differs following exposure to secondhand cigarette smoke
Authors: Gassman Jason R. , Lewis Joshua B , Milner Dallin , Monson Troy , Broberg Dallin , Bodine Jared , Lewis Adam , Dunaway Todd , Egbert Kaleb , Chavarria Bryan , Thomas Daniel , Arroyo Juan A , Reynolds Paul R 
Published: November 2023

The receptor for advanced glycation end‐products (RAGE) is a pattern recognition receptor initially identified in the lung and known to be expressed by many cell types including smooth muscle cells, fibroblasts, macrophages/monocytes and epithelial cells. RAGE is capable of binding a variety of endogenous ligands including advanced glycation end‐products (AGEs), S100/calgranulins, amyloid‐β peptide, and HMGB1. Furthermore, exogenous entities such as tobacco smoke particulates also have been demonstrated to activate RAGE and as such, RAGE signaling has been implicated as a major contributor to a variety of inflammatory states. In the present study we examined the expression patterns of RAGE in pulmonary and systemic tissues from mice exposed to room air (RA) and then compared expression with mice following exposure to daily secondhand smoke for up to 6 months via a nose‐only exposure system (Scireq, Montreal, Canada). Following sacrifice, real time PCR revealed interesting expression patterns for RAGE mRNA in tissues and organs such as eyes, lungs, heart, kidney, spleen, gastrocnemius, liver, brain, small intestine, adipose tissue, and dermis. RT‐PCR analysis revealed that augmented RAGE expression is a cellular response to secondhand smoke exposure. Confirmatory studies aimed at characterizing protein levels were also conducted using immunoblot and immunohistochemistry. In conclusion, our data reveal that as already detected in several studies involving the pulmonary apparatus, elevated RAGE expression is a distinct response to smoke exposure and its expression likely elevates the pro‐inflammatory RAGE signaling axis in cells and tissues responding to exposure.Support or Funding InformationThis work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
Molecular mechanisms underlying angiotensin II‐induced angiotensin converting enzyme 2 internalization and degradation
Authors: Ogunlade Blessing , Sriramula Srinivas , Lazartigues Eric , Guidry Jessie J , Filipeanu Catalin M 
Published: November 2023

Angiotensin converting enzyme II (ACE2), the newest identified member of renin‐angiotensin system (RAS), catalyses the conversion of angiotensin (ang)‐II to ang‐(1‐7) which plays a compensatory role through Mas1 receptor in the cardiovascular system. We previously reported that decreased ACE2 expression and activity leads to the development of ang‐II‐mediated hypertension in mice, that ang‐II stimulated ACE2 internalization and degradation in lysosomes and that inhibition of this process prevents the hypertensive actions of ang‐II. On the other hand, severe acute respiratory syndrome coronavirus is internalized together with ACE2, clearly indicating a regulated endocytotic mechanisms for this enzyme. However, the molecular mechanisms controlling this event remain largely unknown and the aim of the present study was to clarify these aspects.We first performed a proteomic study in HEK293T cells co‐transfected with ACE2 and AT1R and found that ang‐II treatment (100 nM, 4 h) significantly changed the interactions of ACE2 only with fascin, an actin‐bundling protein. Confocal microscopy experiments confirmed that ang‐II strongly enhanced the co‐localization of the two proteins in the perinuclear space. In addition, we demonstrated that ang‐II treatment augmented ubiquitination of ACE2 and amino acid analysis indicated that in the C‐terminus the enzyme has only five putative lysine ubiquitination sites. Six ACE2 mutants were constructed, five in which each individual ubiquitination site was replaced by arginine, and the sixth mutant (ACE2‐L5R) included all five mutations. In basal conditions in HEK293T cells co‐transfected with AT1R, ACE2 wild‐type activity was 3536.88 ±331.63FU/min/μg protein. Ang‐II treatment (100 nM, 4h) significantly attenuated enzymatic activity (3298.60±255.89). Remarkably, every mutant displayed enhanced enzymatic activity in basal conditions of at least 195±45%, with the highest increase observed in the case of ACE2‐L5R mutant being 285±61%. Furthermore, none of these ACE2 mutants displayed decreased enzymatic activity after ang‐II treatment. The present results demonstrate that ACE2 is constitutively ubiquinated and that this posttranslational modification is augmented by increased levels of ang‐II, contributing to lysosomal degradation of the enzyme in fascin‐dependent manner and overall diminishing its compensatory role against overactive RAS. These data indicate that interfering with the mechanisms contributing to ang‐II‐mediated ACE2 degradation may constitute an alternative therapy in cardiovascular diseases.Support or Funding InformationSupported by NIH‐P20‐GM103514 (EL, JJG and CMF), R01‐HL093178 (EL), and startup funds from Howard University (CMF)
Hydrogen sulfide inhibits Orai3‐mediated store‐operated Ca2+ entry by targeting extracellular cysteines
Authors: Fresquez Adriana Maria , White Carl 
Published: November 2023

The store operated calcium entry (SOCE) pathway is activated by interactions between the endoplasmic reticulum localized STIM1 and STIM2 proteins and the Orai 1, 2 and 3 plasmalemmal Ca2+ channels. We recently discovered that the gaseous signaling molecule hydrogen sulfide (H2S) selectively inhibits SOCE mediated by STIM1 and Orai3. We showed that STIM1/Orai3 regulation has physiological importance in the innate immune system where endogenously produced H2S limits maximal activation of SOCE and proinflammatory cytokine production. The purpose of the current study was to define the mechanism by which H2S modifies STIM1/Orai3 function. It is known that H2S impinges on protein function by targeting disulfide bridges. Sequence analysis of human Orai1, 2 and 3 revealed two cysteines (C226 and C232) unique to the human and mouse Orai3 protein. We hypothesized that C226 and C232 are the site of action for H2S on Orai3 and C226 and C232 forms a disulfide bridge. We tested this by assessing the effect of mutating the C226 and C232 residues to serine on H2S‐regulation of SOCE. This was done by transiently transfecting wild‐type or mutant human Orai3 into HEK293 cells stably expressing human STIM1 and measuring SOCE by Ca2+ imaging. Treatment with the H2S donor GYY4137 (500 μM) inhibited the SOCE mediated by STIM1/Orai3. Mutating either C226 or C232 alone had no effect on SOCE under control conditions, however, the ability of GYY4137 to inhibit SOCE was lost in the mutants. A double mutation of both C226 and C232 also had no effect on SOCE under control conditions and GYY4137 again failed to inhibit SOCE. In addition, pre‐incubation with the reductant dithiothreitol (5 mM) completely blocked the inhibitory effect of GYY4137. These data are consistent with a model in which H2S inhibits SOCE mediated by STIM1/Orai3 by targeting a disulfide bridge between C226 and C232.Support or Funding InformationRosalind Franklin University of Medicine and Science
The role of SNARE proteins in targeting KCa3.1 to the basolateral membrane of epithelial cells
Authors: Farquhar Rachel , Hamilton Kirk Lee 
Published: November 2023

Targeting epithelial membrane proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium‐activated, intermediate‐conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. A consequence of mutations of KCa3.1 is impaired ion and fluid transport of erythrocytes resulting in hereditary xerocytosis. Surprisingly, the mechanism of membrane targeting of KCa3.1 is not well understood. Recently, we reported that KCa3.1 is targeted to the BLM of epithelial cells by a Myosin‐Vc‐dependent pathway (Farquhar et al. FASEB J. 30:1224.41, 2016) and by a Rab1‐ and Rab8‐dependent process (Bertuccio et al. PlosONE 9:e92013, 2014). Here, we extend our work to examine the role of the SNARE proteins VAMP3, syntaxin 4 (STX‐4) and SNAP‐23 in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We used our FRT epithelial cell line stably expressing KCa3.1‐BLAP/Bir‐A‐KDEL (cells grown on filters), immunoblot (IB), siRNA and Ussing chamber experiments in this study. Our IB approach was to streptavidin label channels that arrive at the BLM to determine the effects of siRNA knockdown of specific SNARE proteins on the targeting to and function (KCa3.1 current) of KCa3.1 at the BLM of the epithelial cells. Using siRNA, we showed that knockdown of VAMP3 reduced the BLM expression of KCa3.1 by 49 ± 8% (P ≤ 0.05, n = 3) compared with control cells. With Ussing chamber experiments, we used 1‐EBIO (100 μM, an agonist of KCa3.1) to stimulate current via KCa3.1. Knockdown of VAMP3 reduced the KCa3.1 current by 86 ± 9% (P ≤ 0.05, n = 3) compared with control cells. Similarly, knockdown of STX‐4 lowered the BLM expression of KCa3.1 by 84 ± 5% (P ≤ 0.05, n = 3) and reduced the KCa3.1 current by 82 ± 8% (P ≤ 0.05, n = 2). Finally, siRNA knockdown of SNAP‐23 reduced the expression of KCa3.1 at the BLM by 52 ± 11% (P ≤ 0.05, n = 6) and reduced the KCa3.1 current by 1.2 ± 0.3 μA (P ≤ 0.05, n = 6) compared with control cells. These data suggest that VAMP3, STX‐4 and SNAP‐23 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells.Support or Funding InformationThis work was supported by UORG and Dean's Fund grants from the University of Otago and an Aims grant from the Department of Physiology (KLH).