bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of high‐ and low‐carbohydrate diets on glycogen branching enzyme activity in the liver of C57BL/6J mice
Authors: Fuller Scott , Nelson Arnold , Stewart Laura , Henagan Tara , Waldrop Grover
Published: June 2021

INTRODUCTION

Glycogen has a highly branched structure with the branch points formed by glucose molecules being joined by α 1,6‐glycosidic linkages. These linkages are formed by the action of branching enzyme (BE). While glycogen BE has been studied in clinical medicine, modulation of the enzyme has received little attention. This study measured the degree of branching in glycogen from mouse liver in response to low versus high carbohydrate diets.

METHODS

Six week old, male C57BL/6J mice (n = 68) were randomly assigned to either a low‐carbohydrate (35% CHO) or a high‐carbohydrate (70% CHO) diet for 8 weeks. Food consumption, body weight and body composition were evaluated weekly (NMR). At the 8 week time point, mice were euthanized and liver samples were collected and assayed for the degree of glycogen branching. Degree of branching was determined by spectrophotometrically quantifying the ratio of branch points to total glucose residues.

RESULTS

Food consumption did not differ between the groups. However, body weight and body fat percentage were different between the two groups (p < 0.001) at the 8 week time point. Degree of glycogen branching did not differ between the groups.

CONCLUSION

Glycogen BE activity in mouse liver does not appear to be affected by varying carbohydrate intake, despite the observation that both body fat percentage and body weight were higher in mice consuming the LC diet. Further investigation should aim to discover how BE activity is modulated in skeletal muscle.
The effects of TNF‐alpha and Ceramide on Insulin Receptor signaling and its localization to Caveolae in C2C12 Myocytes
Authors: Vallejo Johana , Leung Lana , Tran Jesse , Gordon Christopher , Javid Jason
Published: June 2021

TNF‐α and ceramide are associated with the etiology of insulin resistance. There is evidence for the involvement of caveolae membrane rafts in the modulation of insulin signaling and glucose uptake. Thus, we propose to elucidate the signaling mechanisms by which TNF‐α and ceramide induce insulin resistance in C2C12 myocytes and the role of caveolins in the organization of this pathway within the membrane. We hypothesized that exposure of C2C12 myocytes to TNF‐α and ceramide will result in altered insulin signaling, decreased glucose uptake, and translocation of the insulin signaling cascade away from caveolae. C2C12 myocytes were treated with TNF‐α, ceramide, and insulin, individually and in combination. The treated cells were subjected to Western blot analysis for insulin receptor‐β (IR‐β), phosphorylated IR (p‐IR), Protein Kinase B (PKB or Akt), phosphorylated Akt (p‐Akt), Cav‐3, Glut‐4, and actin (as loading control). The total protein expression of IR, Cav‐3, and Glut‐4 was unaltered with treatment. However, there was a significant increase in the total expression of Akt in cells treated with TNF‐α. In addition, there was a significantly higher activation of Akt and IR in cells treated with TNF‐α after 48 hours exposure. This data suggests that the effect of TNF‐α on insulin signaling in a result of a cross‐talk mechanism. This data will further the identification of novel signaling mechanisms leading to insulin resistance as a prerequisite to improved diagnosis and therapeutic treatments for diabetes.
Comparison of acute supplementation with whey, soy or carbohydrate on lymphocyte responses to resistance exercise
Authors: Freidenreich Daniel Joshua , Hiester Kevin , Mastro Andrea , Volk Brittanie M , Aristizabal Juan Carlos , Kraemer William J , Volek Jeff Scott
Published: June 2021

Little is known about the effects of acute supplementation with soy (Soy), whey (WHEY) or carbohydrate (CHO) on lymphocyte subset responses to resistance exercise. We randomized untrained men and women (23.0 ± 3.2 yrs, 72.8 ± 13.0 kg, 26.3 ± 10.6 %fat) to supplement with WHEY (n = 19), Soy (n = 14) or CHO (n = 18) immediately after performing a squat exercise protocol (6 sets, 10 repetitions, 65% of 1RM, 2 min rest between sets). Changes in circulating lymphocyte (LY) counts prior to (Pre) and 15 and 60 min post exercise (PE) were assessed. Most lymphocyte subsets, except NK cells, showed a decrease below Pre at 60 min PE in all supplement groups. NK cells in the Soy group were elevated above Pre at 15 min PE. Soy demonstrated a smaller decrease below baseline for total CD3 T cells at 60 min PE, most likely the result of a smaller decrease in CD8 T cells at 60 PE in comparison to CHO. CD4 T cells although not different from Pre were more elevated in Soy than CHO at 15 min PE. These differences in the magnitude and temporal response patterns between supplement groups indicate that Soy may contain a component that attenuates the post‐exercise lymphopenia. Funded by the Dairy Research Institute.
Post‐exertional blood pressure response following swim exercise is dependent on training status
Authors: Lakin Robert , Notarius Catherine F , Thomas Scott , Goodman Jack
Published: June 2021

Aerobic exercise is known to elicit a post‐exercise hypotensive (PEH) blood pressure (BP) response in both untrained and endurance‐trained subjects. However, it is not known if swim exercise produces a similar effect. We tested the hypothesis that an acute bout of swimming would fail to elicit a PEH response compared to an equivalent bout of stationary cycling, regardless of training state. 11 untrained and 10 trained healthy normotensive (systolic BP (SBP)/diastolic BP (DBP) < 120/80 mmHg) men and women (age 23 ± 1) underwent 30 min of intensity‐matched cycling and swimming exercise to assess changes in BP during a 75 min seated recovery. Cardiac output, stroke volume, total peripheral resistance, heart rate (HR), and heart rate variability (HRV) were also assessed. In untrained subjects, PEH was similar between cycling (−3.1±1 mmHg) and swimming (−5.8±1 mmHg). Trained individuals did not exhibit a PEH response following swimming (0.3±1 mmHg), yet had a significant drop in SBP at 50 min post cycling (−3.7±1 mmHg) (P < 0.05). The absence of PEH after swimming may reflect slower vagal reactivation (high frequency spectral component of HRV) (25 and 50 min) (P < 0.05), consistent with a significant increase in HR between modalities (P < 0.05). These results suggest that training may limit the potential for an effective PEH response to aerobic swimming. Funding was provided by the University of Toronto.
Effects of Hypoxic Training on Serum Lipoprotein of Obese Rats
Authors: Lu yingli , Zhang li , Feng lianshi , Xu jianfang , He zihong
Published: June 2021

Objective

To study the effects of hypoxic training on serum lipoprotein of obese rats.

Method

130 obese rats were divided into 13 groups at random:control group, normoxic training 1,2,3,4 week group, hypoxia sedentary1,2,3,4 week group, and hypoxic training 1,2,3,4 week group. The rats in training groups ran on a treadmill for 60 min a day at 26 m/min in normoxia or 21 m/min in hypoxia(13.6%), 6 days a week for 1–4 weeks. After corresponding week, the serum level of cholesterol(TC), triglyceride(TG), high‐density lipoprotein(HDL), low‐density lipoprotein (LDL), apolipoprotein AI (ApoAI) and apolipoprotein B(ApoB) were measured.

Results

1) hypoxic training significantly reduced serum TC, TG and ApoB of obese rats respectively in 1–4week,3–4week and 1–2week, and serum TC, TG and ApoB were also lower in above hypoxic training groups compared with the corresponding week groups of normoxic training and hypoxia sedentary. 2) hypoxic training significantly increased serum HDL and ApoAI of obese rats respectively in 3–4week and 1–4week, and serum HDL and ApoAI were also higher in above hypoxic training groups compared with the corresponding week groups of normoxic training and hypoxia sedentary.

Conclusion

Hypoxic training can ameliorate serum lipoprotein in obese rats especially in 3–4 week, the effect is also better than hypoxia sedentary and normoxic training.

(Supported by NSFC: Grant No. 30971415)
Examination of luminal acetylcholine on CFTR activity in porcine airway epithelium
Authors: Dittrich Nikolaus P , Clauss Wolfgang G , Kummer Wolfgang , Fronius Martin
Published: June 2021

The CFTR plays a crucial role in airway fluid homeostasis since mutations are associated with cystic fibrosis lung disease. Luminal acetylcholine (ACh) has been identified to activate Cl− secretion in airway epithelia although the underlying mechanisms and targets are not identified. The present study investigates whether or not the CFTR is a putative target of luminal ACh.

Porcine tracheal preparations were mounted in Ussing‐chambers for short‐circuit‐current (ISC) recordings. ACh (100 μM) was applied from the luminal side. CFTR‐dependent Cl− secretion was assessed by application of IBMX/forskolin (100/10 μM, luminal) in the presence of amiloride (10 μM, luminal). GlyH‐101 (50 μM, luminal) was used to block CFTR currents.

Application of luminal ACh significantly increased ISC. This ACh effect was not changed by amiloride. In the presence of amiloride the IBMX/forskolin‐activated current (ΔII/F) was 32.4±6.4 μA/cm2 (n=6). GlyH‐101 reduced ΔII/F to 13.1±3.7 μA/cm2 (n=6) demonstrating that a substantial portion of that current is CFTR dependent. However, GlyH‐101 did not altered the effect of luminal ACh (ΔIACh: 11.3±2.3 (control); 10.1±2.2 (GlyH‐101); n=5).

From these results it can be concluded that luminal ACh activates ion transport processes in porcine airway epithelium. Nevertheless this neither includes CFTR‐dependent Cl− secretion nor ENaC‐dependent Na+ reabsorption.
Omeprazole inhibits biliary proliferation during extrahepatic cholestasis
Authors: Afroze Syeda , Jensen Kendal , Rahal Kinan , Guerrier Micheleine , Glaser Shannon
Published: June 2021

Omeprazole (OME) has been shown to reduce liver regeneration and enhance gastrin serum levels. Since gastrin inhibits biliary growth, we tested the hypothesis that omeprazole inhibits cholangiocyte proliferation in BDL rats by increasing gastrin serum levels.

Methods

In vivo, male Fischer 344 rats (immediately BDL) were treated with vehicle or OME (40 mg/Kg BW daily) for 1 wk. We measured changes in: liver histology, and intrahepatic bile duct mass in liver sections; H3 histone and PCNA and secretin receptor (SR) gene expression; basal and secretin‐stimulated cAMP levels in cholangiocytes bile and bicarbonate secretion in bile fistula rats. Changes in SR and secretin‐stimulated secretion are key functional tools for evaluating the degree of biliary hyperplasia.

Results

Chronic in vivo administration of OME to BDL rats increased gastrin serum levels and reduced biliary proliferation and hyperplasia and decreased SR expression and significantly inhibited the stimulatory effects of secretin on cAMP levels and ductal secretion compared to vehicle treated controls.

Conclusion

The usage of OME may be important for the management of chronic cholestatic liver diseases.
Cyclooxygenase (COX)‐1/prostaglandins (PGs) and their receptors involved in modulating gastric mucosal integrity under stress conditions
Authors: Amagase Kikuko , Izumi Nahoko , Takeuchi Koji
Published: June 2021

Nonsteroidal anti‐inflammatory drugs themselves are not only ulcerogenic in the gastrointestinal mucosa but also potentiate the gastric ulcerogenic responses to various stimuli including stress. In the present study, we investigated the roles of COX isozymes/PGs and their receptors in the mucosal defense against cold‐restraint stress‐induced gastric lesions. Male C57BL/6 wild‐type (WT) mice and those lacking COX‐1 or COX‐2 as well as those lacking EP1‐, EP3‐ or IP‐receptors were used after 18 h fasting. The animals were restraint in ballman cages and kept in cold room at 10°C for 90 min. Cold‐restraint stress induced multiple gastric lesions in WT mice. These lesions were significantly worsened by pretreatment of indomethacin, loxoprofen or SC‐560, but not celecoxib. Likewise, these lesions were aggravated in the animals lacking COX‐1 but not COX‐2. The gastric ulcerogenic response to cold‐restraint stress was also aggravated in the animals lacking IP‐receptors but not EP1 or EP3 receptors. These results suggest that COX‐1/PGs play a crucial role in gastric mucosal defense during cold‐restraint stress, and this action is mediated mainly by the activation of IP receptors.
Distribution of ghrelin and GHS‐R in the rat gastric mucosa: effects of early weaning and consequences to growth control
Authors: Gama Patricia , Zulian Juliana Guimaraes , Bittar‐Rodrigues Natalia Martins
Published: June 2021

During postnatal development, maternal milk is the main source of nutrients, hormones and factors that promote growth and the functional maturation of the gastrointestinal tract. Early weaning is the abrupt interruption of suckling and it changes the development of the stomach, which represents the major site of ghrelin synthesis. In the present study, we evaluated the short‐term effects of early weaning on gastric ghrelin concentration and conducted a detailed investigation on the distribution of growth hormone secretagogue receptor (GHS‐R) in the rat gastric mucosa during the third postnatal week. In addition, we studied whether ghrelin is part of cell proliferation and differentiation control in gastric epithelium, and to that we used a receptor antagonist. We observed an increase of ghrelin immunostained cells in pups submitted to early weaning. After RT‐PCR, we observed that GHS‐R expression was not altered by dietary change and this result was confirmed through Western blot. We also identified GHS‐R immunolabeled cells along the gastric gland. The antagonist [D‐Lys‐3]‐GHRP‐6 reduced DNA synthesis index, as determined by BrdU incorporation followed by immunohistochemistry, but it did not alter mucous neck cell differentiation. Therefore, we found that ghrelin and GHS‐R are distributed in the gastric mucosa during the third postnatal week and that early weaning increased hormone levels in the gastric epithelium, without changing its receptor. We suggest that ghrelin modulation might be involved in the control of cell proliferation, which is essential for stomach growth. Supported by CNPq and FAPESP (2011/17415–3; 2011/07089–1).
Insight into oxygenation levels within 3D cell models and its impact on cell metabolism
Authors: Papkovsky Dmitri B , Carey Conn , Morten Karl , Dmitriev Ruslan I , Hynes James
Published: June 2021

Appreciation that typical culture conditions reflect nonphysiological, hyperoxic states for most cell types, has spurred a trend to the use of 3D models and controlled O2 conc. to better reflect the in vivo condition. Importance of cellular O2 conc. is underscored by its impact on processes as diverse as stem cell differentiation or tumour drug resistance development. While control of ambient O2 is easily achievable, measurement of oxygenation at the monolayer and analysis of the critical O2‐ sensitive processes has presented significant challenge through lack of robust quantitative tools. We present a novel multiplexed fluorescence method using dedicated O2‐sensitive probes, allowing robust quantitative measurement of O2 conc. in biological specimens. Both intracellular oxygen and glycolytic flux are measured in parallel across range of 3D models and the effect of applied oxygen tension on tissue oxygenation and cell metabolism examined. Data presented illustrates that O2 conc. within 3D structures can deviate significantly from the environmental oxygen conc., and this can impact significantly on the balance between oxidative and glycolytic ATP generating pathways. These new high throughput (plate reader) and high content (imaging) offer the potential for new and detailed insight into the impact of physiological oxygen and its perturbation on metabolism and intracellular signalling.
The application of Chinese Visible Human dataset
Authors: Zhang Shaoxiang , Jie Bin , Tan Liwen
Published: June 2021

The objective of the study was to establish the Chinese Visible Human (CVH) dataset and to explore its application on clinical field and basic research.

We have achieved five CVH dataset up to 2003. The CVH project achieved greater integrity of images, easier blood vessel identification, and free of organic lesion. The establish of CVH dataset was done in a low temperature laboratory, which contributed to prevention of small structures from falling off of the milling surface and better integrity of images.

We're taking our step toward application by acquiring new data sets, performing segmentation and setting up a platform of computer‐assisted medicine. We have established the three‐dimensional (3D) digital model for transjugular intrahepatic portosystemic shunt pathway and model for the craniocervical junction region by using the serial sectional images from CVH dataset. These models correctly reflected the complicated anatomic structural characteristics and spatial adjacency relationships for diagnostics and interventional therapy. We also use the dataset to display anatomic features of the superior mediastinal and the left extraperitoneal space for diagnostic imaging, which is better than CT image at visualizing the small soft tissue structures. In additionally, the CVH dataset was used to construct Chinese adult anatomical models for evaluation of radio frequency electromagnetic field exposures. Further more, we use the CVH sectional image and the Duel‐Sources CT image to establish the 3D visible model of heart to display the third‐level branch of the coronary artery, and combine with the echocardiogram to diagnose the coronary heart disease by Beta ultrasound. Our study on the clinical application of CVH dataset will go on.
Acute intermittent optogenetic stimulation of glutamatergic NTS neurons induces sympathetic long‐term facilitation
Authors: Yamamoto Kenta , Bathina Chandra Sekhar , Rajulapati Anuradha , Franzke Michelle , Mifflin Steve
Published: June 2021

Acute intermittent hypoxia (AIH) induces phrenic and sympathetic long‐term facilitation (LTF). Phrenic LTF can be induced by carotid sinus nerve stimulation, suggesting that hypoxia is not necessary to generate phrenic LTF. To determine if hypoxia is necessary to generate sympathetic LTF, we investigated the effect of acute intermittent optogenetic stimulation (AIO) of glutamatergic NTS neurons on renal sympathetic nerve activity (RSNA) in isoflurane anesthetized, vagotomized and artificially ventilated Sprague‐Dawley rats. AIH was performed by ventilation with 10% oxygen for 1min every 6min for a total of 10 exposures to hypoxia. In a separate group of animals, AAV containing CaMKIIa ‐ hChR2(H134R) ‐ mCherry was injected into NTS to express channelrhodopsin2 in glutamatergic neurons and the same pattern of stimulation as in the AIH protocol was used to stimulate the NTS (20 Hz, 5ms duration and 600mA for 1min every 6min for a total of 10 stimuli). Arterial Pressure (AP), RSNA and heart rate (HR) were compared at baseline (before) and 60min after AIH (n=6) or AIO (n=2). AIH increased AP and HR (P<0.05). RSNA was increased by 85% measured 60min after AIH (P=0.02). AIO increased AP and HR. RSNA similarly increased by 60% measured 60min after AIO. These results suggest that acute intermittent direct stimulation of glutamatergic NTS neurons can induce sympathetic LTF in the absence of hypoxia. Supported by HL‐088052
N‐Terminal Propeptide of Type III Procollagen (P3NP) Responses to Resistance Exercise in Older Adults
Authors: Fragala Maren S. , Jajtner Adam R , Beyer Kyle S , Wells Adam J , Townsend Jeremy R , Emerson Nadia S , Scanlon Tyler C , Stout Jeffrey R , Oliveira Leonardo P , Hoffman Jay R
Published: June 2021

Circulating P3NP, a peptide fragment cleaved from type 3 procollagen during collagen synthesis, is lower in older adults than younger adults and is predictive of changes in lean muscle mass. The purpose of this investigation was to determine whether circulating P3NP changes in response to resistance exercise training and if potential changes correspond to changes in muscle strength or quality (MQ). Twenty‐one older adults (69.4±5.2 y; BMI =28.1±5.4 kg·m−2) completed a 6‐week resistance training program in a randomized, controlled design. Resting circulating plasma P3NP, lean muscle mass, cross sectional area of the rectus femoris, muscle strength, and muscle quality were determined at baseline and after the intervention or control period by ELISA, DXA, ultrasound, leg extension 1‐RM prediction, and relative strength, respectively. P3NP changes were evaluated with repeated measures ANOVA and relationships between variables were evaluated with bivariate correlations. Short term resistance training did not result in uniform changes in circulating P3NP. Interestingly, baseline P3NP (mean ± SD = 4.91 ± 2.5 ng·mL−1) correlated to lean body mass (r=.471, p=.031) and muscle cross‐sectional changes (r=.527, p=.014), but not changes in strength or muscle quality. While further research is needed, P3NP appears to be reflective of muscle mass but not muscle quality.

This research was supported from by grants from the Learning Institute for Elders (LIFE) and the Toni Jennings Exceptional Education Institute.
An in vitro injury model to examine pericyte‐skeletal muscle cell cross talk
Authors: LaBarbera Katherine E , O'Fallon Kevin S , Clarkson Priscilla M , Witkowski Sarah
Published: June 2021

This study aimed to evaluate an in vitro model of cross talk between pericytes and skeletal muscle cells following scrape injury. Nuclear factor‐kappa B (NF‐κB) and monocyte chemoattractant protein‐1 (MCP‐1) are important in the in vivo response to muscle stress and were chosen to evaluate the model. C2C12 cells were co‐cultured with human primary pericytes (HPPs) in transwell inserts. Nuclear NF‐κB/p65 DNA binding activity and MCP‐1 concentration were quantified via ELISA at baseline (BSLN), 3h, 6h, and 24h following scrape injury (INJ) to C2C12 cells. In C2C12 cells, p65 DNA binding activity was significantly elevated at 3h (2.5 fold, p=0.027) and 24h (3.57 fold, p=0.001), relative to BSLN, with no effect of INJ (p=0.698) relative to CON. In HPPs, p65 DNA binding activity was increased relative to BSLN at 6h (2.0 fold, p=0.007), and 24h (2.33 fold, p=0.001). HPPs trended towards greater p65 DNA binding activity in INJ compared to CON (p=0.085) and in comparison to C2C12 cells (p=0.079). At 24h, HPP MCP‐1 secretion was first detected and exceeded C2C12 MCP‐1 secretion (2.1 fold, p<0.001). These data reveal 1) this model is useful for evaluating secreted proteins involved in pericyte‐skeletal muscle cross talk, which are mostly unknown at this time, and 2) novel information regarding HPP MCP‐1 secretion in response to C2C12 scrape injury. This research was supported by an ACSM Foundation Doctoral Student Research Grant.
The activity and molecular interactions of extracellular EMMPRIN
Authors: Kendrick Agnieszka A , Eisenmesser Elan Z
Published: June 2021

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) is highly expressed in multiple cancers, particularly in pancreatic cancer where it stimulates the secretion of entire classes of proteins including matrix matelloproteinases (MMPs) and pro‐inflammatory cytokines. Although, EMMPRIN was initially identified as a cellular transmembrane glycoprotein, extracellular EMMPRIN has recently emerged as a key player in disease progression. The described study here aims to directly characterize glycosylated extracellular EMMPRIN in regard to its stimulatory activity and target interactions, thereby unraveling a critical mechanism that underlies disease progression. We have developed recombinant expression systems to purify both unglycosylated and glycosylated extracellular EMMPRIN. Our biological studies have assessed the difference between unglycosylated and glycosylated extracellular EMMPRIN activity in pancreatic cancer cells. Furthermore, we performed crosslinking pull‐down experiments, followed by mass spectrometry to identify the specific extracellular EMMPRIN interacting partners. Thus, our studies have determined the functional influence of glycosylation on the activity of extracellular EMMPRIN, and have begun to characterize the interaction partners of extracellular EMMPRIN. EZE is supported by NIH application number 1R01g‐01A1.
Temporal Changes in the Acetylation Profile of Skeletal Muscle Proteins during Atrophy
Authors: Ryder Dan James , Judge Andrew R
Published: June 2021

Skeletal muscle is a plastic tissue that changes in response to several different stimuli. Pathologies such as septicemia and cancer lead to extensive skeletal muscle atrophy, causing poor quality of life and death. Modulation of protein acetylation has recently been linked to skeletal muscle atrophy. During muscle disuse, acetyltransferase over‐expression inhibits the expression of several atrophy biomarkers. Moreover, histone deacetylase (HDAC) 4 and HDAC5 knockout mice are resistant to skeletal muscle atrophy. This suggests that protein deacetlyation is an important mechanism for mediating muscle atrophy. The aim of this study was to determine the extent to which acetylation is altered among proteins expressed in skeletal muscle over a time course of atrophy. To induce atrophy, rats were cast immobilized for 0, 2, 4 or 6 days and the soleus muscle was harvested for identification and quantification of acetylated peptides via mass spectrometry. Gene expression profiling confirmed increased expression of MuRF1 and atrogin‐1 transcripts indicating successful induction of the atrophy program. We identified 107 proteins that showed significant changes in acetylation levels in atrophied muscle compared to weight bearing controls. Total acetylation of skeletal muscle protein was significantly decreased during atrophy by 58%, 18%, and 38% at days 2, 4, and 6, respectively. Motor/contractile and mitochondrial proteins were highly enriched for deacetylation. In contrast, hyperacetylation was observed within a subset of chromatin and enzymatic proteins. We conclude that protein deacetylation is a common feature among diverse classes of regulatory proteins during muscle atrophy.
STRA6: A gatekeeper of neuronal vitamin A homeostasis
Authors: Amengual Jaume , Zhang Ning , Maeda Tadao , Palczewski Krzysztof , von Lintig Johannes
Published: June 2021

Mutations in the STRA6 gene are associated with fatal Human Micro‐ophthalmic Syndrome 9. We generated a mouse model to elucidate the biochemical basis of this congenital disease. These mice failed to transport vitamin A across the blood‐retina and ‐ brain barrier and developed blindness, brain anomalies and hydrocephalus. Restriction of embryonic vitamin A supply extended malformations to extra‐neuronal tissues, whereas pharmacological doses of vitamin A mitigated anomalies of STRA6‐deficient offspring. Our study established a critical role of STRA6 for retinoid homeostasis of neuronal tissues and identified the vitamin as stochastic modulator of the phenotypic characteristics of this disease.
The impact of a single dose of resveratrol on intramuscular signaling and whole body fat oxidation in humans
Authors: Williams Cameron Bryce , Zelt Jason G E , Simpson Craig A , Gurd Brendon J
Published: June 2021

Resveratrol's (RSV) mechanism of action in skeletal muscle has been characterized in cellular and murine models, but RSV's in vivo signaling pathways remain unconfirmed in human skeletal muscle. Overweight males (n=8; BMI, 30.5±3.6; VO2peak, 34.0±7.3 ml/kg) reported to the lab on 2 occasions following a standardized dinner and 12 h fast. Respiratory exchange ratio (RER) and a muscle biopsy were taken while fasted, and 2 hours after breakfast supplemented with 0.3g of RSV or a placebo (PL) pill. Experiments were performed in random order using a double blind crossover design. RER increased from 0.81±0.04 to 0.84±0.06 with PL but decreased from 0.85±0.07 to 0.81±0.05 following RSV. The interaction for RER approached significance (p=0.09). A significant effect of RSV treatment was observed for p‐p38 MAPK (interaction, p < 0.05) with the decrease observed after PL (−31±28%) being reversed following RSV (+7±13%). p‐AKT increased (p < 0.05) in a time dependent fashion following both RSV (+111±68%) and PL (+79±19%). While p‐ACC tended to increase with time this change did not reach significance in either group. Significant interactions were not observed for p‐AKT (p=0.12) or p‐ACC (p=0.47). These results suggest that a single dose of RSV can influence intramuscular signaling; specifically pathways involved in the control of mitochondrial biogenesis, and may increase whole body fat oxidation. This study was funded by NSERC
Opioid inhibition of NMDA‐dependent pain sensitization persists long after the resolution of inflammatory injury
Authors: Corder Gregory , Doolen Suzanne , Winter Michelle , He Ying , Wang Zaijie , McCarson Kenneth , Taylor Bradley
Published: June 2021

Endogenous opioids orchestrate an adaptive response that modulates neuronal excitability and fine‐tunes glutamatergic nociceptive transmission. Intraplantar injection of complete Freund's adjuvant (CFA) to C57Bl6 mice decreased mechanical thresholds, which peaked at 3d and resolved within 21d. Upon hypersensitivity resolution, intrathecal injection of mu‐opioid receptor (MOR) blockers, naltrexone (NTX) or CTOP, reinstated mechanical pain and evoked a 3‐fold increase in spinal ERK phosphorylation (pERK). Prior treatment with the NMDA‐R blocker, MK‐801, prevented both NTX‐precipitated pain and pERK. MOR‐G‐protein coupling was assessed by measurement of GTPγ[S35] binding in lumbar spinal cord slices, 21d after sham or CFA injury. Stimulation with the MOR selective agonist DAMGO revealed GTPγ[S35] binding Emax values of 58.02 ± 0.67% or 79.85 ± 7.35% after sham or CFA injection, respectively. Calcium imaging studies in spinal cord slices revealed that either NTX or CTOP potentiated glutamate‐evoked Ca2+ mobilization in CFA but not sham animals. We conclude that NMDA‐Rs drive the spinal sensitization that is masked by up‐regulated, tonically active MORs.
Comparison of plasma AngII and arterial pressure responses to subcutaneous AngII administration by implantable Alzet minipumps or external syringe pump
Authors: Kuroki Marcos Takuya , Osborn John W
Published: June 2021

Hypertension (HTN) induced by subcutaneous (s.c.) infusion of angiotensin‐II (AngII) using implantable osmotic minipumps is a common model of experimental HTN. However, the long‐term performance of these pumps are unclear. Therefore, we compared the temporal profiles of plasma AngII and arterial pressure (AP) induced by 2 week s.c. infusion of AngII (150ng/kg/min, 5uL/hr) with osmotic minipumps (n=7) to those induced using an external syringe pump (n=8) in conscious rats. Venous blood was collected during control, and days 3, 7, 14 of AngII infusion. Plasma AngII was assayed using a commercial ELISA kit. Plasma AngII in the two groups (minipump vs syringe pump) were 9±2 vs 19±6 pg/mL during control (n.s.), and 110±20 vs 70±10 (p=0.022), 40±10 vs 70±10 (n.s.), 35±9 vs 41±9 (n.s.) on days 3, 7, and 14 of AngII infusion. Plasma AngII levels on AngII day 14 was similar to those measured in a separate group of salt deplete rats (52±7 pg/mL), and peak levels were below those measured in salt deplete rats treated with furosemide (170±30 pg/mL). AP was similar between both groups until days 10–14 of AngII. By day 14, AP in the minipump group was lower (132±5) compared to the syringe pump group (148±4 mmHg; p<0.001). We conclude that this dose of s.c. AngII results in physiological levels of AngII but the modality of s.c. administration has an impact on the temporal profile of plasma AngII and AP during AngII‐salt HTN. (HL076312)
Serotonin is possibly involved in the anorexigenic and antidepressant effects of estrogen in ovariectomized rats
Authors: Nishimura Yuri , Mabuchi Kaori , Negishi Hiroko , Morimoto Keiko , Takamata Akira
Published: June 2021

Estrogen attenuates food intake and enhances c‐Fos expression in the suprachiasmatic nucleus (SCN) specifically during the light phase in ovariectomized rats, and also has the antidepressant effect. Serotonin (5HT) neurons play an important role in the food intake, the pathogenesis of depression, and the formation of circadian rhythm. To test the hypothesis that 5HT is involved in the anorexigenic and antidpressant effects of estrogen, we examined the effect of selective serotonin reuptake inhibitor (SSRI) administration on food intake, depression like behavior and c‐Fos expression in the SCN in ovariectomized rats. Rats were ovariectomized and implanted either with an estradiol (E2 group) or a cholesterol (Veh group) containing silicon tubing. SSRI was injected at the beginning of the light and dark phases for 10 days. SSRI attenuated the food intake specifically during the light phase in the both groups, and reduced depression‐like behaviors in the Veh group. The number of c‐Fos like immunoreactive cells in the SCN during light phase was larger in the E2 than the Veh group, and SSRI increased the c‐Fos expression in the ventrolateral SCN only in the Veh group. These suggest that 5HT is possibly involved in the estrogen‐induced anorexigenic and antidepressant effects by changing the response to photic stimulation.
Different hemodynamic responses of normotensive and hypertensive rats to intracerebroventricular infusion of NaHS
Authors: Sikora Mariusz , Jodlowska Maria , Kolaszynska Katarzyna , Ufnal Marcin
Published: June 2021

Hydrogen sulfide (H2S) has been found to be involved in the regulation of the circulatory system. Previously, we found that intracerebroventricular (icv) infusion of sodium hydrosulfide (NaHS), a H2S donor, evokes hypertensive response in normotensive rats. Here, we checked hemodynamic effects of icv infusions of NaHS in hypertensive rats. We measured mean arterial blood pressure (MABP) in male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats at baseline and during 45 min of icv infusions of either vehicle (Krebs‐Henseleit bicarbonate‐buffer) or NaHS solution at a dose of 30, 100 and 300 nmol/h. ICV infusions of vehicle did not cause significant changes in MABP. WKY rats infused with 30 nmol of NaHS showed mild hypotension, whereas the infusion of 100 nmol caused hypotension followed by an increase in MABP. NaHS at a dose of 300 nmol produced a hypertensive response. In contrast, SHR rats infused with all the tested doses of NaHS showed a decrease in MABP in a dose‐dependent manner. The results support our previous findings that H2S is involved in the neurogenic control of blood pressure. Furthermore, this study shows that hypertensive rats have altered responsiveness to H2S, which may suggest disturbances in H2S homeostasis in the brain of hypertensive animals. This work was supported by the National Science Center, grant 3G29 and Medical University of Warsaw, grant 1MA/PM11/12.
Baroreflex mechanism as a model for integration of genotype, phenotype, computational model, and pathway information within a user‐friendly graphical interface
Authors: Munzenmaier Diane H. , Zientek Amy , Carlson Brian , Tabesh‐Saleki Nazanin , Shimoyama Mary , Jacob Howard J.
Published: June 2021

Translational research requires effective integration of physiological and genomic data with informative predictive modeling. Our goal was to develop a tool that would allow physiology, genetics, and computational biology researchers to explore existing data and models within an intuitive pathway framework that allows logical integration of multiple, diverse datasets with physiological processes and mathematical equations developed to describe them.

A multi‐level diagram depicting the baroreflex mechanism was created using a program designed for the Rat Genome Database (www.rgd.mcw.edu) to model physiological pathways. The diagram was integrated with a well‐established baroreceptor model (Bugenhagen, et al, 2010). Images were imported, linked and labeled to identify various components of the process. Processes were linked to multiple datasets, genomic information, and computational model equations. Weekly meetings with physiologists, bioinformaticians, and computational physiologists elucidated the challenges in creating the model and established effective guidelines for enhancement of the tool. Additional communication with the graphical design artist and software developer allowed for refinement of the image quality and improvement in the ease of use and functionality of the tool.

Funding was made possible by NIH HL64541 (RGD) and the APS Frontiers in Physiology program.
Molecular Cloning of Green Fluorescent Protein in Escherichia coli
Authors: Shoneye Temitope , Lee Henry , Church George
Published: June 2021

Molecular cloning of green fluorescent protein (GFP) can be expressed as a functional transgene and enables new avenues of investigation, providing an easily detectable phenotype that can be used for studies. We hypothesized GFP can be expressed in E. coli cells. We sought to test the functional expression of a gene coding for agglutinin, from Vibrio natriegens. We tested for the functional expression of GFP from Aequorea victoria in E. coli because earlier work showed no positive results in the PCR or initial transformation reactions. We ran PCR on protein coding DNA (GFP), cloned GFP into plasmids and transformed the plasmids into E. coli cell by varying the number of templates, and increased the denaturing time from 10 to 20 sec. We also ran reactions with primers designed for blunt and cohesive ends cloning, and “touch‐down” PCR. We obtained a GFP sequence, designed primers and obtained correct products between 0.7 and 0.8 kb. We then digested the GFP product and the host vector with cohesive end cutters, KpnI and HindIII. Following ligation of the cohesive ends, we transformed the plasmids into chemically competent E. coli cells and tested the functional expression of GFP by measuring fluorescence. The results showed E. coli is able to functionally express GFP and that using the appropriate techniques, GFP expression in E coli is possible.
[Ca2+]i oscillations in renal epithelia caused by α‐hemolysin from Escherichia coli require ATP release and P2 receptor mediated signaling
Authors: Christensen Mette G. , Skals Marianne , Fagerberg Steen , Bjaelde Randi G. , Leipziger Jens , Praetorius Helle A
Published: June 2021

Urinary infections are commonly caused by α‐hemolysin (HlyA)‐ producing E. coli. HlyA forms pores in cell membranes that renders the attacked cells permeable to ions and water. In erythrocytes, the effect of HlyA is strongly amplified by purinergic signaling. We hypothesize that HlyA‐induced [Ca2+]i oscillations in renal epithelia is mediated by ATP‐dependent P2 receptor activation. Here we confirm that HlyA, added at a concentration that cause 50% lysis of human erythrocytes, initiate marked [Ca2+]i oscillatory activity in renal epithelia. This was quantified by live cell fluorescence microscopy in both MDCK cells and freshly isolated, murine thick ascending limb. The HlyA‐induced oscillations were reversible and did not cause permanent cell‐damage. These [Ca2+]i oscillations are completely prevented by non‐selective P2‐receptor antagonists (suramin/PPADS) and by ATP‐degradation (apyrase). To confirm these results, we tested the effect of HlyA on ATP‐biosensor cells. In native 132–1N1 that do not express any P2 receptors, HlyA barely caused any changes in [Ca2+]I. Transfection of the cells with a hP2Y2 receptor resulted in an extensive increase in the [Ca2+]i oscillatory activity, which is also sensitive to P2 receptor antagonists. These results suggest that HlyA triggers ATP‐release from renal epithelia, which via P2 receptor activation is responsible for the HlyA‐induced [Ca2+]i oscillations.
Experimental Ischemia/Reperfusion Model in Relation to Apototic Cell Death
Authors: Emre Mustafa , Demirkol Serhat , Dora Devrim D. , Unlukurt Isa , Topcu Zeki , Ozsarlak‐Sozer Gonen
Published: June 2021

Kidney failure is an important health issue all over the world. Therefore effective treatment strategies need to be developed to cure acute kidney failure. Ischemia initiates a series of biochemical reactions that may end up with cellular dysfunction and ultimate cell death via diminishing cellular energy level and accumulating toxic subtances. Tissue and organ death is therefore an inevitable result due to added harms exerted by neutrophils, which arose following reperfusion and released mediators. In this study we estimated the percentage of poliferative, necrotic and apoptotic cell death in a kidney ischemia/reprfusion model using Wistar albino rats. Ischemia was conducted in left kidney while the right ones served as control. The presence of the apoptotic cell was investigated in reperfused kidney. Our study was extended to cover testing the effect of antioxidant agent, N‐acetylcystein on apoptosis. We showed that a reperfusion with a 60 mins duration following a 60 mins of ischemia triggered apoptosis. Moreover, N‐acetylcystein significantly diminished apoptosis. These findings have potential importance in clinical applications because the 60 mins duration was designated as a critical period for kidney surgical operations in both process.
Hydrophobic residues as a key determinant in hexoses exchange urate mechanism mediated by human glucose transporter 9, hSLC2A9
Authors: Long Wentong , Cheeseman Chris I. , Witkowska Kate
Published: June 2021

Human GLUT9 (hSLC2A9) is a urate/hexose transporter expressed predominantly in the liver and kidney where human urate homeostasis is achieved. hSLC2A9 has two splice variants: 9a and 9b differing in their N‐termini and targeted differentially within polarized epithelia. The transporter shows trans‐acceleration between urate and hexoses, however, the mechanism is unknown. Hydrophobic residues in TM7 of GLUTs have been shown to play a role in hexose selectivity.

Objective

To investigate urate/hexose exchange mediated by SLC2A9 isoforms and their mutants.

Methods

hSLC2A9 isoforms and I335V mutants were overexpressed in X. laevis oocytes. Urate induced currents and I‐V curves were obtained using Two Micro‐Electrode Voltage Clamp (TEVC) recordings. Urate transport and hexose trans‐stimulation activities were determined by radiolabelled urate uptake and efflux measurements.

Results

TEVC studies demonstrated that the membrane potential plays a major role in urate transport mediated by hSLC2A9. Fructose was found to trans‐stimulate urate uptake and efflux in wild type hSLC2A9a; whereas it stimulates only urate efflux in the I335V mutant. These finding advance our understanding of how fructose intake is linked with serum urate levels and implicates hSLC2A9 as an important transporter that may contribute to pathophysiological conditions involving urate or fructose. Supported by CIHR.
High fat diet contributes to improper kidney AT1 activation and impaired sodium regulation through ENaC cleavage in a model of insulin resistance
Authors: Minas Jacqueline N. , Rodriguez Ruben , Awayda Mouhamed S. , Ortiz Rudy M.
Published: June 2021

High fat diet (HFD) is known to accelerate diabetic nephropathy (DN) while blockade of the renin angiotensin system (RAS) improves the proteinuria and antinatriuresis associated with DN. However, whether RAS blockade can improve defects in sodium (Na+) handling resulting from HFD induced insulin resistance remains unknown. To assess the effect of HFD on the epithelial Na+ channel (ENaC), which regulates Na+ transport and balance and is regulated by the insulin‐signaling pathway, we performed Western blot and urinary analyses on the following groups (6 weeks of treatment): 1) untreated, lean LETO, 2) LETO + HFD, 3) LETO HFD + ARB, 4) untreated OLETF, 5) OLETF + HFD, 6) OLETF + ARB, and 7) OLETF + HFD + ARB. ARB reduced SBP in both OLETF (114 ± 3 vs 151 ± 3 mmHg) and OLETF HFD (101 ± 6 vs 151 ± 2). Urine Na+ excretion (UNaV) decreased 50% in OLETFs compared to LETOs. These data suggest that impaired UNaV and overactivation of AT1 may contribute to the manifestation of hypertension in insulin resistant rats. Treatment with ARB increased UNaV in all treatment groups by 3 to 4 fold and HFD decreased UNaV by 70 and 30% in LETOs and OLETFs, suggesting that a HFD may contribute to activation of AT1 in the kidney, resulting in impaired Na+ regulation. Levels of cleaved γ‐ENaC (60 kDa) negatively correlated with UNaV indicating that cleavage in vivo is likely a key component in the regulation of UNaV and blood pressure in insulin resistant conditions.
Photoaffinity labeling the lipid binding site of mammalian diacylglycerol kinase
Authors: Petro Elizabeth Jane , Tu‐Sekine Becky , Rowland Meng M. , Eni Sammy Eni , Best Michael D. , DeVine Lauren , Cole Robert N. , Raben Daniel M.
Published: June 2021

We are taking a photoaffinity labeling approach to identifying the lipid binding site on mammalian diacylglycerol kinase (DGK). The probe includes a diacylglycerol(DAG)‐like moiety, except with ether linkages instead of acyl linkages to prevent chain migration, in order to target the probe to the lipid binding site on DGK. The probe also includes a benzophenone moiety, in order to covalently crosslink to whatever amino acids happen to be near it when excited with 360 nm light. In this way, the probe is expected to covalently modify the enzyme near the DAG‐binding site(s). When the probe is incorporated into liposomes, DGK's enzymatic activity is reduced in a probe‐ and UV‐dependent way, but only when the linker connecting the benzophenone moiety and the DAG‐like moiety on the probe is sufficiently short. Probes with longer linkers are able to be used as substrates by the enzyme, suggesting that they are able to access the active site but not to crosslink to the protein. Mass spectrometry studies are currently underway to map the site of crosslinking. Preliminary results show reduced amino acid coverage for in‐gel trypsin‐digested crosslinked enzyme as compared to control: these missing peptides are candidates for sites of crosslinking. This research is supported by Grant GM059251 from the National Institutes of Health.
Role of the Locus coeruleus purinergic system in cardiorespiratory control under normocapnic and hypercapnic conditions in unanesthetized male rats
Authors: Batalhão Luciane Helena Gargaglioni , Biancardi Vivian , Bícego Kênia Cardoso
Published: June 2021

Locus coeruleus (LC) purinergic neuromodulation is of particular interest because adenosine 5′‐triphosphate (ATP) acts as a neuromodulator in brainstem regions involved in cardiovascular and respiratory regulation such as the LC. Thus, the goal of the present study was to investigate the role of LC purinergic neuromodulation in cardiorespiratory responses during normocapnia and hypercapnia in unanesthetized Wistar male rats. We microinjected an ATP P2X receptor agonist (a,ß‐MeATP, 0.5 nmoL/40 nL and 1 nmoL/40 nL) and non‐selective P2 receptor antagonists (PPADS 0.5 nmoL/40 nL and 1 nmoL/40 nL; suramin, 1 nmoL/40 nL) into the LC. Pulmonary ventilation (VE, plethysmography), mean arterial pressure (MAP) and heart rate (HR) were determined before and after unilateral microinjection (40 nL) of a,ß‐MeATP, PPADS, suramin or 0.9% saline (vehicle) into the LC. Under normocapnic conditions, PPADS decreased respiratory frequency (f) and increased MAP and HR. Suramin increased MAP and HR. Hypercapnic conditions induced an increase in ventilation and a decrease in HR in all groups. Under hypercapnic conditions, α,β‐MeATP increased ventilation. PPADS increased MAP. Suramin increased both ventilation and MAP. Thus, our data suggest that purinergic signaling in the LC plays an important role in cardiorespiratory control under normocapnic and hypercapnic conditions in unanesthetized rats.