bluuebunny/crossref_metadata_2025_split huggingface dataset:
Various Physiological Factors for Regulating Activation Speeds in the Delayed Calcium‐Dependent Activation of ANO6/TMEM16F
Authors: Roh Jae Won , Nam Joo Hyun 
Published: April 2020

Anoctamin6/TMEM16F (ANO6) is a transmembrane protein that functions as a Ca2+‐activated lipid scramblase and Ca2+‐activated ion channel. Unlike other ion channels in its family, such as ANO1/TMEM16A or ANO2/TMEM16B, ANO6 shows unique characteristics of delayed ion channel activation in the presence of relatively high intracellular Ca2+. This property raises questions regarding its physiological function as an ion channel. Here, we demonstrate that the delayed activation of ANO6 ion channel activity can be modified by different splicing variant types and various intracellular physiological factors, including temperature, Mg‐ATP, and actin cytoskeleton. Human ANO6 has three splicing variants, designated 1, 2, and 5, with variant 1 showing the longest delayed activation, and variants 2 and 5 showing a relatively short delay in whole‐cell patch‐clamp experiments. Conversely, murine ANO6 has only one splicing variant which corresponds to human variant 1 ANO6, thus exhibiting long‐delayed activation. Using a human whole transcriptome database (e.g., TCGA, ISOexpress), we analyzed mRNA levels of each ANO6 splicing variant in human tissues. Interestingly, variant 5 was predominant in all healthy human tissues. This finding implies profound experimental and clinical importance, given that almost all ANO6 studies are performed using murine ANO6. Furthermore, the present study focused on elucidating the different activation mechanisms of ANO6 splicing variants. Similar to variants 2 and 5, variant 1 showed no delayed activation on the excised inside‐out patch‐clamp experiment, implying the existence of unknown cytosolic factors that modulate and delay the activation of ANO6 variant 1. We also discovered that increasing intracellular ATP and stabilizing the actin cytoskeleton extended delayed activation of ANO6 variant 1. Temperature also participates in ANO6 activation. Increasing the temperature to 37□ markedly reduced the delayed activation and increased the Ca2+ sensitivity to physiological levels. Specific mechanisms through which ATP, actin cytoskeleton, and temperature modulate delayed activation remains to be studied further. Collectively, the data obtained in the present study revealed that humans and mice possess different ANO6 splicing variants with different delayed activation kinetics, and many cytosolic factors modulate delayed activation of ANO6 variant 1 which corresponds to murine ANO6. This finding is important because most studies on ANO6 utilize murine ANO6. We posit that physiological and clinical research data should be considered separately when evaluating human and murine ANO6.Support or Funding InformationThis work was supported by a National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning (MIST) of the Korean government (No. NRF‐2015R1C1A1A02037738).
Characterization of Viraemic Cells in a Murine Model of EHV‐1 Infection
Authors: Awan Aftab , Tulp Orien L. , Einstein George P. , Field Hugh 
Published: April 2020

Equine herpes virus (EHV‐1) causes respiratory infection in equine, abortion, paresis, neonatal death, and retinopathy and the virus may become latent following initial infection. Virus entry is via respiratory route, and the virus replicates in ciliated and non‐ciliated epithelial cells of the respiratory tract and in Type 1 and Type 2 pneumocytes in the lung parenchyma. After viral replication in the respiratory system, the virus is disseminated to other parts of body via viraemic cells. The virus crosses the placenta which leads to abortion of live or dead fetuses without premonitory signs. Infected horses show transient immunity after natural or experimental infection and immune responses to EHV‐1, but the immunoprotective status begins to decline after few months of infection. Due to the transient immune response, recovered horses are not protected and thus are prone to subsequent re‐infection. Immunity is not long lived after experimental or natural infection, and as a result the development of an effective vaccine has remained a challenge. In this study viraemic cells were studied in a murine EHV‐1 infection model. Mice were infected intranasally and viraemic cells were studied on days three and five of infection (peak of infection). This study may help to identify the nature of viraemic cells and their role in the transient immune response to infection. Buffy coat cells were removed and stained with a fluorescent antibody test for EHV‐1 antigen, and lung specimens were subjected to transmission electron microscopy. Both techniques confirmed the presence of viraemic cells. These viraemic cells were further stained for EHV‐1 antigen and for CD4 or CD8 biomarkers and results are discussed re: possible identification of viraemic cells in a murine model and their role in transient immune responses.Support or Funding InformationSupported by Institutional Resources of the University of Cambridge, UK, the University of Health and Humanities, British Virgin Islands, the Einstein Institute, and University of Science, Arts & Technology, Montserrat BWI.
Anomalous AMPK‐Regulated Angiotensin AT1R Expression and SIRT1‐Mediated Mitochondrial Biogenesis at RVLM in Hypertension Programming of Offspring to Maternal High Fructose Exposure
Authors: Chan Julie Y.H. , Chao Yung-Mei 
Published: April 2020

Tissue oxidative stress, sympathetic activation and nutrient sensing signals are closely related to adult hypertension of fetal origin, although their interactions in hypertension programming remain unclear. Based on a maternal high‐fructose diet (HFD) model of programmed hypertension, we tested the hypothesis that dysfunction of AMP‐activated protein kinase (AMPK)‐regulated angiotensin type 1 receptor (AT1R) expression and sirtuin1 (SIRT1)‐dependent mitochondrial biogenesis contribute to tissue oxidative stress and sympathoexcitation in programmed hypertension of young offspring. Increased systolic and diastolic blood pressure, plasma norepinephrine level and sympathetic vasomotor activity were exhibited by young HFD offspring. Reactive oxygen species (ROS) level was also elevated in rostral ventrolateral medulla (RVLM) where sympathetic premotor neurons reside, alongside augmented protein expressions of AT1R and pg91phox subunit of NADPH oxidase, decrease in superoxide dismutase 2; and suppression of transcription factors for mitochondrial biogenesis, peroxisome proliferator‐activated receptor g co‐activator a (PGC‐1a) and mitochondrial transcription factor A (TFAM). Maternal HFD also attenuated AMPK phosphorylation and protein expression of SIRT1 in RVLM of young offspring. Oral administration of a HMG‐CoA reductase inhibitor, simvastatin, or an AMPK activator, metformin, to young HFD offspring reversed maternal HFD‐programmed increase in AT1R and decreases in SIRT1, PGC‐1a and TFAM; alleviated ROS production in RVLM, and attenuated sympathoexcitation and hypertension. Together these results suggest that dysfunction of AMPK‐regulated AT1R expression and SIRT1‐mediated mitochondrial biogenesis may contribute to tissue oxidative stress in RVLM, which in turn primes increases of sympathetic vasomotor activity and blood pressure in young offspring programmed by excessive maternal fructose consumption.Support or Funding InformationThis work was supported by research grants from the Chang Gung Medical Foundation to J.Y.H. Chan (CMRPG8C0053‐33 and OMRPG8G0011). Y.M. Chao was supported by a Postdoctoral Fellowship (MOST107‐2811‐B‐182A‐501) from the Ministry of Science and Technology, Taiwan.
Effects of Exogenous Lactate Intake During Exercise on Energy Metabolism and Energy Substrate Utilization in Mice
Authors: Jang Inkwon , Kim Jisu , Kyun Sunghwan , Lim Kiwon 
Published: April 2020

PurposeIn the past, lactate has been considered a by‐product of lack of oxygen in the skeletal muscle and accumulates in muscles and blood, which causing acidosis and muscle fatigue. However, lactate is always produce, removed or used regardless of presence of oxygen, and it has been identified as an important metabolite that maintain the constancy of glucose, ATP (adenosine triphosphate) in cells, changing the understanding of lactate. Nevertheless, the effects of exogenous lactate intake have not explained and there is no study of the interrelationship between ingestion time and exercise. Therefore, we aimed to investigate the effects of exogenous lactate intake before exercise on energy metabolism during exercise.MethodsThe male 7‐week‐old ICR mice were randomly divided into 2 groups. Con (vehicle) n = 8, Lac (1 g/kg of sodium lactate) n = 8. After 30 minutes oral administration lactate, during exercise metabolism was measured for 30 minutes at 18m/min and 6° slope. A breathing gas was measured for VO2: oxygen consumption, VCO2: carbon dioxide production, RQ: respiratory quotient, fat oxidation, carbohydrate oxidation, EE: energy expenditure.ResultsLac was no longer able to exercise for 30 minutes. From the total during exercise metabolism results, there were no significant differences between the two groups. But when the data were divided by 15 minutes, the overall metabolism of Lac tended to changes as the second half progressed. At 16 to 30 minutes, the RQ in Lac tended to increase, while VCO2, carbohydrate oxidation and EE showed significant differences with Con. In addition, the increase in RQ and carbohydrate oxidation caused the fat oxidation to decrease, but there was no significant difference between the two groups.ConclusionThis study confirmed that exogenous lactate intake 30 minutes before exercise contributes to higher VCO2, carbohydrate oxidation and EE in the second half of the exercise and increase relative intensity. Therefore, our results suggest that lactate intake as an exercise supplement has the potential to increase carbohydrate oxidation and EE relatively, and that it can have a positive effect on improving lifestyle diseases such as diabetes and obesity. However, despite these result, our research shows that acute intakes have increased only in the latter half of exercise and that data have been identified only with open circuit calorimetry system. Furthermore, it could be assessed by the molecular biological approach associated with metabolism in the skeletal muscle.Support or Funding InformationThis work was supported by the Ministry of Education of the Republic of Korea and the National Research Foundation of Korea(NRF‐2019S1A5A8033825)
Insulin‐like Growth Factor 1 Improves Cardiac Function after Acute Myocardial Infarction in a Prediabetic Mouse Model
Authors: Spychala Andre , Heinen Andre , Zimmerhofer Alexandra , Nederlof Rianne , Panjwani Priyadarshini , Gödecke Axel 
Published: April 2020

BackgroundType 2 diabetes mellitus (T2DM) is a major comorbidity worsening the outcome after acute myocardial infarction (MI). Reduced cardiac glucose uptake may be the underlying mechanism compromising cardiac function even under prediabetic conditions. Insulin‐like growth factor 1 (IGF1) is an anabolic hormone similar to insulin in molecular structure and signaling. IGF1 controls proliferation, differentiation and metabolism of cells. In previous studies we have shown that short‐term IGF1 treatment after MI improved cardiac function and reduced scar size in mouse hearts. Here we investigate whether IGF1 can maintain cardiac function also after MI under conditions of prediabetes.MethodsMice were fed standard chow or high fat/high sucrose diet (HFHSD) for 10 weeks to induce obesity and prediabetes. Phenotyping was performed by measurements of blood glucose, insulin levels, glucose (GTT) and insulin tolerance tests (ITT). Mice were subjected to 45 min left anterior descending coronary artery occlusion and 1‐week reperfusion. IGF1 or vehicle were given over 3 days using osmotic mini pumps. Heart function was analyzed echocardiographically before and one week after MI including strain analysis. In addition, a transcriptome analysis was carried out for both the area at risk and the remote area by next‐generation sequencing.ResultsHFHSD led to increased fasting blood glucose levels with up to 200 mg/dl and glucose intolerance. In line with this results, plasma insulin levels were elevated in HFHSD mice with 1.3 ng/L in comparison to 0.4 ng/l in standard chow mice. However, exogenous insulin was able to lower blood glucose in the ITT, indicating prediabetes but no T2DM. Functional analysis by EC indicated a slight improvement of ejection fraction (EF) in HFHSD mice with 71.1% in comparison to 64.0% in the control group. However, cardiac function after MI under prediabetic conditions (EF= 35.3%) worsened to the same extent as in the control group (EF= 34.4%). Treatment of IGF1 after MI preserved cardiac function not only in the control group but also in the prediabetes group, with higher EF (control: 45.5%; IGF1: 48.2%), higher stroke volume and lower end‐systolic volume. Strain analysis revealed that this improvement is rather by an improvement of the remote area than by the area at risk.ConclusionDespite a similar signal transduction mediated by IGF1 and insulin, IGF1 was able to preserve cardiac function after MI in a prediabetic mouse model. Thus, an IGF1‐based therapeutic intervention could be a promising approach to improve the worsened cardiac outcome after myocardial infarction even under prediabetic conditions and may also be protective under T2DM.Support or Funding InformationThis work was funded by the German Research Foundation (CRC 1116 “Master Switches in Cardiac Ischemia”, TP A06).
Acute and Chronic Effects of Prostaglandin E2 on NKCC2, NCC and ENaC in Ex Vivo Renal Tubules
Authors: Esteva-Font Cristina , Krogager Toke P. , Hoorn Ewout J. , Fenton Robert A. 
Published: April 2020

The major renal prostaglandin, prostaglandin E2 (PGE2), is enzymatically generated from arachidonic acid in a series of steps catalyzed by various enzymes including Cox1/2. PGE2 has modulatory effects on renal Na+ handling, but the molecular targets of these effects in the distal segments of the renal tubule (TAL, DCT, CD) are less well characterized. Here, we assessed acute (30 minutes) and long‐term (24 h) actions of PGE2 (0.1 nM to 10 μM) on various Na+ transport proteins in these segments using tubule suspensions isolated from mouse kidney. In the long‐term, no significant effects could be detected on the abundance of the NaCl cotransporter NCC, the Na‐K‐Cl cotransporter NKCC2 or the epithelial Na+ channel ENaC (αENaC). Altered phosphorylation of NCC (T58) and NKCC2 (T105) or proteolytic cleavage of αENaC was not observed. Similar results were obtained in the presence of Cox1/2 inhibition and no sex differences were observed. Acutely, no effects on NKCC2 or αENaC were observed, but phosphorylation of NCC was decreased. A similar decrease in NCC phosphorylation was detected in NCC‐expressing MDCK cells. Data mining of online kidney RNAseq databases suggested that prostanoid receptor EP4 was expressed in the DCT, and ongoing studies are centered on determining if the actions of PGE2 on NCC are via this receptor.Support or Funding InformationThis project has received funding from The European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska‐Curie grant agreement no 754513, The Aarhus University Research Foundation and NNF Laureate Research Grant ‐ Novo Nordisk Fonden
Structural analysis of determinants for high‐affinity binding of G proteins to muscarinic and histaminic receptors
Authors: Buenemann Moritz , Jelinek Volker 
Published: April 2020

GPCRs typically couple to a preferred G protein family. The question is, how receptors recognize their cognate G protein and how this selectivity is mechanistically achieved. Due to the enormous progress in resolving structures of GPCR ‐ G protein complexes, there is overwhelming evidence that the C‐terminus of Ga subunits binds into the cavity of GPCRs that is opened upon outward movement of helix 5 and 6 and thereby is intimately involved in the mechanism responsible for G protein activation. However, other structures of the G protein must contribute as well, as switching C‐termini results only partially in a corresponding switch of the G protein selectivity. Based on M3 receptors we previously found a correlation of the stability of the ternary complex under nucleotide‐free conditions with the binding affinity to the receptor and the potency of receptors to activate G proteins (Ilyaskina et al. PNAS, 2018).The muscarinic M3receptor and the histaminic H1 receptor were chosen as a model system to investigate possible structures of the G protein because they are able to bind to two different G protein classes with different affinities. Chimeric Gqo and Goq subunits were created, in which several parts of the G protein were exchanged with the respective ones of the other G protein class. The relative binding affinities to the receptors were then compared with the native Gq and Go subunits by using a FRET based assay performed in single permeabilized HEK293T cells. Our results show that the binding affinities of certain Gqo and Goq chimeric subunits to M3 and H1 receptors were similar to the native G proteins, carrying the same backbone. For other chimeras, the binding affinities were significantly changed, which could be attributed primarily to alterations in the dissociation kinetics. Specifically, the high affinity receptor binding properties of Gq proteins could be transferred onto Go‐chimeras by exchanging structures at the N‐ or the C‐terminus or in the middle of the G protein. Furthermore, the combination of several chimeric structures showed even stronger effects. In contrast, exchanging similar regions using Gαq as the backbone it was much more difficult to impair the high affinity of chimeric Gq proteins.According to these results, the very end of the C‐terminal α5 helix of the G protein seems not to be the crucial part in generating the high‐affinity binding to M3 and H1 receptors. However, it is still important, as in combination with other exchanged structures, the binding affinity was significantly altered. Furthermore, the N‐terminal region of this α5 helix might be more important than previously thought, as it had a pronounced impact on the binding affinity in our experiments.
Squalene Monooxygenase at the Nexus between Cholesterol Homeostasis and Proteostasis
Authors: Chua Ngee Kiat , Howe Vicky , Jatana Nidhi , Hart-Smith Gene , Scott Nicola A. , Thukral Lipi , Brown Andrew J. 
Published: April 2020

Cholesterol homeostasis is governed by cholesterol uptake, synthesis and efflux. Cellular cholesterol synthesis relies on numerous enzymes. One of these enzymes is squalene monooxygenase (SM), an endoplasmic reticulum localized rate‐limiting enzyme in the cholesterol synthesis pathway, previously implicated as another target for cholesterol‐lowering and recently gained increasing attention in cancer metabolism. Excess cholesterol stimulates the proteasomal degradation of SM, a classic example of negative feedback regulation to lower cholesterol production. The cholesterol‐accelerated degradation of SM requires the first 100 amino acids of SM (SM N100), which represents the cholesterol‐responsive degron of SM. How the SM N100 cholesterol‐responsive degron operates to fine‐tune SM levels is not well‐understood. Here, we dissected degron features of SM N100, revealing key mechanistic insights into degron features and protein factors mediating the degradation of this degron. We identified (1) a 12‐amino acid amphipathic helix (Gln62 – Leu73) that allows SM N100 to sense membrane cholesterol levels, (2) serine ubiquitination as a non‐canonical mode of ubiquitination to trigger protein degradation, and (3) valosin‐containing protein/p97 as a protein that facilitates the removal of the hydrophobic SM N100 degron from the endoplasmic reticulum membrane. Our findings highlight concerted actions of cholesterol‐sensing and protein degradation mechanisms in regulating SM protein abundance. We also discuss our observations in the context of degron features and sterol‐sensing mechanisms reported by other studies – defining general themes that make proteins amenable to destruction in response to fluctuating lipid levels.Support or Funding InformationThis work was supported by the National Health and Medical Research Council Grant 1060515, Australian Research Council Grant DP170101178, and a Gold Star award from University of New South Wales Sydney (to A. J. B). N.J. was supported by a Department of Biotechnology, Government of India, Research associateship. L.T. was supported by DST‐INSPIRE Faculty Grant LSBM‐45. G.H.S. was supported by the Australian Research Council Grant DP170100108. N.K.C. was funded by a University International Postgraduate Award (UIPA) from UNSW Sydney.
Muscle Fiber Atrophy With Single‐Limb Immobilization is Associated With a Significant Decrease in Muscle‐Derived α‐Klotho of Male Mice
Authors: Parker Emily , Khayrullin Andrew , Mendhe Bharati , Yu Kanglun , Hamrick Mark 
Published: April 2020

Loss of muscle mass associated with aging, known as sarcopenia, is attributed to an increased risk of fractures and falls among the elderly population. The mechanisms underlying the development and progression of sarcopenia are, however, not well‐understood. Recent studies suggest that the anti‐aging factor α‐Klotho (α‐KL) plays a role in muscle regeneration, though its relevance for muscle disuse atrophy and atrophy has not been examined. This study aimed to investigate the response of α‐KL and inflammatory factors after immobilization. Twelve‐month C57/Bl6 mice were subjected to two weeks of hindlimb immobilization, consisting of wrapping surgical tape from the pad of the foot, connecting to cast padding and winding several times around the hindlimb. A layer of vetwrap was put on top followed by multiple layers of tape for protection with a drop of super glue applied on the top of the foot. This immobilization technique induced muscle atrophy through significant reduction of relative quadriceps weight (p<0.005) and a significant reduction in fiber size (p<0.005) in the immobilized legs of wild type mice. A significant increase in body weight was seen in the male mice (N=9, p=0.006), while no difference was seen in the females. PCR investigation showed that immobilized muscles had significantly increased TNF‐alpha expression (p<0.05) by a mean change of 67.37%. α‐KL ELISA analysis of muscle lysate revealed that muscle α‐KL was significantly decreased by a mean change of 44.23% in immobilized male muscles compared to controls (p<0.05), while female muscles showed the same trend but was not significant (p=0.2). RT2 profiling of myogenesis and myopathy in mouse skeletal muscles revealed that ten genes were upregulated (Dysf, Dmpk, Prkag3, Akt1, Musk, Myf6, Acvr2b, Prkab2, Hdac5, Myh1) and six were downregulated (Igfbp3, Tnnt1, Myat, Crayb, Trim63, Mb). Transcription factor Sp1, which regulates α‐KL expression, is also involved in regulating three of these genes (Tnnt1, Dmpk, Acvr2b, Cryab). Our data reveal that immobilization simulated disuse atrophy resulting in increased TNF‐α expression which lead to decreased α‐KL in muscle cells in male mice. This indicates that perhaps treatment with α‐KL could alleviate atrophy seen with acute muscle injury.Support or Funding InformationThis research was funded by the National Institutes of Aging (NIA P01 AG036675).