bluuebunny/crossref_metadata_2025_split huggingface dataset:
Uncoupling protein (UCP)1 and exercise induced ‘browning’ of white adipose tissue is absent at thermoneutrality
Authors: Aldiss Peter , Lewis Jo , Ebling Fran , Bookcock David , Budge Helen , Symonds Michael 
Published: June 2021

Background and aimThe induction of thermogenic genes in white adipose tissue (WAT) of exercise‐trained rodents is commonly seen in animals housed at standard temperatures (e.g. ~20°C) despite not being seen in exercise‐trained humans1,2. Here, we hypothesised that raising and exercise training rodents at thermoneutrality (e.g. ~28°C) thus mimicking human physiology would negate the induction of thermogenic genes typically seen at standard housing temperatures.MethodsTwelve weanling (i.e. 3 weeks old) Sprague‐Dawley male rats were housed at thermoneutrality (28°C) and fed a high‐fat diet (HFD) to induce weight gain with (Ex) or without (C) exercise training. At 12 weeks of age Ex was commenced by swim‐training (i.e. 1 hour/day, 5 days per week) for 4 weeks. Metabolic assessment was undertaken in metabolic cages during the last 48h to assess energy intake, expenditure and physical activity in addition to the acute response to administration of a β3‐agonist (Mirabegron, 0.75mg/kg/d). Then, at 16 weeks of age following an overnight fast, tissues were taken for analysis of thermogenic genes and quantitative proteomics.ResultsEx attenuated weight gain, but did not affect food intake, energy expenditure, final body weight, fat mass, plasma insulin or glucose or HOMA‐IR. There was also no increase in energy expenditure in response to administration of Mirabegron with UCP1 mRNA in IWAT remaining undetectable in EX despite an upregulation of genes considered to be classical ‘browning’ inducers i.e. PGC1a, ADRB3 and DIO2. We found 189 proteins were differentially regulated in IWAT following exercise. Among the most significant were those regulating nuclear factor kappa B signalling (LRRFIP2), the mitochondrial electron transport chain (NDUFS6) and oxidative stress (SOD3). Gene ontology analysis demonstrated these enriched 51 groups based on their biological process including ‘regulation of alternative mRNA splicing, via spliceosome’, ‘DNA damage checkpoint’ and ‘regulation of type 2 immune response’ whilst only 13 groups were enriched based on their molecular function including ‘metallodipeptidase activity’, ‘ATPase regulator activity’ and ‘pre‐mRNA binding’.ConclusionUnlike at standard housing temperatures, exercise induced ‘browning’ of IWAT was absent in animals raised at thermoneutrality corroborating human data. Proteomic analysis further highlights alternate novel pathways in adipose tissue regulated by exercise training that could have functional relevance.Support or Funding InformationBritish Heart Foundation [grant number FS/15/4/31184/]This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sonic hedgehog binds to Patched molecules on sensory stem cells to initiate smell and taste function
Authors: Henkin Robert 
Published: June 2021

Smell and taste function are tripartite systems involving sensory receptors, nerves and brain. Pathological changes in these systems depend primarily on impaired receptor function. Receptors in these systems are unique in that they contain neither blood vessels nor lymphatics and do not exhibit mitosis. For their growth and development, sensory receptors depend upon stem cell stimulation with growth/transcription factors secreted into saliva for taste and nasal mucus for smell. There are many of these growth/transcription factors but one of the major ones is sonic hedgehog (SHH) which causes receptor growth and development. Many pathological entities inhibit smell and taste function and do so by inhibition of these growth/transcription factors including SHH. Treatment with phosphodiesterase inhibitors activates SHH secretion which corrects these various pathologies. However, the molecular mechanism by which SHH acts on these stem cells to activate them is unclear. Recent studies have demonstrated that the efficacy of SHH at a molecular level depends upon its binding to two Patched molecules at a distinct site on the cell membrane of these sensory stem cells. This binding yields a signaling competent complex which, in the taste and smell systems, initiate growth and perpetuation of olfactory and gustatory receptors and results in their growth and development. This binding initiates the olfactory and gustatory signals which initiate smell and taste function.Support or Funding InformationThis institution did not receive any funding from agencies in the public, commercial, or not‐for‐profit sectors.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMPK ablation aggravates colorectal tumorigenesis via epigenetic suppression of antioncogenes
Authors: Zhu Meijun , Sun Xiaofei , Du Min 
Published: June 2021

BackgroundThe incidence of colorectal cancer (CRC) is closely related to metabolic diseases. AMP‐activated protein kinase (AMPK) is known as a key metabolic regulator. The objective of study is to assess the regulatory role of AMPK in metabolic alteration, epigenetic modification and colorectal tumorigenesis.MethodsAMPKα1‐floxed mice was cross‐bred with Lgr5Cre mice to specifically knockout AMPK α1 gene in intestinal stem cells and their derived epithelial cells. The wild‐type and AMPK knockout mice were subjected to azoxymethane (AOM)/DSS induced‐CRC induction.ResultsAMPK stem cell specific knockout caused colonic hyperproliferation and pathological features in mice without CRC induction; augmented adenocarcinoma number and size as well as dysperplasia in AOM/DSS‐indiced mice model. The aggravated colorectal tumorigenesis induced by AMPK ablation likely resulted from alteration of metabolites, as indicated by reduced production of acetyl‐CoA, a substrate required for histone acetylation, and α‐ketoglutarate, an obligate substrate for ten‐eleven translocation hydroxylases (TETs) mediated DNA demethylation, in colonic tissues. Consistently, α‐ketoglutarate producing isocitrate dehydrogenase 1 (IDH1) content and the activity TET1 were markably decreased in AMPK deficient mice, correlated with reduced metastasis inhibitors, Adamts1 (A disintegrin and metalloproteinase with thrombospondin motif 1) and Mal (myelin and lymphocyte), as well as hypermethylation of Mal and Mgmt (O‐6‐methylguanine‐DNA methyltransferase) promotor regions.ConclusionData suggested critical roles of AMPK in integrating metabolism with epigenetic modifications and colorectal tumorigenesis.Support or Funding InformationUSDA‐NIFA#2018‐67017‐27517 and WSU ERI competitive grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caspases from Scleractinian coral show unique regulatory features
Authors: Clark Clay , Shrestha Suman , Tung Jussica , Swartz Paul , Grinshpon Robert , Mydlarz Laura 
Published: June 2021

Coral bleaching is an immune response that rejects compromised symbionts. Porites astreoides and Orbicella faveolata are coral species that exhibit different responses to stress. Disease‐resistant P. astreoides employs adaptive autophagic mechanisms when immunocompromised, while disease‐sensitive O. faveolata activates caspase‐mediated apoptosis. We characterized homologs of caspases from O. faveolata (O.fav‐3a and O.fav‐3b) and P. astreoides (P.ast‐7 and P.ast‐3). O.fav‐3a and P.ast‐7 contain a human initiator/inflammatory CARD‐like domain despite high sequence similarity to effector caspases. Experimental results from substrate‐phage display show that O.fav‐3a and P.ast‐7 prefer aspartate in the P1 position, which is corroborated by activity against canonical substrate, DEVD‐AFC. In contrast, substrate‐phage display studies showed that O.fav‐3b and P.ast‐3 prefer valine at the P1 position, but the enzymes are largely inactive on the canonical substrate VEID‐AFC, suggesting additional requirements for the active enzyme. We determined the structure of P.ast‐7 bound with DEVD‐FMK inhibitor to 1.57Å resolution by X‐ray crystallography. The data reveal a hydrogen‐bonding network with a unique insertion (RYP) in one active site loop and suggest that the sequence may be autoinhibitory when substrate is not bound. In addition, the structure reveals an N‐terminal peptide bound near the active site that may serve as a regulatory exosite.Support or Funding InformationUT ArlingtonThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Purification and enzymatic assay of BmaI1, a quorum sensing signal synthase
Authors: Kellner‐Rogers Julian , Ricciardi Meghan , Ulrich Scott Michael 
Published: June 2021

Quorum sensing is a bacterial cell‐cell communication system that functions through the synthesis, secretion, and detection of signaling molecules called autoinducers. Quorum sensing enables bacteria to assess their cell density and coordinate group behaviors that are advantageous at high cell density. Many bacteria that cause acute and chronic infections in humans use quorum sensing to control expression of virulence factors and formation of biofilms, so inhibitors of quorum sensing hold significant promise as novel antibacterial agents. By targeting bacterial virulence as opposed to growth and survival, quorum sensing inhibitors avoid the selective pressure for drug resistance that is inherent to traditional antibiotics.The main quorum sensing system in Gram‐negative bacteria is based on the synthesis and detection of acyl homoserine lactone (AHL) autoinducers. Efforts to inhibit AHL‐mediated quorum sensing have focused on developing synthetic AHL analogs that act as antagonists of AHL receptors. These compounds have been shown to block virulence controlled by quorum sensing, validating quorum sensing as an antibacterial drug target.The overall goal of the project is to develop inhibitors of AHL synthase enzymes, an alternative quorum sensing drug target that remains under‐explored. Towards this goal, we cloned and purified BmaI1, an AHL synthase from Burkholderia mallei. AHL synthases use S‐adenosyl methionine and acylated ACP protein as substrates. Generating Acyl‐ACP requires purification of ACP, and several sequential enzymatic modifications to make it suitable as an AHL synthase substrate. We have developed a small, synthetic substitute for acyl ACP that is utilized by BmaI1, avoiding the difficult and time‐consuming production of acyl‐ACP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Single particle cryo‐EM studies of membrane proteins
Authors: Cheng Yifan 
Published: June 2021

In the last few years, major technological advancements, particularly the development of new direct electron detection cameras and associated technologies, have enabled single particle electron cryo‐microscopy (cryo‐EM) to become the technique of choice for high‐resolution structure determination of many challenging biological macromolecules. We pioneered some of the technologies now being routinely used, and applied them to determine atomic structures of a number of integral membrane proteins, such as some major members of transient receptor potential (TRP) channel superfamily. Some of our studies paved the way of applying this method to a wide range of integral membrane proteins. Nowadays, atomic structures of membrane proteins that are refractory to crystallization are being determined by this method in a rapid pace.Technologically, we further aimed to enable high‐resolution structural studies of membrane proteins in near native lipid bilayer environment. To this end, we pioneered a number of approaches, including reconstituting membrane protein into lipid nanodisc or into saposin based lipid nanoparticles, and developed methodology of using conformational specific monoclonal Fab or other designed scaffoldings to facilitate image alignment of very small integral membrane proteins reconstituted into lipid nanoparticles. In a number of recent studies, we observed specific lipid‐protein interactions that play critical role in regulating the channel functions. In TRPV1 ion channel, as an example, certain specific lipid‐protein interactions enhance binding of a spider toxin to the channel through formation of a toxin‐lipid‐TRPV1 tripartite complex, and an endogenous phosphatidylinositol lipid occupies the capsaicin‐binding site of the channel. These observations provide important clues about physiological mechanisms of channel regulation.Support or Funding InformationThis work is funded by NIH grants: R01GM098672, S10OD020054 and S10OD021741.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chaperone interactions of human α‐crystallin homo‐oligomers with their physiological substrate γD‐crystallin
Authors: Acosta‐Sampson Ligia Isabel , King Jonathan 
Published: June 2021

The chaperone α‐crystallin, an ATP‐independent small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses, along with the γ and β family of Greek key crystallins. αA‐ and αB‐crystallin (αA‐ and αB‐crys) subunits (~ 20 kDa) form an hetero‐oligomeric, polydisperse complex of ~ 800 kDa in the lens. The chaperone function involves suppressing aggregation by binding aggregation‐prone species. Aggregates isolated from mature‐onset cataracts, the major cause of sight loss worldwide, contain damaged and misfolded forms of β γ‐crystallins. Human γD‐crystallin (γD‐crys) is a stable and long‐lived major structural crystallin localized in the lens nucleus. Human γD‐crys refolds in vitro through population of a partially folded intermediate with the C‐terminal domain folded and the N‐terminal domain disordered. This species partitions between productive refolding and competing aggregation pathways. The γD‐crys intermediates were populated by dilution out of GuHCl at 37°C, and subsequently incubated with α‐crys. Turbidity assays show that a 1:20 or 1:10 (monomer/monomer) ratio of γD‐crys to αA‐ or αB‐crys, respectively, suppressed aggregation by 90%. α‐Crys homo‐oligomers presumably suppress γD‐crys aggregation by binding γD‐crys partially folded intermediates formed during refolding. The α‐crys/γD‐crys complex were stable for many hours after initial incubation. It is not clear if the long‐lived nature of the complex reflects internalization of the substrate into the lumen of the α‐crys oligomer, or whether partially unfolded crystallin species are tightly bound to the chaperone surface.Supported by NIH Grant GM 17980 and NEI grant EY 015834 (to J.K.)
The sHSP and molecular chaperone, aB crystallin, regulates microtubule assembly
Authors: Clark John I , Houck Scott A. , Ghosh Joy G. 
Published: June 2021

Purpose:The small heat‐shock protein (sHSP) αB crystallin, which protects against unfolding and aggregation of proteins has a regulatory effect on microtubule assembly. The sequences responsible for αB crystallin ‐ microtubule interactions were identified and tested.Methods:Five interactive sequences in αB crystallin were identified and studied for their effects on the assembly of microtubules using synthetic peptides and mutants of αB crystallin. Assembly of microtubules was measured using a fluorescence DAPI assay (PloS one 6:3498).Results:αB crystallin inhibited microtubule assembly at molar ratios >2:1 αB crystallin:tubulin and promoted microtubule assembly at molar ratios between 1:4 – 2:1. The synthetic peptide corresponding to the αB crystallin interactive sequence, 113FISREFHR120, partially inhibited microtubule assembly. In contrast, the peptides 131LTITSSLSSDGV142 and 156ERTIPITRE164promoted microtubule assembly. Mutations in these sequences altered the nature of the interactions with microtubules.Conclusion:Three interactive sequences on the surface of human αB crystallin modulate microtubule assembly through the dynamic subunit model of small heat shock proteins.
REDOX PROPERTIES OF ATP‐SENSITIVE K+ CHANNELS IN BRAIN MITOCHONDRIA
Authors: Fornazari Maynara , de Paula Juliana , Castilho Roger Frigerio , Kowaltowski Alicia Juliana 
Published: June 2021

Brain mitochondrial ATP‐sensitive K+ channel (mitoKATP) opening by diazoxide protects against ischemic damage and excitotoxic cell death. We studied here the redox properties of brain mitoKATP. MitoKATP activation during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of reactive oxygen species (ROS) and cell death. Furthermore, mitoKATP activation in isolated brain mitochondria significantly prevented H2O2 release by these organelles, but did not change Ca2+ accumulation capacity. Interestingly, the activity of mitoKATP was highly dependent on redox state. Thiol reductant mercaptopropionylglycine prevented mitoKATP activity, while exogenous ROS activated the channel. In addition, the use of mitochondrial substrates that lead to higher levels of endogenous mitochondrial ROS release closely correlated with enhanced K+ transport activity through mitoKATP. Altogether, our results indicate that brain mitoKATP is a redox‐sensitive channel that controls mitochondrial ROS release.Supported by FAPESP, CNPq, Redoxoma, Guggenheim Foundation and the NIH.
The calcium dependent cysteine protease μ‐calpain is located in the mitochondrial intermembrane space
Authors: Garcia Matthew , Badugu Rama Krishna , Bondada Vimala , Joshi Aashish , Geddes James W 
Published: June 2021

The ubiquitous proteases, m‐ and μ‐calpain, are thought to be localized in the cytosolic compartment, as is their endogenous inhibitor calpastatin. As a result, it is unclear how calpains become activated. We previously demonstrated that μ‐calpain was found enriched in mitochondrial fractions isolated from rat cerebral cortex and SH‐SY5Y neuroblastoma cells (Garcia, et. al. 2005. BBRC 338, 1241–1247), but the submitochondrial localization of μ‐calpain was not determined. In this study, we localized the large catalytic calpain 1 subunit along with the regulatory calpain small subunit 1, which together form μ‐calpain, to the mitochondrial intermembrane space. The results further demonstrate that calpain 1 is imported into mitochondria and contains a mitochondrial targeting sequence in its NH2‐terminus. This targeting sequence is not present in the large subunit of m‐calpain, which is not imported into mitochondria. The localization of μ‐calpain to the mitochondrial intermembrane space is consistent with a role for μ‐calpain in the cleavage of intermembrane space proteins including apoptosis inducing factor, providing a potential mechanism of mitochondrial μ‐calpain activation contributing to caspase independent cell death.This work was supported by the NIH, USPHS grants; P01NS058484, P01AG010836, R01NS045726, T32DA022738 and by the Kentucky Spinal Cord and Head Injury Research Trust.
Enrichment of Phosphopeptides using Phos SpinTrap Fe, a new IMAC kit
Authors: Albenius Marianne , Risberg Gabriella , Salomonsson Inger , Hedlund Helena , Sjödahl Marika 
Published: June 2021

There are many different types of post‐translational modifications (PTM) identified till now of which protein phosphorylation is one of the most important in eukaryotic cells and plays an essential role for regulating various cellular processes.Immobilized Metal Affinity Chromatography (IMAC) is a common technique used for purification and enrichment of phosphopeptides and is based on the affinity of the metal ion for the phosphate group. Phos SpinTrap™ Fe is a new sample preparation kit based on Fe Sepharose™ 6 Fast Flow. The kit is designed for single use small‐scale phosphopeptide enrichment. A mixture of different phosphorylated proteins was spiked into a background of E. coli extract. The protein mixture was cleaved with trypsin and purified on a Phos SpinTrap Fe column. Several phosphopeptides were detected with masspectrometric methods after the enrichment. Conclusion: The Phos SpinTrap Fe kit shows high specificity for phosphopeptides and can be used for enrichment of phosphopeptides from complex samples.
A Fragment of Cartilage Collagen, Chondrostatin, Inhibits Migration of Breast Cancer Cells
Authors: Wang Zhepeng , Bryan Jennifer , Franz Carl , Siegel Adam , Wagenseil Jessica , Mecham Robert , Sandell Linda J 
Published: June 2021

Cell migration is a prerequisite for cancer invasion and metastasis, suggesting cell motility as a potential therapeutic target for cancer treatment. We have studied the interaction of procollagen type IIB N‐propeptide (chondrostatin) with several tumor cell lines. We found that chondrostatin binds to integrins αvβ3 and αvβ5 via RGD. In order to investigate downstream consequence of chondrostatin‐integrin interaction, we applied live cell imaging technology and phalloidin staining to investigate effects of chondrostatin on breast cancer cell migration and actin cytoskeleton reorganization.MB231 breast cancer cells were incubated with or without chondrostatin, and visualized on a Leica DMI 6000B scope. Images of 12 pre‐selected microscopic fields were captured every 10 min using a Q Imaging Retiga Exi Fast 1394 camera. The cells were tracked using ImageJ software. The length of track over time was graphed, and the velocity of cell movement was calculated by linear regression analysis. The cell migrating velocity was reduced by 30%.To begin to investigate the mechanism of the inhibition, the actin stress fiber was examined. Cells were treated with chondrostatin and actin stress fibers were stained with Alexa Fluor 546 Phalloidin and visualized by fluorescence microscopy. Actin fibers accumulated in cytoplasm when cells were incubated with chondrostatin.These results show that chondrostatin can inhibit MB231 cell migration and suggest that rearrangement of the actin cytoskeleton may be involved. NIH R01 AR36994
Insulin Induces Intracellular Calcium Transients through Activation of NADPH Oxidase in Myotubes
Authors: Espinosa Berta Alejandra , Hidalgo Cecilia , Jaimovich Enrique 
Published: June 2021

In cultured myotubes, insulin triggers intracellular calcium increases measured as fluo‐3 fluorescence. We occasionally see a fast calcium transient (first signal), but around 80s later, myotubes produce a second, oscillatory calcium signal. It is known that insulin signaling involves reactive oxygen species (ROS) production in many cell types. The aim of this work is to study the role of ROS, via NADPH oxidase activation in the mechanism that generates the second intracellular calcium increase. The second calcium signal was inhibited by two drugs (xestospongin B and U73122) that interfere with IP3 pathway. Calcium increase was also blocked by bisindolylmaleimide (BIM), and rottlerin, both inhibitors of protein kinase C. Using the probe 2′, 7′‐dichlorofluorescein diacetate (DCFH‐DA) we showed that insulin induced ROS production in myotubes. Both calcium increase and ROS production were abolished by diphenyl iodonium (DPI), NADPH oxidase inhibitor apocynin and siRNA against p47phox; a regulatory subunits of NADPH oxidase. These results point to a role of insulin‐induced ROS in triggering calcium increase via IP3 receptor. NADPH oxidase is probably activated by a PKC and then ROS modulate IP3 receptors to increase intracellular calcium.FONDAP 15010006