bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effects of Menkes Syndrome on the Immune System
Authors: Engelhart Morgan , Zhang Jessica , Gotschall Jennifer , Huffman David , Miller Keith Russell 
Published: June 2021

Menkes syndrome is an inherited disorder that affects copper levels in the body because of a mutation in the enzyme ATP7A. This protein is found in all cells throughout the body, except liver cells, and is essential for shuttling copper between two cellular locations. ATP7A can be found within the Golgi apparatus to supply copper to certain enzymes, but if copper levels are elevated in the cells environment, it can move to the cell membrane and shuttle copper out of the cell. When this protein is mutated, copper becomes poorly distributed throughout the cells of the body and can lead to reduced activity of many copper containing enzymes. This leads to brain abnormalities, bone weakness, degeneration of retina, and slowed development and growth. However, effects on the immune system and specifically macrophages are still unclear. These cells can become stimulated by foreign pathogens and secrete a cytokine, called Interferon‐gamma (IFN‐γ), which activates and recruits surrounding immune cells for an immune response. IFN‐γ stimulates production of reactive oxygen species (ROS) from metals within the cell to kill pathogens after phagocytosis. Due to its capability to aid in an immune response, it is hypothesized that IFN‐γ coupled with copper—a producer of reactive oxygen species—would activate macrophages and improve the cell's bactericidal effects. However, if ATP7A is mutated, and copper cannot be transported into the cell, its capacity to kill will decrease. Before experimentally determining the effects of IFN‐γ and copper on the immune system, the cytokine itself was synthesized. IFN‐γ encoding cDNA was amplified using PCR, and then annealed to the pET‐32‐Xa/LIC vector. The newly formed vector was successfully produced as determined by restriction endonuclease digestion, and then transformed into BL21‐DE3 E. coli. Induction of protein expression using IPTG was completed and visualized using SDS‐PAGE. IFN‐γ was isolated and purified using a HisTrap Nickel affinity column, and a SDS‐PAGE showed two bands, at 39 kDa (IFN‐γ) and 22 kDa (contaminated band). These results suggest a lack of purity due to an absence of protease inhibitors in solution, or low binding affinity to the nickel affinity column. Ongoing studies are observing macrophage phagocytosis and killing of bacteria in the presence and absence of IFN‐γ and copper to further understand the impact of Menkes syndrome on the immune system.Support or Funding InformationFunded by The Giving Well Family Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beneficial Metabolic Consequences of Acyl‐CoA Synthetase ACSVL3 Knockout in Glioma Cells
Authors: Watkins Paul , Shi Xiaohai , Kolar Elizabeth , Clay Emily , Liu Yanqiu 
Published: June 2021

We previously reported that knockdown or knockout (KO) of the fatty acid metabolic enzyme Very Long Chain Acyl‐CoA Synthetase 3 (ACSVL3; SLC27A3) in glioblastoma cells was beneficial. KO U87 glioma cells grew at a slower rate, became adherence‐dependent, and produced fewer, slower‐growing subcutaneous and intracranial tumors when implanted in NOD‐SCID mice. To understand the mechanisms underlying these changes, we investigated several possibilities. We initially demonstrated that lack of ACSVL3 did not slow the synthesis of membrane phospholipids necessary for the rapid proliferation of tumor cells in culture. We then investigated the possibility that ACSVL3 KO affected either apoptosis or autophagy. However, no differences in these processes between control and KO U87 cells were found. We next looked at cell cycle, and found that KO produced significant changes. In non‐synchronized cells, control U87 cells were mainly in G0/G1, while ACSVL3 KO cells were mainly in S‐phase. Both control and KO cells synchronized by serum starvation started out mainly in G0/G1, but by 12 hours KO cells shifted significantly toward S‐phase. Identification of the cyclins and cyclin‐dependent kinases that may be involved is in progress. Proteomic and metabolomic profiling indicated that in addition to effects on lipid metabolism, ACSVL3 KO produced changes in glucose and energy metabolism. Although glucose transporters GLUT1 and GLUT3 protein levels were reduced by KO, cellular uptake of labeled 2‐deoxyglucose was unaffected. However, oxidation of glucose to CO2 was reduced 40% by ACSVL3 depletion, and the cellular glucose level was 25% higher in KO cells. Glycolytic enzymes were upregulated by KO, but metabolic intermediates were essentially unchanged. Interestingly, lactate production and the levels of both LDHA and LDHB were elevated by ACSVL3 KO, and pentose phosphate pathway activity was lower in KO cells. Citric acid cycle enzymes, electron transport chain complexes, and ATP synthase protein levels were all reduced by ACSVL3 depletion. Mitochondria had a more punctate morphology in U87 cells, but were more elongated in KO cells. In conclusion, depletion of ACSVL3 affects many metabolic processes not directly related to lipid metabolism in human glioblastoma cells.Support or Funding InformationKennedy Krieger Research Administration fundsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lower Progesterone Receptors on Mast Cells Do Not Effect Blood Vessel Replication in Lipedema
Authors: Rosas Victoria , Al‐Ghadban Sara , Herbst Karen 
Published: June 2021

IntroductionLipedema (meaning edema in fat) and Dercum Disease (DD) are fat disorders in which accumulation of painful subcutaneous adipose tissue (SAT) affects more females than males, especially at times of female hormone change. Patients with both fat disorders are often misdiagnosed as obese. The purpose of this study was to determine if estrogen (ER) and progesterone receptors (PR) are different in lipedema versus DD in SAT and skin versus controls. These receptors are on mast cells that produce histamine causing leakage from blood vessels inducing hypoxia and angiogenesis. Progesterone is known to activate histamine release from mast cells. We aim to determine levels of ER and PR in SAT and if blood vessels replicate at a higher rate in lipedema and DD versus controls to help further understand these conditions and work towards finding a cure.Materials and MethodsImmunohistochemistry (San Diego Pathology, San Diego, CA) was used to test for the presence of PR, ER, Ki67 (marker of replicating cells), and CD117 (marker of mast cells).Results and DiscussionMast cell numbers were similar in control, DD and lipedema SAT (Figure). ER were not different in control, DD and lipedema SAT. PR were significantly lower in lipedema SAT. There was no difference in Ki67 in lipedema or DD blood vessels compared to controls.ConclusionLower numbers of PR in our data suggest mast cell secretions (histamine and others) could be higher inducing leakage from vessels and fluid collection in SAT. Fluid in the tissue should induce hypoxia and growth of more blood vessels. Despite higher PR on mast cells, lipedema blood vessels did not appear to be replicating at a higher level. With further research and additional samples, the relevance of elevated PR in lipedema tissue may become apparent.Support or Funding InformationResearch reported in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Special acknowledgement to the Minority Health Disparities summer research program at the University of Arizona, the College of Medicine Tucson Treatment, Research, and Education of Adipose Tissue Program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interorganellar Phosphatidylserine Transfer by Sec14 Family Protein Sfh1 in Saccharomyces cerevisiae
Authors: Mizuike Aya , Kobayashi Shingo , Horiuchi Hiroyuki , Ohta Akinori , Fukuda Ryouichi 
Published: June 2021

Interorganellar phospholipid transfer is critical for the generation and maintenance of uneven distribution of phospholipids in membranes and for the phospholipid synthesis through CDP‐DAG pathway in the yeast Saccharomyces cerevisiae. While involvement of lipid transfer proteins (LTPs) in directional lipid transport has been actively discussed, the precise mechanisms of interorganellar phospholipid transfer remain unsolved. We carried out a screening to identify genes involved in aminophospholipid transfer in Psd2 branch of the CDP‐DAG pathway and obtained SFH1, a SEC14 homolog. Overexpression of SFH1 recovered the growth of psd1Δ on non‐fermenting carbon sources without ethanolamine supplementation, and partially restored the mitochondrial and cellular PE levels. Sfh1 was able to transfer fluorescence‐labeled phosphatidylserine (PS) between liposomes in vitro. In addition, binding of Sfh1 to PS in vivo was suggested by the analysis of phospholipid species copurified with Sfh1 from yeast cells by ESI‐MS/MS. Subcellular fractionation suggested that a portion of Sfh1 is localized in the endosome and the Golgi where Psd2 is reported to be localized. Together with the results that overexpression of SFH1 did not affect the protein abundance of Pss1 and Psd2, or the in vitro Psd2 activity, it is conceivable that Sfh1 enhance PE synthesis by PS transfer from the endoplasmic reticulum to the endosome. Our findings propose a novel function for Sec14‐family proteins in yeast.Support or Funding InformationThis work was partly supported by JSPS KAKENHI Grant Number 17K07710 and 15J10871.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HspA1A, a 70‐kDa heat shock protein, contains several distinct lipid‐binding sites
Authors: Daniels Amanda Jo , Smulders Larissa , Nikolaidis Nikolas 
Published: June 2021

HspA1A is a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70). This molecular chaperone plays critical roles in protein homeostasis and cellular survival. In addition to its role in protein homeostasis, HspA1A also localizes at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although these interactions have major physiological consequences their mechanistic details remain elusive and unexplored. We have recently hypothesized that the lipid‐binding sites for PS and BMP are distinct and that the lipid‐binding and chaperone functions do not overlap. To test these hypotheses, we used a mutation from Tryptophan to Phenylalanine (W90F), which has been described to affect the binding of HspA1A to BMP, and a Lysine to Alanine (K71A), which is known to abolish the hydrolysis of ATP. We first determined whether these mutations alter HspA1A's stability, intracellular and membrane localization, as well as its ATPase and refolding functions using recombinant proteins and ex vivo experiments. Our results revealed that the W90F mutation does not significantly alter any of the tested functions, but the K71A abolishes both the ATPase and refolding activities, as expected. We then used the lipid vesicle sedimentation (LVS) method and quantified the binding of the wild‐type and mutated HspA1A to both PS and BMP. These experiments revealed that the W90F‐HspA1A binding to BMP was 50% less than the WT, when 0.4 mM of lipids were used, while the loss‐of‐binding was only 10% when 1 and 4 mM of lipids were used. Differently, we found that the W90F mutation does not affect the binding to PS under any of the conditions tested. Furthermore, the K71A mutation did not significantly change the binding of HspA1A to PS or BMP under any of the conditions tested. We verified these results using Surface Plasmon Resonance for both mutations and determined that the W90F‐HspA1A associates with BMP five times faster and dissociates 18 times faster than the WT. These results reveal that the W90F mutation alters the kinetics of the interaction with BMP, but does not alleviate the binding, while the K71A does not affect the binding to any of the lipids tested. Together these results strongly support the prediction that the lipid‐binding sites for PS and BMP are distinct and that the lipid‐binding and chaperone functions do not overlap.Support or Funding InformationNIGMS SC3GM121226This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phospholipid molecular species profile and functionality of nicotinic acetylcholine receptor detergent complex from Torpedo californica solubilized with lipid‐like detergents
Authors: Quesada Orestes , Colón José , González Carol , Maldonado Rafael , Rosado Irvin D. , Acevedo Jesús A. , Lasalde‐Dominicci José A. 
Published: June 2021

One of the limiting factors in the crystallization of membrane proteins is the process of solubilization mediated by detergents. During this process, preservation of the native protein structure and adjacent lipid environments is crucial for appropriate activity and stability. The detergent disruption of lipid–lipid and lipid‐protein interactions alters the aggregation state, stability, and functionality of the native structure. The effect is more severe when the protein activity is lipid‐dependent as is the case with the nicotinic acetylcholine receptor (nAChR) from Torpedo californica, which is required to maintain a functional conformation for crystallization. This study compares the efficiency of three families of detergents; alkylphosphocholine (FC), lysofoscholine (LFC), and Cyclo Foscholine (CF), in terms of stability, lipid composition and functionality of the nAChR‐detergent complexes (nAChR‐DCs). The molecular composition of all detected phospholipid classes from nAChR‐DCs solubilized with these detergents were analyzed by Ultra Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI‐MS/MS). To investigate the possible delipidation effect of the above‐mentioned detergents, we performed a functional assessment of these nAChR‐DCs in Xenopus oocytes using a two‐electrode voltage clamp (TEVC). The stability was monitored by fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase of the Alexa labeled nAChR. A qualitative cross‐correlation analysis of the lipid species present in each of these nAChR‐DCs cannot dissect which lipid species is essential for nAChR function in the soluble state, however, the partially functional nAChR‐FC12 detergent complex lack for lipid species (i.e. PC 18:0/20:4, PC20:4/20:5 and PC20:5/20;4) that are present in the fully functional, nAChR‐LFC16s. The nAChR‐FC16 not only produces the higher fractional mobility, but sustains the mobility throughout the whole 30 day period which is essential for protein nucleation crystallization but still contains some ATPase. In contrast, the nAChR‐FC12 complex is only partially functional but is almost free of ATPase.Support or Funding InformationThis work was supported by the NIH Grant #1R01GM098343 and UPR‐RP RISE Program NIH Grant #5R25GM061151This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulating a G Protein‐Coupled Receptor by Topological Inversion through Regulated Alternative Translocation
Authors: Denard Bray 
Published: June 2021

BackgroundWe have recently reported the first example that ceramide stimulates the topology inversion of a newly synthesized transmembrane protein. This novel regulation, which is designated as Regulated Alternative Translocation (RAT), requires a GXXXN motif in the first transmembrane domain. Utilizing a bioinformatics approach, we have discovered over 100 proteins that contain a GXXXN motif in the first transmembrane domain and therefore can potentially undergo RAT. Greater than 90% of these proteins are G Protein‐Coupled Receptors, including nearly all of the C‐C and C‐X‐C Chemokine Receptors. In the current study, we determine whether C‐C Chemokine Receptor 5 (CCR5) can be regulated through RAT.Study SummaryWe demonstrate though multiple approaches that CCR5 undergoes topological inversion via RAT in response to exogenously added ceramide in transfected cells. In primary macrophages, we demonstrate Lipopolysaccharide (LPS) treatment induces dihydroceramide to catalyze the RAT of CCR5. LPS‐induced RAT of CCR5 is the mechanism by which LPS inhibits chemotaxis of macrophages towards C‐C Motif Chemokine Ligand (CCL5).ConclusionsThis study demonstrates that LPS‐induced dihydroceramide causes the topology inversion of CCR5 through RAT in primary macrophages. The resulting topology inversion of CCR5 prevents macrophage migration towards CCL5. This finding provides a novel regulation of macrophage chemotaxis and suggests that topological inversion through RAT is a general mechanism to regulate G Protein‐Coupled Receptors.Support or Funding InformationNIH GM‐116106 and HL‐20948This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Unsuspected Role for Organic Cation Transporter 3 in the Actions of Amphetamine
Authors: Mayer Felix Paul , Owens Anthony W. , Boehm Stefan , Gether Ulrik , Koek Wouter , Daws Lynette C. , Sitte Harald H. 
Published: June 2021

OBJECTIVEPsychostimulants such as amphetamines exert their actions by targeting the “classical” transporters for monoamine neurotransmitters, the dopamine (DAT), norepinephrine (NET) and serotonin (SERT) transporter. However, whether organic cation transporter 3 (OCT3), a transporter recently found to play an important role in the homeostasis of monoamine signaling, contributes to the actions of amphetamine remains unknown.METHODSBidirectional radiotracer flux studies and real‐time measurements of fluorescent substrate accumulation were applied to monitor the effects of drugs on transporter mediated uptake and release, respectively, in vitro. Furthermore, we performed high‐speed chronoamperometry experiments to determine amphetamine induced dopamine release in vivo. Locomotor activity was measured to determine the effects of amphetamine on behavior.RESULTSHere we show that a major component of amphetamine‐induced neurotransmitter efflux is mediated by OCT3 in primary neuronal cultures. High‐speed chronoamperometry revealed that the OCT3‐inhibitor decynium‐22 (D22) markedly attenuated amphetamine‐induced dopamine release in striatum. This effect was lost in mice deficient in OCT3. Finally, systemically administered D22 reduced amphetamine‐induced locomotion in wild‐type but not in OCT3‐knockout mice.CONCLUSIONOur findings reveal that amphetamine induces release via OCT3. Hence, this study identified an additional transporter, aside from DAT, NET and SERT, that contributes to the actions of amphetamine, which might serve as a new target for the treatment of psychostimulant abuse.Support or Funding InformationAustrian Science Fund/FWF (grants F3506 and W1232 to H.H.S.)NIH MH093320 (L.C.D. & W.K.), NIH MH106978 (L.C.D.),NIH P01 DA12408 (U.G.), Danish Council for Independent Research – Medical Sciences (U.G.),Lundbeck Foundation Center for Biomembranes in Nanomedicine (U.G.),F.P.M. is a recipient of a DOC‐fellowship of the Austrian Academy of Sciences.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex differences in measures of anxiety‐like behaviors following chronic intermittent ethanol and/or stress exposures using a zebrafish model system
Authors: Devaud Leslie , Sano Madeleine , Trupp Laura , Cabuang Aaron , Lally Jessica 
Published: June 2021

Studies on ethanol use have found that stress has a greater impact on responses in females than males. To improve our understanding of these differences, we used zebrafish to assess behavioral activities in male and female fish following treatment with chronic intermittent ethanol exposure and/or repeated stress exposure. Treatments were: Experiments A: once‐daily 30 min 1% ethanol and/or 3X 20 sec stress, or Experiments B: twice daily 30 min 1% ethanol and/or 3X 40 sec stress). We used a custom light/dark tank to assess behaviors at 24 hr after the final treatments. Zebrafish were tested individually on every 10 seconds, including light vs. dark preference, crossings between light and dark, preference for tank top vs. bottom and general activity levels (freezing or hyperactivity). Time spent in these activities were summated across the 8‐min testing period for each animal and averaged by sex within treatment condition. Overall, control females spent more in the dark (10–15%) for both sets of experiments compared to males. The milder treatments (Experiment A) found reduced time spent in the dark for females but not males, with Experiments B treatments significantly reducing time spent in the dark by 80% for ethanol and 55% for ethanol+stress in females. Only the Experiments B ethanol+stress condition reduced time in the dark, by 48% in males. Most of the additional Experiment A behavioral measures found an anxiolytic‐like effect for females, suggestive of resiliency, whereas the expanded treatments of Experiment B uncovered more anxiogenic‐like responses in females than males. Male zebrafish displayed greater increased levels of activity than females across treatment conditions, enhanced in Experiments B compared to Experiments A. These data support the utility of zebrafish as an animal model to study the impact of stress on ethanol responses and found that behavioral responses vary both by measure and by sex.Support or Funding InformationSupported by a PUSOP award to LLDThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.