bluuebunny/crossref_metadata_2025_split huggingface dataset:
Angiotensin‐converting enzyme (ACE) up‐regulates metabolic function in myeloid‐derived cells
Authors: Cao DuoYao , Khan Zakir , Peng ZhenZi , Giani Jorge F , Veiras Luciana C , Chang Corey , Bernstein Ellen , Bernstein Kenneth 
Published: June 2021

While most studies of ACE focus on the blood pressure effects of angiotensin II, it is now recognized that ACE also plays an important role in both the innate and adaptive immune responses1. To study the mechanism of this effect, we made two mouse strains: ACE 10/10, which over‐express ACE in macrophages, and NeuACE, which over‐express ACE in neutrophils. NeuACE and ACE 10/10 mice have a better immune response in several different disease models (e.g. bacteria, tumor, Alzheimer's disease)1,2. We hypothesized that ACE affects myeloid function by inducing changes in cellular energy metabolism. To examine this, metabolic intermediates were studied by mass spectrometry and chemical methods in ACE over‐expressing cells.MethodsNeutrophils were isolated from bone marrow; macrophages were collected from the peritoneum 4 days after thioglycolate injection. Cells were purified by Percoll gradient.ResultsMass spec. showed more than 32 differences, with virtually all affected metabolites increased in the ACE over expressing cells. In ACE 10/10 macrophages, cellular ATP, ADP and AMP was 3.4‐, 2.7‐, and 2.7‐fold the levels in WT cells. In NeuACE neutrophils, the pattern was the same but numerically less than in macrophages. Chemical analysis showed a 2.8‐fold and a 1.5‐fold increase of cellular ATP in ACE 10/10 macrophages and NeuACE neutrophils. In ACE 10/10 macrophages, the ATP/ADP ratio was 2.1 vs. 1.4 for WT cells. For NeuACE neutrophils, the ATP/ADP ratio was 3.0 vs. 1.9 in WT neutrophils. The increase of cellular ATP is due to the catalytic activity of ACE; when animals were treated with an ACE inhibitor, peritoneal macrophages or bone marrow derived neutrophils showed ATP levels that were no different from similarly treated WT mice. The effect of ACE is not due to increased angiotensin II.ConclusionsACE overexpression in macrophages or neutrophils of mice upregulates ATP concentrations in these immune cells. This is due to the catalytic activity of ACE but not the production of angiotensin II. Increased cellular ATP plays an important role in the enhanced innate and adaptive immune ability of myeloid cells over expressing ACE.Support or Funding InformationNIH P01 HL129941 and R01 AI143599
imageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
S‐sulfhydration of SIRT3 by hydrogen sulfide attenuates mitochondrial dysfunction in cisplatin‐induced acute kidney injury
Authors: Yuan Yujia , Li Lan , Zhu Lingling , Chen Younan , Liu Jingping , Lu Yanrong , Cheng Jingqiu 
Published: June 2021

Clinical use of cisplatin, one of the most widely used common and effective chemotherapeutic agents, is limited by its side effects, particularly tubular injury–associated nephrotoxicity. Mitochondrial dysfunction is one of the main mechanisms contributing to cisplatin‐induced acute kidney injury (AKI), and sirtuin 3 (SIRT3) is closely associated with mitochondrial function and oxidative stress. Hydrogen sulfide (H2S) has powerful anti‐oxidative and anti‐inflammatory properties. We report that H2S alleviated cisplatin‐caused renal toxicity via SIRT3 activation and subsequent mitochondrial bioenergetics. Using a biotin switch assay, we show that H2S increased S‐sulfhydration of SIRT3 and induced deacetylation of its target proteins (OPA1, ATP synthase β and SOD2). These effects of H2S were associated with a reduction of mitochondrial fragment, an increase in ATP generation, and less oxidative injury. Notably, the S‐sulfhydration of SIRT3 induced by NaHS (a H2S donor) was abrogated when Cys256, Cys259, Cys280, and Cys283residues on SIRT3 (2 zinc finger domains) were mutated. Collectively, we demonstrate that H2S attenuates cisplatin‐induced AKI by preventing mitochondrial dysfunction via SIRT3 sulfhydrylation. Thus, SIRT3 may represent a new therapeutic target and H2S may be a potentially effective agent to limit cisplatin‐induced nephrotoxicity by activating SIRT3.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China (Grant number 81370824 and 31571474), and China Postdoctoral Science Foundation (2017M620428).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Facilitating fructose‐driven metabolisms increases a capability to resist anoxic stress in Drosophila
Authors: Kim Man Su , Kim Hongjo , Kim Misun 
Published: June 2021

Hypoxic stress is linked to various cardiovascular disorders including stroke and myocardial infarction. A key pathophysiological process upon hypoxic stress is a reduction in ATP generation. However, a fructose‐driven glycolysis has been proposed as an alternative biochemical pathway, which could sustain ATP production even in anoxic stress. Here we tested the hypothesis that facilitating fructose‐driven ATP generation increases organisms' capability to withstand anoxic stress using a Drosophila as an in vivo animal model. Genetically modified flies that contain human fructose transporter (GluT5) and ketohexokinase (KHK) sequence downstream of UAS were constructed. Using a GAL4‐UAS system that allows us to express GluT5 and KHK in a tissue‐ and time‐dependent manner, it was found that overexpression of both genes in whole flies (with Act5c‐gene switch GAL4 driver) or in neurons (with elav‐gene switch GAL4 driver) reduces flies' mortality upon anoxic stress. Besides, it was found that spontaneous locomotor activity of the GluT5/KHK overexpression flies was higher than that of control flies upon anoxic assaults. Taken together, these data suggest that enhancing fructose metabolisms could be utilized as a potentially clinically relevant approach to minimize hypoxia‐induced cellular damages.Support or Funding InformationThis work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and Technology (NRF‐2017R1E1A2A01079316)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of the Escherichia coli Lysophospholipase PldB in Determining Cell Length
Authors: Nikpour Naveed , Garrett Teresa A 
Published: June 2021

Headgroup acylated glycerophospholipids (GPLs) are proposed to carry out an essential function in cell division in the cell membranes of Escherichia coli. Lysophospholipase L2 (PldB) has been shown to transfer the acyl group from 2‐acyl‐lysophospholipids to the headgroup of phosphatidylglycerol (PG) to form acyl PG. Cells overexpressing PldB have been found to show an elongated cell phenotype. As the cause for this phenotype is not entirely known, we aim to develop a robust assay to test PldB activity so as to understand what aspect of PldB activity is implicated in the cell elongation phenotype. We are developing a radiochemical thin‐layer chromatography assay for PldB using 14C PG and 14C (bis‐monoacylglycerol)phosphate to the study this mechanism. Using liquid chromatography‐mass spectrometry‐based assays, we are testing cells with mutations to PldB's catalytic triad to determine a catalytically dead mutant. This mutant would allow us to assess if cell elongation requires PldB activity or if PldB's association with the inner membrane impacts the activity of cellular division machinery. This project aims to undercover more information about the functioning of PldB and headgroup acylated GPLs. Due to PldB's role in cell membrane integrity, further analysis of its mechanism may further our understanding of how the cell membrane functions during cell division. Future research can focus on localizing PldB within the membrane to help assess PldB's function during cell division.Support or Funding InformationThis work was funded by National Science Foundation Research at Undergraduate Institution Award #1516805 to T. A. G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancement of Lipid Signal in MALDI MS imaging obtained from Formalin Fixed Human Brain Tissue
Authors: Harris Aaron , Mor Rahul , Whitehead Shawn , Yeung Ken 
Published: June 2021

Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric analysis directly on biological samples providing accurate visual/anatomical spatial information of molecules within the tissue. A current limitation of MALDI‐IMS is that it is largely performed on fresh frozen tissue whereas clinical tissue samples stored long term are fixed in formalin. It has been shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI‐IMS procedure display an increase in observed signal intensity and sensitivity for lipid molecules detected in the brain while maintaining the special distribution of molecules throughout the tissue. In this work we investigate the effectiveness of this AF wash on post‐fixed rat and human brain tissue sections in an effort to increase the viability of formalin fixed tissue imaging in a clinical setting. Results herein demonstrate that the AF wash significantly improved MALDI‐IMS spectra for gangliosides, including GM1 in fresh frozen rat brain, formalin‐fixed rat brain and formalin fixed human brain samples. AF wash also demonstrated improvements in MALDI‐IMS image quality while retaining the spatial distribution of molecules. Results indicate that this method will allow analysis of gangliosides from formalin‐fixed clinical samples, which can open additional avenues for neurodegenerative disease research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Kinetics of the Arabidopsis thaliana Acyltransferase At1g78690
Authors: Jahns Max , Garrett Teresa A 
Published: June 2021

Headgroup acylated glycerophospholipids (GPLs) are a class of membrane phospholipids containing an acyl‐chain attached to the headgroup of the molecule that make up a relatively small portion of the cell envelope but are implicated in important functions such as the cell stress response and cell division. At1g78690 is an acyl‐transferase native to Arabidopsis thaliana that converts mono‐acylated GPLs to diacylated GPLs in vitro. Interestingly, overexpression of At1g78690 in E. coli leads to an accumulation of the headgroup acylated GPL acyl‐phosphatidylglycerol (acyl‐PG) in vivo. While At1g78690 is known to be capable of transferring an acyl chain from acyl‐CoA to two isoforms of bis(monoacyl glycerol) phosphate (BMP), 3,1’ BMP and 3,3’ BMP, to form acyl‐PG, it does not acylate 1,1’ BMP suggesting the enzyme possesses some stereoselectivity that needs to be further investigated. We reevaluate the extent to which At1g78690 is substrate selective, by further characterizing the ability of At1g78690 to acylate PG and 1,1 BMP. We also estimate the Km of At1g78690 on lyso‐PE, lyso‐PG, lyso PS, lyso‐PI, and 3,1’ and 3,3’ BMP. The kinetics of At1g78690 are measured via an acyl‐chain acceptor (either lyso‐PG, lyso‐PS, lyso‐PI, 3,1’ BMP or 3,3’ BMP) receiving a 13C‐labeled acyl chain from acyl‐CoA. The kinetics of an enzyme can give insights into its substrate specificity, its catalytic and energetic properties, and its role in cellular phospholipid synthesis.Support or Funding InformationThis work was funded by National Science Foundation Research at Undergraduate Institution Award #1516805 to T. A. G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Gylcerophosphocholine Acyltransferase Homolog, Gpc1, in Candida albicans
Authors: King William Ray , Patton‐Vogt Jana 
Published: June 2021

The maintenance of membrane composition through regulated synthesis and turnover of phospholipids is crucial to cellular homeostasis. Phosphatidylcholine (PC) is the most abundant phospholipid in most eukaryotic membranes. We have identified a glycerophosphosphocholine acyltransferase (GPCAT), Gpc1, that expands our understanding of PC biosynthesis and acyl chain remodeling in Saccharomyces cerevisiae. Gpc1 acylates glycerophosphocholine (GPC), a product of complete deacylation of PC, to form lysophosphotidylcholine (LysoPC). LysoPC can subsequently be converted to PC by Ale1. This PC deacylation reacylation pathway (PC‐DRP) defines a novel route for PC biosynthesis and provides a potential mechanism to alter the acyl chain composition of a PC molecule. Changes in acyl chain composition can, in turn, affect physical properties of a membrane, such as fluidity, width, thickness and shape. Gpc1 displays no homology to known acyltransferases and defines a new gene family with members in animals, plants and fungi. The pathogenic fungus Candida albicans displays robust transport of GPC into the cell. We have previously shown that the inability of C. albicans to transport GPC results in decreased virulence in a mouse model of disseminated candidiasis implying this metabolite may play an important role during pathogenicity. Here we report on the creation of a C. albicans diploid knockout of Gpc1 homolog (orf19.998) using a transient CRISPR Cas‐9 system. Radiolabeling coupled with mass spectrometry are being employed to determine if the caGpc1 is similar to the scGpc1 in terms of biochemical function. Initial studies to examine the physiological role of caGpc1 are being performed.Support or Funding InformationNIH R15GM104876 (to JPV)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Membrane Microdomains on a Human Granulosa Cell Line by Atomic Force Microscopy
Authors: Brogie Molly , Cohen Brian D 
Published: June 2021

Signaling via the Hypothalamic‐Pituitary‐Gonadal (HPG) axis regulates fertility in both men and women. Follicle stimulating hormone (FSH), secreted from the anterior pituitary, binds to a G protein coupled receptor (GPCR) on ovarian granulosa cells. The activation of this receptor triggers a cellular signaling pathway that leads to the promotion of follicle development and estrogen secretion. Our lab has previously demonstrated that the human FSH receptor (hFSHR) shows an affinity for residing in lipid rafts of HEK293 cells which may play a pivotal role in its signaling. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids, with higher stiffnesses than bulk membrane domains. To characterize lipid rafts on the human granulosa cell line hGrC1 we used atomic force microscopy. Atomic force microscopy is a technique used to produce nanoscale resolution topographical images. It can also be used for determining mechanical characteristics, such as stiffness and strain. Here, using contact and force‐volume mode atomic force microscopy, we successfully generated topographical maps of hGrC1s and recorded membrane stiffness measurements. Areas of the granulosa cell membrane showed variable stiffness on a scale of 58.998 nm / pixel, providing the first direct evidence of lipid rafts in human granulosa cell membranes. Demonstration of lipid rafts contributes to the implications of the role of lipid rafts hFSH signalling. By understanding membrane dynamics on granulosa cells we hope to gain insight into how to manipulate hFSHR function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DPMS is Essential for Tumor Angiogenesis
Authors: Collazo‐Irizarry Diego , Pabon Andrea Firpo , Ramirez‐Alameda Ricardo , Baksi Krishna , Banerjee Dipak K. 
Published: June 2021

Dolichol phosphate mannose synthase (DPMS) an inverting GT‐A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). It catalyzes the transfer reaction Dol‐P + GDP‐Man < == > Dol‐P‐Man + GDP in endoplasmic reticulum (ER) for the synthesis of dolichol‐linked tetra‐decasaccharide [Glc3Man9GlcNAc2‐PP‐Dol; lipid linked oligosaccharide (LLO)], a pre‐requisite for the glycosylation of asparagine‐linked (N‐linked) glycoproteins. N‐Glycosylation provides structural and functional stability, and adds flexibility to glycoproteins. The gene for DPMS has been cloned from many species, i.e., from archaea to human. The amino acid sequence identifies the presence of a metal binding (i.e., DAD) signature and a cAMP‐dependent protein phosphorylation (PKA) motif. Phosphorylation enhances the DPMS catalytic activity and consequently increases the synthesis and turnover of LLO. In capillary endothelial cells, it means increased proliferation, i.e., angiogenesis. Angiogenesis is a hallmark for breast cancer progression. Therefore, we have hypothesized that overexpression of DPMS in capillary endothelial cells could mimic the cAMP signaling and independently increase angiogenesis. To test the hypothesis, we have used a cell line that stably overexpresses DPMS and evaluated the role of D(+)mannose on their proliferation by MTT assay. Capillary endothelial cells harboring the DPMS overexpressing plasmid exhibited increased expression of DPMS when examined by immunofluorescence microscopy, western blotting, and QPCR. There was increased expression of cell surface N‐glycans as judged by Texas‐Red conjugated WGA‐binding. The cell proliferation as well as the cellular mitotic activity were also enhanced. To evaluate if mannose is required for the observed effect, we have monitored the cellular proliferation as a function of mannose concentration and the time of incubation by MTT assay. The results indicate mannose supplementation did not support the growth of neither the DPMS overexpressing cells nor the control cells. Triple negative human breast cancer cells also showed a similar result. We therefore conclude DPMS overexpression induces angiogenesis. Mechanistically it is not due to upregulation of mannose transport. The environmental factors increasing intracellular cAMP level increase DPMS catalytic activity metabolically or through gene expression and enhance angiogenesis for breast tumor progression. DPMS, thus could be an excellent target for developing a new generation glycotherapy against breast cancer.Support or Funding InformationInstitutionalThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of an antifungal peptide, Diapausin, from Manduca sexta
Authors: Li Miao , Al Souhail Qasim , Veerapandian Raja , Vediyappan Govindsamy , Kanost Michael 
Published: June 2021

Antimicrobial peptides, a first line of defenses against the infectious microbes, are present in bacterial, fungal, plant and animal kingdoms. They are typically cationic and are less than 50 amino acid residues long. Most known insect antimicrobial peptides are bactericidal, and a few target both bacteria and fungi with relatively broad spectrum, but only a small group is specifically active against fungi. Manduca sexta, the tobacco hornworm, is an established model for biochemical studies of insect immunity. Eighty‐six antimicrobial peptide genes from 12 families were identified in the M. sexta genome. We recently discovered diapausins as a family of antifungal peptides in M. sexta hemolymph, active against Saccharomyces cerevisiae and some ascomycete fungi. Diapausin concentration increases in hemolymph after injecting the larvae with yeast or bacteria. Purified diapausin‐1 and ‐2 recombinant proteins expressed in E. coli block growth of S. cerevisiae with IC50 of 30uM and 60uM, respectively. Diapausin‐1 and ‐2 disrupt daughter cell separation after cell division, resulting in cell clusters. Diapausin‐1 is active against the human fungal pathogen Candida genus as well. The growth of Candida albicans and Candida krusei is inhibited by diapausin‐1, with IC50 of 60uM and 20uM respectively, and diapausin‐1 treatment alters the morphology of these fungi. Diapausin‐1 impairs the germination of spores and hyphal growth of Beauveria bassiana, an insect pathogenic fungus. FITC‐labeled diapausin‐1 binds to the surface of S. cerevisiae, and pull‐down experiments show that diapausin‐1 binds to □‐1,3‐glucan, a component of fungal cell walls. We are investigating the mechanisms of diapausin action using □‐1,3‐glucan synthase (FKS1) mutants from S. cerevisiae and C. albicans. Diapausin‐1 may disrupt fungal growth by binding to □‐1,3‐glucan and/or impairing □‐1,3‐glucan synthesis. This study advances our understanding of an insect immune response to fungal infections and may contribute to the identification of new targets for the development of antifungal drugs.Support or Funding InformationNIH grants GM041247, Kansas State University Division of Biology's startup funds, AHA grants 4SDG18910036This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Microstructure Detection by Scanning Acoustic Microscope (SAM) using Special and Antigen Staining
Authors: Miura Katsutoshi , Fujie Michio , Yamashita Kanna 
Published: June 2021

ObjectiveA SAM detects and visualizes cellular components and extracellular matrix (ECM) proteins. However, which materials contribute to the values of speed‐of‐sound (SOS) or attenuation‐of‐sound (AOS) is unknown. SOS and AOS indicate tissue stiffness or softness because greater SOS and AOS areas correspond to stiffer portions of tissues. The aim of this study is to clarify the role of each component in SAM observation.MethodsTo detect each component, special staining for ECM and cellular components such as collagen, elastin and DNA were used. To recognize specific antigens, immunostaining with Ni‐DAB solution as horseradish peroxidase substrate was applied. SAM images before and after staining were compared.ResultsFor nuclear detection, both hematoxylin and Giemsa staining resulted in increased SOS and AOS values of the nuclei (Fig. 1 and Fig. 2). To distinguish ECM components, aniline blue (Fig. 3) and Sirius red for collagen and resorcin‐fuchsin (Fig. 4) for elastin stained well to increase SOS and AOS values according to duration of incubation. After collagenase digestion, SOS and AOS values at the positive‐staining areas decreased. Immunostaining with Ni‐DAB solution raised SOS and AOS values of antigen location (Fig. 5).ConclusionsSpecial and immunostaining used in light microscopy are also available to certain the antigen location for SAM. Collagens and smooth muscles contribute to raise the values of SOS and AOS.Support or Funding InformationThe study was supported by a grant from the Japan Society for the Promotion of Science KAKENHI, Scientific Research Grant Number (c) 15K08375. There are no conflicts of interest to declare.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.