bluuebunny/crossref_metadata_2025 huggingface dataset:
The Dose‐Related Effects of Doxorubicin Chemotherapy on Interstitial Amino Acid Concentrations in Skeletal Muscle
Authors: MacLean David , Fabris Sergio
Published: June 2021

Doxorubicin (DOX) is a frontline anticancer therapeutic which has proven effective in the treatment of various cancers. However, very little is known regarding the effect that DOX has on SM metabolism, specifically its influence on interstitial amino acid (AA) concentrations. In SM, the interstitial space represents an important active compartment located between the vasculature and the tissue and it has been shown to play a functional role in the regulation and integration of various metabolic substances. The purpose of the current study was to determine the effect of DOX on interstitial AA concentrations in SM. Rats received a 1.5 or 4.5 mg/kg dose of DOX i.p. and grouped into endpoints every 24 hrs up to 192 hrs. A control group was administered a sham i.p. injection of saline. Total amino acids (TAA), essential amino acids (EAA) and branched‐chain amino acids (BCAA) were quantified in the interstitial space. The administration of DOX resulted in a dose‐related difference in interstitial AA. The 1.5 mg/kg dose resulted in a transient increase (P<0.05) in TAA, EAA and BCAA from 24–96 hrs as compared to control. Contrarily, TAA, EAA and BCAA decreased (P<0.05) 24–192 hours following the administration of 4.5 mg/kg DOX as compared to control. This study by way of the microdialysis technique, represents the first time that amino acid concentrations have been quantified in the interstitial space following the administration of a chemotherapeutic drug. In addition, the possibility of a dose‐related effect of DOX on amino acid transport mechanisms from the skeletal muscle compartment into the interstitium/vasculature is of particular interest as this may represent a regulatory mechanism that has not previously been considered.

Support or Funding Information

Supported by NOSMFA

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Catabolism of Nicotinic Acid and Nicotine and the Role of a Putative Monooxygenase in Bacillus niacini
Authors: Fine Sydney R. , Zoretich Kaeli M. , Snider Mark J.
Published: June 2021

N‐Heteroaromatic compounds (NHACs) comprise a class of molecules that are commonly found in pharmaceuticals, personal care products, and pesticides and are known to have toxic effects on the environment. Intriguingly, certain species of soil‐dwelling bacteria have the innate ability to degrade particular NHACs. A better understanding of how these soil bacteria degrade NHACs has important implications in the development of bioremediation techniques to remove toxic compounds from the environment. This investigation focuses on developing preexisting hypotheses in regards to how Bacillus niacini, a ubiquitous species of soil bacteria, degrades nicotinic acid (NA) and nicotine, both of which are NHACs. It has been hypothesized that the non‐canonical NA degradation pathway in B. niacini involves a reaction in which 2,6‐dihydroxynicotinic acid (2,6‐DHNA) undergoes oxidative decarboxylation to form 2,3,6‐trihydroxypyridine by an enzyme termed NicC‐like. Two methods have been utilized to establish the identity of the nicC‐like gene. First, a novel transformation technique was developed to generate a B. niacini knock out variant lacking the putative nicC gene. Second, the substrate for the NicC‐like enzyme, 2,6‐DHNA, was synthesized with a 60% yield. The substrate was utilized to assess the kinetic activity of the NicC‐like enzyme. Though there are hypotheses regarding the identity of the nicC‐like gene and its involvement in NA catabolism, sequence analysis indicates that the nicC‐like gene is only 21% similar to the nicC gene involved in NA degradation in Pseudomonas putida. In contrast, the nicC‐like gene is 24% similar to HspB, which encodes an enzyme involved in nicotine degradation. Additionally, the structure and the associated catabolic processes of nicotine and NA are similar, suggesting that the novel nicC‐like gene in B. niacini might play a role in either nicotine or NA degradation. Preliminary HPLC evidence indicates that B. niacini does not degrade nicotine. Furthermore, the NicC‐like enzyme is not kinetically active when incubated with the substrate of the HspB enzyme, 6‐Hydroxyl‐3‐succinoyl‐pyridine. Thus, the genes currently hypothesized to encode the enzymes necessary for NA catabolism do not code for nicotine degradation enzymes. Characterizing the reaction catalyzed by the putative NicC‐like enzyme and determining whether or not B. niacini catabolizes nicotine has led to an improved understanding about the genes necessary for NA degradation by this particular species of pervasive soil bacteria. Continued contributions to this field may eventually lead to the development of techniques to remove NHACs from the environment.

Support or Funding Information

Henry J. Copeland Fund for Independent Study at the College of Wooster

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cross‐talk among biochemical defense pathways in Arabidopsis thaliana
Authors: Peterson Alexandra , Bien Erica , Olson‐Manning Carrie , Matzner Steven
Published: June 2021

Cellular metabolism is a complex set of interwoven interactions. That complexity challenges the prediction of how metabolism responds to change. To try to increase our ability to predict how single‐gene changes result in global metabolic shifts, we studied how changing expression in the indolic glucosinolate chemical defense pathway in Arabidopsis thaliana influences production in the related aliphatic glucosinolate pathway.

Aliphatic glucosinolates are synthesized in a pathway parallel to the indolic pathway and share a number of enzymes (SUR1, GGP1). Research on the aliphatic pathway has shown aliphaticspecific and shared gene knockdowns have no control over the indolic pathway. However, by knocking down activity of each step in the indolic pathway and measuring the production of all glucosinolates via High Performance Liquid Chromatography (HPLC), our results suggest crosstalk between these pathways.

Preliminary results reveal that when the gene GTSF9 of the indolic pathway is knocked down, the production of aliphatic glucosinolates is reduced, but the production of indolic glucosinolates is not affected. These results are puzzling given that GSTF9 is not thought to be shared between the pathways like the enzymes SUR1 and GGP1. Indeed, previous research suggests GSTF9 is predicted to act in the aliphatic pathway exclusively. To further study this pathway cross‐talk, we will extract and sequence RNA from single‐gene knockouts of the genes in the indolic pathway to track how changes in gene expression of individual enzymes change global patterns of expression. These results will allow us to begin to understand the genetic mechanisms that mediate cross‐talk between these pathways.

Support or Funding Information

SD BRIN P20GM103443 SD EPSCOR IIA‐1355423

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Transferrin‐1 from Drosophila melanogaster
Authors: Gorman Maureen J. , Brummett Lisa M. , LeSuer Leslie A. , Weber Jacob J. , Kanost MIchael R.
Published: June 2021

Transferrins are secreted proteins that bind iron. Mammalian serum transferrin is involved in iron transport, while mammalian lactoferrin functions as an immune protein. Both proteins are thought to protect against oxidative stress. Insect transferrin is similar to both serum transferrin and lactoferrin, and its functions are not well characterized. Previous studies have demonstrated that insect transferrin can function as an immune protein, and limited data suggest that it may have a role in protection against oxidative stress and in iron transport. The goal of this study was to identify the functions of Drosophila melanogaster transferrin‐1 (DmTsf1). Our approach was to observe changes in DmTsf1 concentration in response to immune challenge, oxidative stress, metal stress, and dietary iron availability; test the antimicrobial activity of apo and holo forms of DmTsf1; and evaluate phenotypes associated with RNAi‐mediated knockdown of DmTsf1. We estimated the concentration of DmTsf1 in hemolymph to be 0.5 μM in larvae, 4 μM in adult females and 20 μM in adult males. We found that DmTsf1 concentration increased after inoculation of bacteria, and that DmTsf1 inhibited bacterial growth by sequestering iron. These findings indicate that DmTsf1 can protect flies against bacterial infection. In contrast to its response to immune challenge, DmTsf1 was not upregulated in response to paraquat‐induced oxidative stress, metal stress, or changes in dietary iron availability. We used genetic crosses to generate insects with strong or weak knockdown of DmTsf1 and observed that strong knockdown resulted in mortality in the early larval stages, while 75% knockdown resulted in insects that eclosed as normal‐looking adults. We found that knockdown of DmTsf1 had no effect on an insect's susceptibility to paraquat; therefore, DmTsf1 does not seem to protect flies from paraquat‐induced oxidative stress. Likewise, knockdown of DmTsf1 had no effect on the susceptibility of insects to a high iron diet. Additional experiments to test the effect of knockdown on susceptibility to iron‐induced oxidative stress are ongoing. Our findings demonstrate that DmTsf1 inhibits bacterial growth by iron sequestration, that it does not protect against at least some types of oxidative stress, and that it plays an essential but still unknown role in development.

Support or Funding Information

NIH NGMS R37 GM041247, NSF IOS 1656388

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Expression of Glutathione S‐Transferase isotopes, alpha, mu and pi, in Mice (Mus Musculus) Chronically Exposed to Atrazine
Authors: Schadler Analiese , Martin Debra
Published: June 2021

Atrazine is one of the most heavily used herbicide in North America to control weeds in crops. The Environmental Protection Agency deemed a safe maximum level of atrazine to be 3 parts per billion (ppb) under the Safe Drinking Water Act. Many Midwest states in the United States have annual mean concentrations of atrazine far greater than 3 ppb. Numerous studies have concluded that atrazine negatively affects growth, development, immune and endocrine system functions in diverse organisms. Of interest is a family of detoxification enzymes found in hepatic tissues, glutathione S‐transferases (GST). There are three main isotypes of GST: μ, π and α. Previous research in fish showed chronic exposure to atrazine decreased the enzymatic activity for the μ isotype of GST. The current study utilized male and female CD‐1 mice (Mus musculus) (n = 20/group). Mice were chronically exposed to atrazine (0, 3, 30 ppb) via drinking water for 11 weeks. Analysis of GST enzyme activities was performed and for both the GSTμ and GSTα isotypes, statistically significant decreases in enzyme activity were observed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of a Sulforaphane and Maitake Mushroom Extract Combination on the Expression of Glutamate Cysteine Ligase
Authors: Erwin Sharis A. , Ownby Stacy L. , Cornblatt Grace A.
Published: June 2021

Oxidative stress contributes to numerous health issues including neurodegenerative diseases and cancer. Reduced glutathione (GSH) is a major antioxidant that neutralizes reactive oxygen species (ROS) occurring both endogenously through respiration and exogenously during exposure to environmental stressors. Increasing levels of GSH to combat ROS is critical to reducing oxidative stress. The rate‐limiting step of glutathione synthesis is catalyzed by the enzyme glutamate cysteine ligase, a heterodimer consisting of a Glutamate‐Cysteine Ligase Catalytic Subunit (GCLC) and a Glutamate‐Cysteine Ligase Modifier Subunit (GCLM). In this study, we focus on the effect the combination of sulforaphane and maitake mushroom extract has in elevating the production of GSH as a means of preventing cellular damage caused by oxidative stress. HepG2 liver cancer cells were treated over a time course of 24h with maitake mushroom extract (250 μg/mL) and a physiologically achievable level of sulforaphane (0.5 μM). The combination induced gene expression of both GCLC and GCLM an average of 2‐fold as early as 6h. Additionally, we examined the levels of GSH in HepG2 cells treated with the combination.

Previously, our laboratory demonstrated that the combination induced the expression of Phase 2 detoxifying enzymes NAD(P)H quinone dehydrogenase 1(NQO‐1), UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1), and heme oxygenase 1 (HMOX‐1/HSP32) as well as reduced the expression of pro‐inflammatory markers interleukin‐6 (IL‐6) and cyclooxygenase‐2 (COX‐2). These results show that the sulforaphane and maitake mushroom extract combination possesses anti‐oxidative properties that may promote cellular health.

Support or Funding Information

Research supported by Nutramax Laboratories

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of Yeast Nem1‐Spo7 Protein Phosphatase Complex by Protein Kinase C
Authors: Dey Prabuddha , Su Wen‐Min , Han Gil‐Soo , Carman George M.
Published: June 2021

Pah1 phosphatidate phosphatase in the yeast Saccharomyces cerevisiae catalyzes the dephosphorylation of phosphatidate to produce diacylglycerol. This is the penultimate step in the synthesis of triacylglycerol in yeast and higher eukaryotes. Pah1 is primarily found in the cytosol as a physiologically inactive phosphoprotein, but becomes active through its translocation to the nuclear/ER membrane, where its substrate phosphatidate resides. The membrane translocation of Pah1 occurs via its dephosphorylation by the membrane‐associated protein phosphatase complex Nem1 (catalytic subunit)‐Spo7 (regulatory subunit). In this work, we showed that both Nem1 and Spo7 were phosphorylated by protein kinase C on serine residues. The phosphorylations of each subunit were dependent on their concentrations, phosphatidylserine, diacylglycerol, the amount of protein kinase C, ATP, and the time of the reactions. Peptides derived from Nem1 and Spo7 that contained putative protein kinase C target sites were tested as substrates for the kinase. The Nem1 peptide containing Ser‐201 and the Spo7 peptide containing Ser‐22 and Ser‐28 were substrates for protein kinase C. That Ser‐201 and Ser‐22 and Ser‐28, respectively, were targets in the Nem1 and Spo7 peptides were confirmed by a combination of mass spectrometry and mutagenesis to alanine residues. Full‐length Nem1 S201A and Spo7 S22A and S28A mutant proteins isolated from yeast were defective in their phosphorylations by protein kinase C.

Support or Funding Information

Supported by NIH grant GM050679.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of phosphorylation state on perilipin 5 localization and trafficking.
Authors: Bailey Hannah M. , Tansey John T.
Published: June 2021

Perilipins are a family of five proteins found on the surface of lipid storage droplets in nearly all tissues. These proteins act as cofactors for lipases and scaffolding for other proteins involved in lipid metabolism. In addition to the lipid droplet surface, members of the perilipin family have been found in the cytosol, endoplasmic reticulum, plasma membrane and mitochondria. The localization of these proteins is in part determined by their phosphorylation state. Recently perilipin 5 has been shown to localize to the nucleus when phosphorylated by PKA on serine 155 and interact with the peroxisome proliferator‐activated receptor gamma coactivator alpha (PGC‐1a). In silico analysis of a phosphomimetic mutant (Ser155Asp) indicate that this site may contain a nuclear localization sequence. Using a CHO model cell line we observed that translocation to the nucleus could be stimulated by treatment with forskolin and isobutylmethylxanthine (IBMX) and occurred independent of treating cells with oleic acid to promote triacylglycerol deposition. We are currently investigating the affects of stimulating other phosphorylation pathways to determine if they also phosphorylate perilipin 5 and affect subcellular localization. These data have the potential to gain insights to the regulation of perilipin 5 trafficking into the nucleus and therefore interacting with PGC‐1a regulating mitochondrial biogenesis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CB1 BUT NOT CB2 CANNABINOID RECEPTOR INCREASES NEURITE EXTENSION IN HUMAN NEUROBLASTOMA
Authors: Lyons Erica Lynn , Kabler Sandra , Howlett Allyn , Kovach Alexander L. , Thomas Brian
Published: June 2021

Despite neurodegenerative disease affecting more than five million Americans, there is not yet sufficient understanding of the underlying mechanism of disease to cure or even halt progression. Transplanted neurons may represent a potential treatment if they could successfully integrate with host brain tissue. Both CB1 and CB2 receptors are located in neuronal growth cone membranes, implying that cannabinoid signaling may play a role in neurite extension or guidance (Harkany et al. 2007). CB1 cannabinoid signaling has been shown to be active in neuronal extension, yet the differences between CB1R and CB2R signaling remain unclear (Berghuis et al. 2007, Duff et al. 2013). We have created a novel stable overexpression model to investigate the role of cannabinoid signaling in human neuronal extension. We quantified the endogenous production of cannabinoid ligand 2‐Arachidonoyl Glycerol (2‐AG) and its isomer 1‐AG in SHSY5Y human neuroblastoma at an abundance of 4,000 nanograms Total AG per million cells. This AG was likely 2‐AG produced by DAGL enzyme activity because calcium stimulation by Oxotremorine M increased Total AG abundance to 6,000 nanograms per million cells while inhibition of DAGL by THL decreased Total AG abundance to less than 100 nanograms per million cells. We transfected SHSY5Y cells with either CB1 or CB2 receptors, purified and maintained the transfection using geneticin selection, and isolated clonal cell lines that stably overexpress either CB1R (CB1XS) or CB2R (CB2XS). Stable overexpression of cannabinoid receptors did not alter mRNA abundance of the enzymes that synthesize and degrade the endogenous cannabinoids 2‐AG and AEA. Stable overexpression of CB1R but not CB2R increased the extension length from 30 to 200 micrometers per neurite. Our research suggests a role for CB1R but not CB2R mediated cannabinoid signaling in neurite development that may provide insights in development of therapies that support functional integration of transplanted neurons into host tissue.

Support or Funding Information

Support from NIDA grant DA03690 and T32 grant HL091797 is appreciated.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Vacuolar H+‐ATPase Inhibition on Activation of Cathepsin B and Cathepsin L Secreted from MDA‐MB231 Breast Cancer Cells
Authors: Hinton Ayana M. , Uhlman Andrew , Folkers Kelly , Liston Jared , Pancholi Harshida
Published: June 2021

Studies indicate secreted cathepsins are involved in metastasis. V‐ATPases, which are necessary for activating intracellular cathepsins, also play a role in metastasis and are targeted to the plasma membrane of metastatic breast cancer cells. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. We investigated whether V‐ATPase inhibition would reduce the activity of secreted cathepsin B and cathepsin L. Using cell lysates and conditioned media, we measured cathepsin B and L activity within and outside of the cells. We found different forms of cathepsin B and L were secreted representing the pre‐pro, pro and active forms of the proteases. Cathepsin B activity was higher than cathepsin L in conditioned media and in cell lysates. V‐ATPase inhibition by concanamycin A decreased cathepsin B activity in conditioned media and significantly decreased cathepsin B activity in cell lysates. Cathepsin L activity showed a slight decrease in cell lysates. Changes in the activity of secreted and intracellular cathepsins following V‐ATPase inhibition were supported by changes in the amounts of pro and active forms of cathepsins B in conditioned media and cathepsin B and L in cell lysates. Overall, our data shows that inactive forms of cathepsins B and L are secreted from the MB231 cells and V‐ATPase activity is important for the activation of secreted cathepsin B. This indicates a connection between cell surface V‐ATPases in metastatic breast cancer cells and the function of secreted cathepsin B.

Support or Funding Information

Denison University Anderson Fellowship, Denison University Research Fund

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nanodiscs: A Novel Approach to Studying the Methionine ABC Transporter System
Authors: Winslow Michael G. , Yang Janet G.
Published: June 2021

The cell membrane is a formidable yet flexible boundary that separates the interior from the exterior of the cell. The tightly regulated, selective movement of molecules across the membrane is crucial to maintain appropriate intracellular levels of metabolites. Concentrations gradients across lipid bilayers are generated and maintained by specialized integral membrane proteins known as transporters. The long‐term goal of the Yang Group is to understand how transporters catalyze substrate transport across cellular membranes.

Toward this goal, we are conducting quantitative analyses of a bacterial methionine transport system. The E.coli MetNI transporter uses the energy of ATP binding and hydrolysis to drive methionine import against its concentration gradient. Unlike the majority of in vitro transporter studies conducted using detergent‐solubilized proteins, we aim to examine the MetNI reaction cycle in a native membrane environment. Here we present our preliminary work on preparing methionine transporters in nanodiscs, a discoidal phospholipid bilayer encircled by a protein helical belt.

Support or Funding Information

No external funding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biomechanical control of lysosomal secretion via the VAMP7 hub: a tug‐of‐war mechanism between VARP and LRRK1
Authors: Galli Thierry , Wang Guan , Nola Sébastien , Bovio Simone , Coppey‐Moisan Maïtté , Lafont Frank
Published: June 2021

The rigidity of the cell environment can vary tremendously between tissues and in pathological conditions. How this property may affect intracellular membrane dynamics is still largely unknown. Here, using atomic force microscopy, we found that cells deficient in the secretory lysosome v‐SNARE VAMP7 were impaired in adapting to substrate rigidity. Conversely VAMP7‐mediated secretion was stimulated by more rigid substrate and this regulation depended on the Longin domain of VAMP7. We further found that the Longin domain bound the kinase and retrograde trafficking adaptor LRRK1 and LRRK1 negatively regulated VAMP7‐mediated exocytosis. Conversely, VARP, a VAMP7‐ and kinesin 1‐interacting protein, further controlled the availability for secretion of peripheral VAMP7 vesicles and response of cells to mechanical constraints. We propose a mechanism whereby biomechanical constraints regulate VAMP7‐dependent lysosomal secretion via LRRK1 and VARP tug‐of‐war control of the peripheral readily‐releasable pool of secretory lysosomes.

Support or Funding Information

Work in our group was funded by grants from INSERM, CNRS, Association Française contre les Myopathies (Research Grant 16612), the French National Research Agency (NeuroImmunoSynapse ANR‐13‐BSV2‐0018‐02), the Ecole des Neurosciences de Paris (ENP), the Fondation pour la Recherche Médicale (FRM), Who am I? Labex (Idex ANR‐11‐IDEX‐0005‐01), awards of the Association Robert Debré pour la Recherche Médicale to T.G. and fellowship from the Region Ile‐de‐France in the framework of DIM Cerveau&Pensée and from FRM (FDT20150532766) to G.W. We acknowledge the ImagoSeine facility, and the France BioImaging infrastructure supported by ANR (10‐EQPX‐04‐01) and the EU‐FEDER (12,001,407) as part of “Investments of the future” 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.
AP‐3‐dependent Mechanisms Regulate the Trafficking of ATP8a1 to Lamellar Bodies in Alveolar Type 2 Cells
Authors: Kook Seunghyi , Wang Ping , Meng Shufang , Hanby Hayley A. , Jaume Alexa , Goetzl Laura , Marks Michael S. , Guttentag Susan H.
Published: June 2021

Alveolar type 2 cells (AT2) are involved in the maintenance of epithelial homeostasis and repair after injury. Lamellar bodies (LB) in AT2 play a critical role in lung health and disease as the site of surfactant synthesis, packaging and secretion. LB biogenesis is poorly understood, especially mechanisms that foster membrane and protein trafficking to LB. In this study, we found that ATP8a1, a member of the P4 subfamily of P‐;type ATPases (P4‐;ATPases), was highly enriched in the ABCA3‐positive LB fraction derived from human fetal lung explants. In AT2 from mice lacking the adaptor protein 3 complex (AP‐3), the pearl mouse model of Hermansky Pudlak syndrome type 2 (HPS2), ATP8a1 was largely depleted from ABCA3‐positive LB and accumulated in Rab11‐positive recycling endosomes (REs). Using targeted mutagenesis, we identified a di‐leucine‐based sorting signal (ExxxLL) present in the C‐terminal cytoplasmic domain of ATP8a1 that is necessary for its proper targeting to LB of AT2. To dissect the relative roles of AP‐1 and AP‐3 in the transport of ATP8a1 to LB, we have developed MLE‐15 cell lines in which a subunit of AP‐1 (Ap1γ1) and/or AP‐3 (Ap3β1) has been inactivated using CRISPR/Cas9 gene editing and tested the targeting of ATP8a1 in these mutated cell lines. The lack of AP‐3 in MLE‐15 reduced efficient LB delivery of ATP8a1, while deletion of the AP‐1 subunit had no effect on ATP8a1 delivery to LB. P4‐;ATPases typically flip aminophospholipids, such as phosphatidylserine (PS), to the cytosolic leaflet of organelle membranes. PS is highly enriched in REs and is essential for endosomal membrane traffic. Here, we show that the cytoplasmic leaflet of LB in MLE‐15 and human AT2 are highly enriched in PS using a GFP‐LactC2 probe; in contrast, AP‐3‐deficient cells showed impaired distribution of PS, favoring RE locazliation. These data provide evidence that ATP8a1 is dependent on AP‐3 for trafficking from RE to LB in AT2, and suggest that loss of ATP8a1 targeting to LB in HPS2 impairs PS localization to LB in AT2 cells.

Support or Funding Information

National Institutes of Health (GM108807/SG, MM and LG). SG is the Julia Carell Stadler Professor of Pediatrics.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tale of the two domains in twinfilin: Deciphering phagocytosis through actin dynamics
Authors: RATH PRAGYAN PARIMITA , KUMAR NITESH , BABUTA MRIGYA , BHATTACHARYA ALOK , GOURINATH SAMUDRALA
Published: June 2021

Phagocytosis is an indispensable cellular function in phagocytic cells or protists. It is choreographed by actin and its surfeit of regulatory proteins. This process is critical in Entamoeba histolytica’s lifecycle as it relies primarily on phagocytosis for its nutritional uptake as well as its pathogenesis. In our studies we have tried to puzzle out the role of Twinfilin‐ an actin binding protein in E. histolytica's pathogenesis. Twinfilin is an Actin Depolymerizing Factor (ADF)/cofilin family member, containing two ADF‐homology domains, one each as N‐terminal (NTD) and C‐terminal (CTD).

We have solved a 1.5 Å resolution crystal structure of EhTwinfilin NTD and determined that it contains typical ADF homology fold i.e., central β sheets surrounded by α helices. With the help of fluorimetric and SPR studies we have shown the actin binding activities of both the domains and interestingly we found that NTD stabilized F‐actin whereas CTD depolymerized it. NTD has stronger G‐actin binding while CTD binds to F‐actin with much higher affinity; this is rather role reversal of the studies published on twinfilin from other organisms. Structural and actin binding studies of EhTwinfilin provides explanation for some of the functional differences observed with respect to the human and Plasmodium falciparum's ADF such as the ability to bind G‐actin. Cellular localization studies established its significant role during phagocytosis. GFP tagged EhTwinfilin was visualized using confocal imaging, it was found to colocalize with F‐actin at phagocytic cups. Down‐regulation of EhTwinfilin expression leads to an increased rate of phagocytosis. This is the second protein so far that was seen to be present throughout the phagocytosis, up till phagosome maturation and first protein to perform actin severing activity in E. histolytica.

Support or Funding Information

SERB, Department of Science and Technology, India

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Seeking new molecular targets to control mitochondrial biogenesis
Authors: Silveira Leonardo R. , Pauletti Bianca A. , Paes Leme Adriana F. , Lima Tanes I.
Published: June 2021

Mitochondrial number and shape are constantly changing in response to increased energy demands. The ability to synchronize mitochondrial signaling pathways with environmental stimuli is a central aspect against obesity in mammals. The binding and regulation of transcriptional complexes at the promoter of mitochondrial genes are critical components of the mitochondrial biogenesis process. Specifically, these promoters contain cis‐regulatory elements that lead to the recruitment of selected nuclear receptors (NR) and transcriptional factors (TF). Although different molecular regulators of this system have been described, only a small number of nuclear receptors and transcription factors have been associated with the mitochondrial biogenesis process. In this study, therefore, we proposed to seek new targets for the regulation of the mitochondrial biogenesis process in C2C12 cells during differentiation through the integration of proteomic and transcriptomic analysis. As expected. mitochondrial function and biogenesis were strongly induced during C2C12 cell differentiation (Figure 1). By evaluating the signaling pathways and molecular networks during myogenesis using RNAseq data extracted from GEO datasets (GSE20846), we revealed the enrichment of several signaling pathways associated with mitochondrial biogenesis and oxidative metabolism such as OXPHOS and TCA cycle (Figure 2). These results showed a strong enrichment in the PPAR signaling which was validated through PPRE transactivation experiments and qPCR (Figure 2). Accordingly, Venn diagram analysis showed an overlap of 170 genes associated with mitochondrial function from upregulated genes during myogenesis and PPAR targets suggesting the involvement of PPAR activity in mitochondrial biogenesis (Figure 3). In addition, proteome analysis during C2C12 differentiation exhibited 609 proteins positively regulated, from which 246 overlapped with PPAR targets and also presented enrichment of mitochondrial signaling pathways. To investigate the involvement of other uncharacterized transcription factors and nuclear receptors in mitochondrial biogenesis accompanying skeletal muscle cell differentiation, we performed a over‐represented conserved transcription binding sites analysis (oPOSSUM 3.0) in the set of upregulated mitochondrial associated genes and proteins previously analyzed using GO‐term of cellular components (DAVID). These analyses revealed the role of several unrelated NRs and TFs in mitochondrial biogenesis accompanying skeletal muscle cell differentiation (Table 1).

Support or Funding Information

This work was supported by grants from Fundação de Amparo à Pesquisa do estado de São Paulo – FAPESP (2013/22733‐0; 2016/23008‐5) and CNPq (444323/2014).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The genetic origin of a rare mitochondrial disorder
Authors: Bartl Lucas , DeVries Brooklyn , Reider Joshua , Vitiello Seasson P.
Published: June 2021

A pediatric patient with hypotonia, cerebellar atrophy, psychomotor delay, and increased blood‐lactate underwent whole exome sequencing. The patient harbors two novel variants of the TARS2 gene, which encodes the mitochondrial threonyl‐tRNA synthetase (mtThrRS). The aminoacylation catalyzed by mtThrRS is necessary for the translation of proteins encoded by mitochondrial DNA. One variant in the patient contains a putative splice‐site mutation, while the other variant contains a missense mutation. Both mutations are predicted to cause loss of mtThrRS function, suggesting that these novel variants of the TARS2 gene cause mitochondrial dysfunction and are therefore causal for the disorder. We aimed to assess the effects of these variants on mitochondria and energy production, and to determine the molecular effects of the TARS2 splice‐site mutation. This work contributes to the understanding of the connections between tRNA synthetase function and health.

Support or Funding Information

NIH/INBRE (NIGMS) South Dakota INBRE Program (P20GM103443)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation
Authors: Yang Loretta , Wu Shengcheng , Cooper J. Christopher , Paul Mallory K. , Cummings Amanda L. , Eletr Ziad M. , Ben‐Arye Shani Leviatan , Padler‐Karavani Vered , Samli Kausar N. , Woods Robert J.
Published: June 2021

Glycans have distinct properties that make them appealing as disease biomarker targets, and novel highly specific reagents are essential to overcome current limitations in the discovery and exploitation of disease‐related glycans.

Lectenz® Bio is engineering glycan‐processing enzymes into catalytically inactive, high affinity glycan binding reagents with tunable specificities. These novel lectin‐like, enzyme‐derived reagents called Lectenz® are being developed for a variety of glycan detection and enrichment applications including affinity chromatography, Western blot, and immunohistochemistry. The conversion of such enzymes into affinity reagents is facilitated by computationally‐guided directed evolution. Using site‐directed mutagenesis and yeast display selection of a site‐saturation mutagenesis library, multiple Lectenz® candidates can be identified.

Here we present two novel sialic acid recognizing Lectenz® engineered from a sialidase enzyme: 1) the pan‐specific sialic acid reagent, Sia‐PS1 Lectenz®, which recognizes sialic acid in a linkage independent manner; and 2) the Sia‐3S1 Lectenz®, which is specific for α2,3‐linked sialoglycans. The data demonstrate that the Sia‐PS1 Lectenz® reagent recognizes glycans terminating in α2,3‐, α2,6‐, and α2,8‐linked sialic acid sequences, but not Gal‐terminating sequences. With the Sia‐3S1 Lectenz®, we demonstrate binding to Neu5Acα2,3Galβ1,4Glc, but not Neu5Acα2,6 Galβ1,4Glc or Galβ1,4Glc.

Support or Funding Information

National Institutes of Health (R41GM113351)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Boston Naming Test Predicts Deterioration Of Cerebrospinal Fluid Biomarkers In Pre‐Symptomatic Alzheimer's Disease
Authors: Wilder Charleen , Moncrieffe Kristina , Nolty Anne , Arakaki Xianghong , Fonteh Alfred , Harrington Michael
Published: June 2021

Background and Objectives

Identifying predictors of Alzheimer's disease provides an opportunity for early treatment, which can minimize the negative impact of the disease. The current longitudinal study examines neuropsychometric changes in cognitively healthy pre‐symptomatic elderly participants using cerebrospinal fluid (CSF) amyloid and tau measurements, established biomarkers of Alzheimer's disease pathology. Participants were classified as having normal amyloid/tau (NAT) or pathological amyloid/tau (PAT). By examining the neuropsychological differences between cognitively healthy individuals with normal biochemistry (NATs) and those who convert over time to abnormal biochemistry (PATs), early neuropsychological predictors may be identified. Our hypothesis is that the differences in neuropsychological data will predict the change in biochemistry in participants who convert from NATs to PATs.

Methods

A total of 63 elderly participants (70–95 years) were classified using the Uniform Data Set, Version 2, as cognitively healthy. They were tested at two time points: at an average initial age of 80, and again 40 months later. At each visit, participants underwent a series of clinical, biochemical, and neuropsychological assessments.

Results & Discussion

Out of the 23 NATs that were reassessed clinically, all remained cognitively healthy as assessed by consensus conferencing and extensive neuropsychological evaluation, while 40% of the PATs had cognitive decline. From their CSF, 14 NATs remained NAT (NAT‐NAT) while 9 converted to PATs (NAT‐PAT) at the second time point. Mann Whitney U‐test analyses indicated that there was a significant difference between those who on reassessment were NAT‐NAT vs. NAT‐PAT on the Boston Naming Test (BNT) at the first time point. Our neuropsychological data suggest that cognitively healthy individuals who convert to abnormal CSF biochemistry performed worse on the initial BNT than cognitively healthy individuals who have persistently normal CSF biochemistry. Small population size is a limitation of this study. We propose that performance on the BNT may predict changes in amyloid and tau biochemistry in cognitively healthy individuals; 40% of these CSF converters (compared to none of the non‐converters) have declined clinically in 4 years.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Cyclin A2 In Aging Hippocampus And rRna Dynamics
Authors: Alves Michele Joana , Goksel Mustafa , Fair Summer , Nunez Samara Mendez , Kaya Behiye , Mustafa Hasnaa , Czeiler Catherine , Otero Jose
Published: June 2021

Background

Novel functions of cyclin A2 (CCNA2) have been demonstrated including DNA synthesis initiation and progression, DNA repair, and protein translation through RNA binding. Previously, we reported that loss of CCNA2 leads to hippocampal defects characterized by abnormal DNA repair and impaired performance on behavioral tests due to memory and learning deficits. Abnormalities in RNA processing and synaptic plasticity are mechanisms associated with deficits in memory and aging. This study was addressed to investigate effects of cyclin A2 loss on modulation of RNA processing in the hippocampus of aged mice.

Methods

All work involving animals was performed under the auspices of a protocol approved by the Ohio State University Institutional Animal Care and Use Committee. CamkIIα‐cre mice were interbred with CCNA2fl/fl mice to achieve adult ablation of CCNA2 in the hippocampus and cerebral cortex. To evaluate brain morphology, animals were sacrificed at 8 months. After fixed in 4% paraformaldehyde, crysections were obtained at 12 μm. Electron microscopy was performed and number of synapses and polyribosomes were quantified.

Results

Ultrastructural analysis of Stratum Moleculare (SM) and Reticulare (SR) in 8mo mices showed a decrease of the synapse density in CamkIIαcre CCNA2 fl/fl mices as compared with control. However, quantity of ribosomes was increased only in SM of the hippocampus in mutant mices. Immunostaining for ribosomal RNA (rRNA) revealed an accumulation of rRNA granules in dentate gyrus and Cornu Ammonis (CA) both regions of the hippocampus. Quantitative analysis resulted an increased rRNA in the SR. To elucidate if mechanisms of translational regulation are modified by the lack of CCNA2 we screened for alterations of 40S ribosomal protein S6 (S6), and translational repressor eukaryotic initiation factor 4E binding protein (ELF4E) and cytoplasmic polyadenylation element binding protein 1 (CPEB1).

Conclusions

Our data shows a distinct role for CCNA2 in adult hippocampus in the regulation of synaptic density as well as the prevention of rRNA granule formation. Alterations in RNA dynamics caused by loss of CCNA2 may be implicated in neuronal disorders and neurodegeneration.

Support or Funding Information

This work was sponsored by NIH/NHLBI grants RO1 HL132354

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prion Deposition in the Retina of Sporadic Cruetzfeldt‐Jakob Disease
Authors: Goodwill Vanessa , Lin Jonathan , Sigurdson Christina , Geschwind Michael , Sanchez Henry
Published: June 2021

Introduction

Human spongiform encephalopathies, also known as prion diseases, are a rare group of infectious, heritable, and acquired diseases leading to rapid cognitive decline. Prion diseases are considered a serious public health threat due to the high level of infectivity and delayed presentation. Up to 50% of patients with prion disease develop visual symptoms during their clinical course, possibly secondary to abnormal prion protein (PrPSc) accumulation within the eye or within the visual pathway in the brain. Few studies, however, have investigated the presence and deposition pattern of PrPSc within the eye of patients with sporadic Creutzfeldt‐Jakob disease (sCJD). Knowing the presence or levels of PrPSc in the eye is important to assess the potential risk of transmission from common ophthalmic surgical procedures (cornea transplantations and cataract extractions). Additionally, the current gold standard of diagnosis for prion disease remains brain autopsy. If PrPSc expression is present consistently in the eye, ocular imaging modalities with PrPSc‐binding compounds may potentially enable earlier diagnosis. Here, we investigate the expression of PrPSc within retinas of confirmed prion disease cases collected from post‐mortem enucleations.

Methods

From December 2015 to May 2017, 28 eyes were collected from 14 autopsies of clinically suspected prion disease and 6 of non‐prion disease at a single institution. sCJD was confirmed in all cases by brain autopsy and genetic analysis of the PRNP gene. Immunohistochemistry was performed with antibodies against PrP and PrP deposition within the retina was evaluated and compared with normal controls. Subjects included both male and female patients with ages ranging from 51–80 years old. The clinical disease course ranged from 2–24 months.

Results

In twelve of the fourteen prion disease cases, PrPSc deposits were observed in the retina by immunohistochemistry. PrPScwas limited to the outer and inner plexiform layers of the retina and was most prominent in the outer plexiform layer, with the majority of cases exhibiting a coarse, 5–10 um well‐aligned ovoid deposits. Controls showed no PrPSc staining in any retinal layers. In the sclera, cornea, lens, and optic nerve, no deposits were observed.

Conclusion

PrPSc deposits were present in a majority of retinas of patients with confirmed prion disease, primarily within the outer plexiform layer, and these deposits can be detected immunohistochemically. Our findings confirm previous isolated reports of retinal PrPSc deposition in the largest series of documented prion cases to date. We suggest that the retina potentially serves as a diagnostic target in clinically suspected prion disease. Additionally, retinal involvement may contribute to visual symptoms commonly associated with prion disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Astrogliosis and downregulation of EAAT2 in rats infused with Nef transfected astrocytes.
Authors: Valles Ortiz Jaydie M. , Cruz Myrella L. , Rodriguez Yarelis L. , Cintron Kevin M. , Noel Richard J.
Published: June 2021

Astrocytes play a major role in homeostasis and neuronal function by preventing excitotoxicity in the CNS. During HIV infection, astrocytes are reported to produce early viral proteins such as Nef, which is known to cause neurotoxicity. Excitatory amino acid transporter 2 (EAAT2) is one of the major astroglial transporters involved in the regulation of extracellular glutamate concentration. We investigated astrocytes transfected with Nef to understand the effect on learning, astrocyte morphology and glutamate transporter expression. Primary rat astrocytes expressing Nef or GFP were infused in the right hippocampus of 30‐day‐old male Sprague Dawley rats. Learning was assessed by Morris water maze five days after infusion. Brain tissues were collected for immunofluorescence staining and the morphological assessment of astrocytes and EAAT2 expression was assessed in different areas of the hippocampus (CA3 and dentate gyrus) using Image J software. In astrocytes, glial fibrillary acidic protein (GFAP) area fraction was significantly increased (P<0.05), while EAAT2 expression was significantly decreased (p<0.05) in the Nef‐treated group when compared to GFP controls. No differences in the length of processes in astrocytes were found, however the number of secondary processes was increased in the Nef‐treated animals. The increase of astrogliosis as measured by GFAP staining, the number of secondary processes, and the decrease of EAAT2 correlates with the learning impairment shown by the Nef‐treated group. These results suggest that there might be an astrocyte dysfunction that affects glutamate uptake at the synaptic cleft leading to neuronal toxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.