bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoimmune Nature of Type I Diabetes Mellitus
Authors: Janvier Darlyne L. , Tulp Orien L. , Einstein George P.
Published: June 2021

Type 1 diabetes Mellitus (T1DM) is a chronic autoimmune disease in which pancreatic β‐cell destruction results in loss of endogenous insulin production. Already one of the most prevalent chronic childhood diseases, the incidence of T1DM in children and young adults is projected to continue to increase over the next several decades. Although advances in technology have allowed for improved glucose monitoring and insulin delivery, no curative therapy is currently available for this growing patient population. Moreover, patients continue to experience long‐term complications such as blindness and kidney failure, even with the most intensive insulin regimens currently available.

T1DM predominantly originates when insulin‐producing beta (β)‐cells in the pancreas are destroyed or suppressed due to an autoimmune response caused by the auto‐reactive T‐helper 1 (Th1) cells. Interestingly, the antigens responsible for this immune response, glutamic acid decarboxylase, are normally present in β‐cells; however, their destructive capabilities are restrained by the regulatory mechanisms of the body. When a person has certain genetic factors (e.g. the absence of certain protective histocompatibility locus antigen) and exposure to certain environmental factors, they are predisposed to immunologic injury of β‐cells. Furthermore, the odds of presenting with T1DM diminish with treatment with immune‐suppressive regimens that specifically target T‐cells. As a result, Th1 cells dominate the protective regulatory T‐cells. In turn, Th1 cells secrete cytokines, interleukin 2, and interferon gamma, which initiate inflammation of the islet cells that appears to result in the development of T1DM.

In 2001 it was found that diabetes might be the first disease for which stem cell therapy is an option, when insulin‐producing embryonic stem cells of R1‐mice were transplanted into streptozotocin‐induced diabetic animals and normal blood glucose levels were achieved, and in other studies implantation of embryonic Sertoli cells into diabetic db mice also resulted in amelioration of the diabetic state. In contrast, infusion of cord blood derived stem cells in the absence of immunomodulation was without lasting effect on the diabetic state. Minor complications in R1‐mice aside, this event once again renewed the quest to find more practical and functional means to create human islets to treat diabetes.

Can pancreatic stem cell transplant be used as a cure for T1DM, an autoimmune disease, and how does it stand against other cell transplants and other treatment modalities in the US? The bulk of preliminary evidence to date suggests encouraging potential for such approaches in the treatment of T1DM.

Support or Funding Information

Supported by Institutional Resources of USAT, the Einstein Institute, and the University of Massassachusetts.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the Structure and Function of Phosphoserine Phosphatases
Authors: Brousseau Merissa C. , Ray Leah C. , Allen Karen N.
Published: June 2021

With the ever increasing availability of new genome sequences every year, there is a growing need for functional annotation both on a structural and cellular level. In the case of large superfamilies of proteins with a great deal of sequence divergence, assignment of function based on sequence alone remains difficult. For instance, although Bacillus subtilis is the model gram positive bacterium, only 30% of the genome has assigned activity. YsaA (BSU28940), a B. subtilis protein discovered in a knockout screen for metabolic mutants, has been confirmed to have phosphoserine phosphatase activity by determining the steady‐state kinetic constants. Despite this, YsaA shares only 12% sequence identity to the known E. coli phosphoserine phosphatase SerB. We are currently working to determine the molecular structure of YsaA, along with a Bacillus halodurans protein BH2155 (60% similarity with YsaA) which will provide better understanding of the structure‐function relationship between these new proteins and SerB. It is very possible that although YsaA and SerB are within the same super family, they may have independently evolved the same function.

BH2155 has been over‐expressed in E.coli, purified, and subjected to sparse‐matrix high throughput screens yielding initial crystallization conditions. Additionally, experiments have been performed using a phosphate assay to obtain the steady state kinetic constants for BH2155 and compare them to YsaA and known phosphoserine phosphatase SerB. By determining the structures of these proteins as well as profiling their activity we hope to gain insight into the structure‐function relationship and the possibility of pseudo‐convergent evolution in phosphoserine phosphatases.

Support or Funding Information

We thank the Undergraduate Research Opportunities Program at Boston University for funding supporting this project (to MCB).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure/Function Analysis of a Novel Myeloperoxidase Inhibitor from Staphylococcus delphini
Authors: Ploscariu Nicoleta T. , de Jong Nienke , van Strijp Jos , Geisbrecht Brian V.
Published: June 2021

Staphylococcus aureus and closely related species are highly adapted to their hosts and have evolved many strategies to resist opsonization and phagocytosis. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium can actively evade specific intracellular killing mechanisms used by neutrophils. We recently discovered a highly conserved S.aureus protein (SPIN for Staphylococcal Peroxidase INhibitor) that specifically binds and inhibits myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils [de Jong et al., PNAS, 2017]. S. aureus cells deleted for SPIN gene showed decreased survival compared to wild‐type bacteria after phagocytosis by neutrophils. A crystal structure of SPIN bound to MPO suggests that it acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. To further our understanding of the structure/function relationships of SPIN proteins, we cloned, overexpressed, purified, and characterized a series of SPIN orthologs of Staphylococcal origin.

We found that SPIN originating from S. delphini although shares only ~53% identity to SPIN‐aureus, it still retained a strong affinity and inhibitory capacity for human MPO. A 2.4 Å crystal structure of SPIN‐delphini bound to human MPO provided a basis for comparing the similarities and differences between SPIN‐aureus and SPIN‐delphini. Together, these studies shed light on the molecular features which determine the specificity of SPIN proteins for MPO, and suggest potential avenues for using this information toward the design of synthetic MPO inhibitors.

Support or Funding Information

NIH Grants AI111203 and GM121511

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantification of total HIV‐1 DNA in seminal and blood samples in men under cART
Authors: Torres‐Strubbe Rafael J. , Godoy‐Muñoz Lenin J. , Figueroa Lysmarie J. , Hill Martin J.
Published: June 2021

Although antiretroviral therapy (ART) exists there are some anatomical sites that serve as drug‐sanctuaries for HIV‐1 treatment. Semen is the principal vector for HIV‐1 transmissions worldwide. Infectious HIV‐1 is found in seminal plasma and in seminal leukocytes. Previous studies have shown that copies of total HIV‐1 DNA are higher in blood than in semen; however, these studies only focus in men with less than two years living with the disease. Studies suggest that semen can be a reservoir promoting the transcription of the virus when it reaches non‐detectable levels. Therefore, we hypothesize that patients undergoing antiretroviral therapy and with non‐detectable viremia should present higher HIV‐1 DNA copies in semen than in blood. Patients with paired semen and blood samples were selected from a previous longitudinal study based on the inclusion criteria. Patient selection criteria included non‐detectable viremia and more than two years under ART. Peripheral blood mononuclear cells and seminal cells were isolated from blood and semen samples from each patient to extract genomic DNA. rtPCR was used to assess total HIV‐1 DNA on each compartment. Our results show higher number of HIV‐1 copies in blood that in seminal cells. Despite the theory of semen being a drug sanctuary and a reservoir for HIV‐1, there may be other factors involve in the ongoing viral replication in blood.

Support or Funding Information

This work was supported by NIGMS‐NIH grants: 5SC3GM088032‐08 (MH), UPR‐Ponce Research Initiative for Scientific Enhancement (R25GM096955) The authors also acknowledge the support from the Molecular and Genomics Core (MAGIC) at Ponce Health Sciences University (NIMHD Grant MD007579).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRAPPC6AΔ, TIAF1 and SH3GLB2 are initiators for amyloid beta plaque formation and tau aggregation in vivo
Authors: Chang Nan‐Shan , Chang Jean‐Yun , Yap Ye Vone , Sze Chun‐I
Published: June 2021

Substantial evidence shows that tumor suppressor WW domain‐containing oxidoreductase, designated WWOX, FOR or WOX1, is involved in neurodegenerative diseases, neural disorders, cancer progression, and metabolic diseases. WWOX protects neurons from degeneration by binding tau and tau‐hyperphosphorylating enzymes GSK3β, ERK, and JNK1, and promotes neuronal differentiation. WWOX is frequently downregulated in the hippocampi of patients with Alzheimer's disease (AD). Indeed, WWOX may start to downregulate in the cortex and hippocampus in mid‐aged normal individuals. Both in vivo and in vitro evidence show that upon WWOX downregulation, a cascade of protein aggregation occurs, involving spontaneous relocation of TRAPPC6AΔ (trafficking protein particle complex 6A delta, TPC6AΔ) and TGFβ1‐induced anti‐apoptotic factor 1 (TIAF1) to the mitochondria. TPC6AΔ aggregates first and then TIAF1, followed by activating caspases and leading to amyloid precursor protein (APP) degradation and formation of amyloid beta (Aβ) plaques and tau tangles. Here we identified SH3GLB2 (SH3‐Domain GRB2‐Like Endophilin B2 ), whose aggregation is downstream that of TIAF1. The aggregates can be found in the AD hippocampi. A 31‐amino‐acid protein Zfra is highly effective in blocking and preventing the aggregation of TPC6AΔ, TIAF1, SH3GLB2, Aβ, and Tau. To characterize how the BAR domain‐containing SH3GLB2 aggregates, here we show that alteration of Tyr77 to Phe77 abolishes TGF‐β‐mediated polymerization, suggesting that Tyr77 phosphorylation is needed for SH3GLB2 aggregation. Activation of p53 by PRIMA‐1 abolishes SH3GLB2 aggregation, whereas inhibition of p53 activation by Pifithrin‐μ induces the aggregation. Induction of autophagy by thapsigargin accelerates SH3GLB2 aggregation. Transiently overexpressed SH3GLB2 causes apoptosis. Neurotoxin MPP+ induces neuronal death in vivo due in part to SH3GLB2 aggregation. Tumor suppressors p53 and WWOX are partners in cancer suppression. Loss of WWOX causes p53 instability. Conceivably, dysregulation of both p53 and WWOX results in initiation of the TPC6AΔ/TIAF1/SH3GLB2 aggregation cascade and leading to the formation of Aβ plaques and tau tangles of the neurodegeneration pathway.

Support or Funding Information

MOST, Taiwan, and DoD, USA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
P21‐Activated Kinase 1 is Necessary for Autophagy and Mitophagy in Cardiomyocytes
Authors: Clements Kevin , Tin Kathy , Kobayashi Tamayo , Huang Yuan , Kobayashi Satoru , Liang Qiangrong
Published: June 2021

P21‐Activated Kinase 1 (PAK1) is a serine‐threonine kinase that regulates multiple signal transduction pathways in cardiomyocytes. Despite the large body of evidence indicating a key role for PAK1 in cardiac protective signalling, the mechanisms underlying the cardioprotective effects of PAK1 remain elusive. Autophagy is a cellular process in which unwanted organelles and proteins are degraded and recycled to maintain cellular homeostasis. Mitophagy is a form of selective autophagy that eliminates damaged mitochondria from the cell via the lysosome system to control mitochondrial quality. Autophagy and mitophagy are essential for cell survival in response to starvation, but may become detrimental under other conditions. How autophagy and mitophagy are regulated for adaptation in different contexts is under intense investigation. In this study, we explored whether PAK1 regulates autophagy and mitophagy in cardiomyocytes. PAK1 was knocked down by using siRNA in H9c2 cardiac myoblasts and then the expression levels of the autophagosome marker LC3‐II were analysed by Western blot. Interestingly, downregulation of PAK1 reduced LC3‐II levels in the total cell lysates, which was not significantly increased by the lysosomal protease inhibitors pepstatin A (pepA) and E64d, suggesting that autophagy flux is reduced by downregulation of PAK1. Similarly, PAK1 knockdown reduced LC3‐II expression levels in mitochondria either in the presence or absence of pepA and E64d, indicating limited mitochondrial degradation or mitophagy in the absence of PAK1. These results were reproduced in mouse hearts lacking PAK1. Together, our results suggest that PAK1 is essential to maintain the activities of autophagy and mitophagy in cardiomyocytes in vitro and in vivo. Current studies in our lab are investigating the functional significance of autophagy and mitophagy in PAK1‐induced myocardial protection.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiac remodeling in obesity is associated with high gene expression of TLR‐4 and TNF‐α
Authors: Alves Pedro Henrique Rizzi , Hasimoto Fabiana , Kitawara Koody , Francisqueti Fabiane , Ferron Artur , Bazan Silméia Garcia Zanati , Campos Djon , Ferreira Ana Lúcia , Correa Camila Renata
Published: June 2021

Aim

Evaluate the correlation between the gene expression of inflammatory markers and cardiac remodeling in obese animals.

Methods

16 male Wistar rats were assigned to receive water plus standard diet (group C, n = 8) and Ob diet with water plus 25% sucrose (Ob group, n = 8). Weekly body weight (BW) of the animals was assessed and, after 30 weeks of experiment, adiposity index (AI). Cardiac structures measured: diastolic diameter (LVDD); diameter of the aorta (AO), left atrium (LA); The MVE index (LVMI) was calculated by normalizing to body weight estimated LV mass. The LV systolic function was assessed by cardiac output; The LV diastolic function was evaluated by the following indices: peak velocity of early diastolic filling (E wave); peak velocity of late diastolic filling (A wave); ratio between the E and A waves (E/A); deceleration time of E wave (DTE); The study was supplemented by evaluation by tissue Doppler early diastolic (E′) and late (A ′) of the mitral annulus (arithmetic average travel speeds of lateral and septal walls), and the ratio the waves (E/E′ and E′/A′). Left ventricular fragment of the animals was homogenized in TRIzol® for extraction of ribonucleic acid (RNA). After wards, the RNA was submitted to reverse transcription, conversion of RNA to complementary deoxyribonucleic acid (cDNA). cDNA was used in the polymerase chain reaction (PCR) using ready‐made assays containing TaqMan MGB (FAM) probe and primer specific for tumoral necrosis factor‐ alpha (TNF‐α) and toll‐like receptor 4 (TLR‐4).

Results

The Ob group showed significantly higher weight (C = 493.008 ± 50.775, Ob = 562.95 ± 53.69; p < 0.05) and higher adiposity index (C = 4.79 ± 0.73, Ob = 8.68 ± 1.76; p < 0.05). Gene expression of TRL‐4 (C = 1.00 ± 0.337, Ob = 1.584 ± 0.559; p < 0.05) and TNF‐α (C = 1.0 ± 0.485, Ob = 2.897 ± 2.596; p < 0.05) presented significant increase in ob group. Results of cardiac evaluation are described in Table 1 followed by the graphs of correlation.

Conclusion

High gene expression of TLR‐4 and TNF‐α in heart is correlated with cardiac remodeling in obese animals.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of the Prostaglandin E2 EP3 Receptor Does Not Affect Beta Adrenergic Signaling in the Heart
Authors: Bryson Timothy D. , Pandrangi Teja S. , Khan Safa Z. , Xu Jiang , Peterson Edward , Harding Pamela
Published: June 2021

High blood pressure is a major risk factor for cardiovascular disease. Prostaglandin E2 (PGE2) is a metabolite of arachidonic acid and is known to mediate inflammation and blood pressure regulation. Previous data from our lab have shown that PGE2 reduces contractility when signaling via its EP3 receptor subtype and that EP3 expression increases in the Angiotensin II (Ang II) model of hypertension. Furthermore, we demonstrated in vivo that the hypertensive effect of Ang II could be abrogated using an EP3 inhibitor (EP3i), suggesting an interaction between the two pathways. Since stimulation of the sympathetic nervous system increases Ang II and is an important regulator of cardiovascular function, we therefore hypothesized that inhibition of EP3 would also antagonize beta adrenergic signaling. To test our hypothesis, we studied 4 groups of male 10–12 wk. old C57Bl/6 mice receiving: (1) vehicle, (2) the beta adrenergic agonist Isoproterenol, ISO 30 mg/kg/d S.C., (3) the EP3i L798,106 40 μg/kg/d, (4) ISO + L798,106 for 10 days. ISO or its vehicle were given via osmotic mini pump and L798,106 or its vehicle were administered via daily injections S.C. Systolic blood pressure (BP) was measured every 2 days via tail cuff and cardiac function was assessed by echo at the end of the study period. At baseline, there were no differences in BP between the 4 groups. As expected, ISO infusion significantly increased both BP (105 ± 3.4 mmHg vs. 130 ± 4.1 mmHg, p<0.01) and heart rate (600 ± 21 bpm vs. 749 ± 16 bpm, p<0.005) at end of study, however, there was no difference in BP between the ISO group and the ISO + EP3i group (130 ± 4.1 mmHg vs 125 ± 6.1 mmHg, respectively) nor heart rate (749 ± 16 bpm vs 729 ± 26 bpm). Consistent with the blood pressure data, myocyte cross sectional area increased with ISO infusion from 195.5 ± 9.1 μm2 to 234.0 ± 10.4 μm2, p < 0.05 and this was unchanged at 219.9 ± 13.1 μm2 in the ISO+EP3i group. Similar results were noted in the heart weight to body weight ratios. Echocardiography in conscious animals demonstrated no significant differences in either ejection fraction (EF), shortening fraction, or left ventricle dimensions between the ISO group and the ISO + EP3i group. In conclusion, our data suggest that contrary to our hypothesis, there is no interaction between the EP3 receptor signaling pathway and the beta adrenergic pathway after ten days of ISO infusion. Thus, EP3 receptor signaling appears to be uniquely linked to the angiotensin II pathway.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of Clinically Relevant Non‐Small Cell Lung Cancer Model in Athymic Nude Mice using 3D Spheroids of A549‐iRFP Cells
Authors: Huang Yingbo , Guo Xin
Published: June 2021

Background

Lung cancer is the top killer cancer in the US. Monolayer cultures of lung cancer cells and their animal xenografts are major models for testing therapeutic modalities against lung cancer. Injection of suspended cancer cells into the lung is one of the most common approach to build an orthotopic lung cancer model but carry several major disadvantages, including the omission of a primary solid tumor and the artificial metastasis generated from the premature leakage of the injected cell suspension [1]. All such drawbacks could severely mislead the development of new therapies against lung cancer. Compared to cell monolayers or cell suspensions in culture, three‐dimension (3D) cancer cell spheroids better mimic the tumor microenvironment [2].

Purpose

The purpose of this study is to build a more clinically relevant, orthotopic lung cancer model by inoculating 3D spheroids of lung cancer cells into the mouse lung.

Method

The 3D spheroids were cultured by seeding human lung cancer cell line A549‐iRFP in corning 96 multicellular microplate. The cells stably expressed infrared fluorescent protein, whose signal was detected on Odyssey CLx imaging machine (λex = 685 nm, λem = 700 nm) to image the tumor growth in vivo. Tumor inoculation was performed by transpleural surgery on the left side of the athymic nude mice [3].

Results

The fluorescence of the spheroids was proportional to their volume and viability (R2=0.9845 and 0.9305). Early in Day 7, the mice inoculated with the 3D spheroids (right 2 and 3) showed confined tumor growth at the inoculation side of the lung whereas those inoculated with cancer cell suspension (left 1) displayed perfused signal on both sides of the lung, indicating premature leakage of the inoculated cancer cells.

Conclusion

The mice inoculated with the cancer cell suspension displayed artificial metastasis due to premature leakage of the injected cells inside the lung, which decreased its clinical relevance of mimicking lung cancer progression. In contrast, mice inoculated with the 3D cancer cell spheroids better mimic the progression of non‐small cell lung cancer in clinic.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A rare, autism‐associated in‐frame deletion in the dopamine transporter exhibits profound functional deficits
Authors: Shekar Aparna , Campbell Nicholas , Aguilar Jenny , Peng Dungeng , Erreger Kevin , Matthies Heinrich , Sutcliffe James , Mchaourab Hassane , Galli Aurelio
Published: June 2021

The neurotransmitter dopamine (DA) mediates fundamental behaviors such as reward, motivation, attention, and cognition. Important to dopamine neurotransmission is the dopamine transporter (DAT), a member of the solute carrier family 6 encoded by the SLC6A3 gene. DAT is a presynaptic membrane protein responsible for the reuptake and recycling of DA following calcium‐mediated vesicular release. DAT dysfunction has been associated with neuropsychiatric disorders including ADHD, schizophrenia, bipolar disorder, and autism spectrum disorders (ASD). DAT is also the target of commonly abused psychostimulants and controlled substances, namely cocaine and amphetamine (AMPH).

Recently, our laboratory identified a novel, SLC6A3 variant identified in an ASD proband from whole exome sequencing. The genetic variant is an in‐frame deletion of three nucleotides resulting in a deletion of amino acid Asn336 (ΔN336). Located in the third intracellular loop, N336 is conserved from human to Drosophila, and in silico algorithms predict that ΔN336 confers a functionally damaging effect to DAT. Expression of human DAT (hDAT) ΔN336 in Chinese Hamster Ovary (CHO) cells revealed a near absence of DAT‐dependent DA uptake relative to wildtype DAT, yet surface expression was unchanged. This SLC6A3 variant is permissible to AMPH‐induced efflux, but exhibits diminished DAT‐mediated inward currents promoted by rapid application of AMPH. To understand the basis of the ΔN336 variant's effects on transporter conformational dynamics, we conducted electron paramagnetic resonance (EPR) spectroscopy analyses by introducing the correspondent deletion in the leucine transporter, LeuT. LeuT ΔV269 promotes a closed conformation of the extracellular gate and facilitates the intracellular gate to reside in a “half‐open and inward‐facing” conformation.

The results of this study provide new structural and functional insights into the role of DAT structural domains controlling DA transport. In addition, they define how disruption of specific DAT structural interactions and gate conformations translate to deficits in molecular function. Most importantly, this work adds to the growing body of literature implicating altered regulation of DA homeostasis/transport as a potential biological mechanism underlying liability to ASD.

Support or Funding Information

American Heart Association Predoctoral Award 17PRE33410145 (A.S), NIH T32 Training Programs T32NS007491 (N.G.C.) and 4TR32GM007628‐29 (J.I.A.) and NIH grant DA038058 (A.G.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reinforcing, Antinociceptive, and Pruritic Effects of a G Protein‐Biased Mu Opioid Receptor Agonist, PZM21, in Primates
Authors: Ding Huiping , Kiguchi Norikazu , Perrey David A. , Nguyen Thuy , Czoty Paul W. , Zhang Yanan , Ko Mei‐Chuan
Published: June 2021

PZM21 is a newly discovered G protein‐biased mu opioid peptide (MOP) receptor agonist which exerts antinociceptive effects with fewer side effects in rodents. The aim of this study was to determine the functional profile of systemic and spinal delivery of PZM21 in non‐human primates. Effects of PZM21 were compared with those of MOP agonists, such as oxycodone and morphine, in a series of behavioral assays established in rhesus monkeys (Macaca mutatta). Following systemic administration, PZM21 (1–6 mg/kg) and oxycodone (0.1–0.6 mg/kg) dose‐dependently produced antinociceptive effects against an acute noxious stimulus. Similar to a prescription opioid oxycodone, PZM21 produced reinforcing effects in primates under fixed ratio (FR30) and progressive‐ratio (PR) schedules of intravenous drug self‐administration. Following intrathecal administration, PZM21 (0.03–0.3 mg) and morphine (0.003–0.03 mg) dose‐dependently produced antinociceptive effects. Although intrathecal PZM21 has a slower onset for eliciting itch scratching activities, intrathecal PZM21 (0.3 mg) and morphine (0.03 mg) both elicited robust scratching responses lasting for more than 5 hours. In addition, intrathecal PZM21 (0.03–0.3 mg) dose‐dependently attenuated capsaicin‐induced thermal allodynia. Anti‐allodynic effects of intrathecal PZM21 or morphine could be blocked by a MOP receptor antagonist, naltrexone. Taken together, these pharmacological studies in primates indicate that PZM21 displays similar functional efficacy as an analgesic like clinically used MOP agonists, oxycodone and morphine. Although structure‐based discovery of PZM21 opens a new, exciting chapter for G protein‐biased MOP agonists, the abuse potential and pruritic effect of PZM21 are not significantly different from those of clinically used opioid analgesics in primates.

Support or Funding Information

The US‐PHS grants DA044775, DA032568, and DA040693.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endometriosis‐associated vaginal hyperalgesia is mediated by the balance of reactive aldehyde production and metabolism.
Authors: McAllister Stacy , Sinharoy Pritam , Gross Eric
Published: June 2021

Background

Endometriosis affects ~176 million women worldwide and is characterized by the growth of endometrial tissue outside the uterus (known as endometrial ectopic cysts). How endometriosis becomes associated with painful symptoms is not clear. Here we hypothesize that endometriosis‐associated vaginal hyperalgesia is mediated by an imbalance of reactive aldehyde production and metabolism.

Methods

To test this hypothesis, we used female Sprague‐Dawley rats (n=5/group) and subjected them to a cutting‐edge model of behavioral psychophysics to assess for rat vaginal nociception. Prior to subjecting the rodents to this model, we treated rodents with the reactive aldehyde, acetaldehyde (9 mg/kg intraperitoneal) to mimic an increased production of reactive aldehydes in the intraperitoneal space. To modulate ALDH2 activity, we administered to rodents either the ALDH2 activator, Alda‐1 (1 mg/kg, diluted in DMSO, given intraperitoneal) or the ALDH2 inhibitor disulfiram (15 mg/kg, diluted in DMSO, given intraperitoneal). The ALDH2 activator were delivered 10 minutes before or after acetaldehyde (Alda‐1 + aldehyde or aldehyde +Alda‐1). Disulfiram was given 10 minutes before acetaldehyde (Disulfiram + aldehyde). As controls Alda‐1, disulfiram, saline, and DMSO were delivered alone. Changes in vaginal nociception were expressed as area‐under‐the‐curve (AUC) units and calculated using standard trapezoid methods. For statistical analysis, a one‐way ANOVA with Bonferroni's multiple comparison test was used. Data presented as mean ± SEM.

Results

Acetaldehyde‐induced vaginal hyperalgesia (44±4* vs. saline 28±2, *P<0.05). Alda‐1 markedly attenuated hyperalgesia when given either before (26±3, *P<0.05 vs. acetaldehyde) or after (25±3, *P<0.05 vs. acetaldehyde) acetaldehyde treatment. Conversely, pretreatment with disulfiram before acetaldehyde increased vaginal hyperalgesia (61±5, *P<0.05 vs. acetaldehyde). Alda‐1, DMSO, and disulfiram alone produced no significant changes in vaginal nociception.

Conclusions

These data suggest that the mechanism of endometriosis‐induced hyperalgesia is mediated by changes in reactive aldehyde production and metabolism. Increasing ALDH2 activity in rats, both prevented and alleviated reactive aldehyde‐induced vaginal hyperalgesia. These data suggest a potential novel therapeutic for treating endometriosis by targeting reactive aldehyde metabolism.

Support or Funding Information

This work was supported by the National Institutes of Child Health and Human Development K99HD093858 (SLM), a Stanford Women & Sex Differences in Medicine (WSDM) Seed Grant, and an Endometriosis Foundation of America Research Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EPAC1 Boosts Macrophage Appetite: Implication of EPAC1 in Foam Cell Formation during Atherosclerosis
Authors: Robichaux William Gene , Mei Fang C. , Sun Hua , Wang Hui , Yang Wenli , Teng Ba‐Bie , Cheng Xiaodong
Published: June 2021

With the increasing age of the global population, cardiovascular diseases (CVD) is the leading contributor of morbidity and mortality in growing patient populations. Thus, further elucidating the pathophysiology and developing novel treatments for CVD are critical to combat this urgent unmet medical need. The cAMP second messenger, is a major stress‐response signal found to play an important role in cardiovascular functions. Moreover, the downstream contribution of the exchange protein directly activated by cAMP 1 (EPAC1) in the development of atherosclerosis has yet to be explored. Genetic ablation of EPAC1 in atherosclerosis prone LDb (Ldlr−/−/Apobec1−/−) mice were developed as a model to investigate the functional consequences attributable to loss of EPAC1 in the pathogenesis of atherosclerosis. Remarkably, absence of EPAC1 protected against atherosclerotic lesion development compared to mice expressing EPAC1. One prevalent source attributing to atherosclerotic pathologies is chronic arterial inflammation, implicating innate immunological responders, such as macrophages. Within the lesion, macrophages are subject to a multitude of inflammatory cytokines and initiate engulfment of oxidized lipids that trigger lipid‐laden foam cell formation. Although the presence of foam cells is observed to further perpetuate macrophage aggregation and subsequent lesion advancement, the precise molecular mechanisms governing formation of foam cells and initial progression of atherosclerosis are still not fully understood. Recent evidence demonstrates EPAC1 participating in functions downstream of cAMP during phagocytosis, and thus may be involved in macrophage accumulation of oxidized‐LDL. Therefore, we hypothesize that loss of EPAC1 could attenuate atherosclerotic lesion development, in part by disrupting physiological endocytosis of oxidized‐LDL in macrophages. Indeed, further investigation demonstrates reduced phagocytosis and lipid‐burden in bone‐marrow derived macrophages not expressing EPAC1 when exposed to oxidized‐LDL. These results depict an unidentified functional role for EPAC1 in endocytosis of oxidized lipids, and suggest targeting EPAC1 may be beneficial in attenuating atherosclerotic lesion development in patients.

Support or Funding Information

This work was supported by the U.S. National Institutes of Health (NIH) Grants R01‐GM066170 and R35‐GM122536.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of the Plasma Membrane Monoamine Transporter and Organic Cation Transporters with Meta‐iodobenzylguanidine (mIBG)
Authors: Lopez Quinones Antonio J. , Wagner Dave , Wang Joanne
Published: June 2021

Meta‐iodobenzylguanidine (mIBG) is a radiopharmaceutical used as both a diagnostic imaging agent (123I‐mIBG) and a targeted frontline radiotherapy (131I‐mIBG) for neuroblastoma. Scintigraphic imaging of mIBG in human and biodistribution studies in xenografted animal models have shown that alongside its uptake into neuroblastoma tumor tissues, mIBG is also significantly transported into normal organs and tissues including the liver, salivary glands, and kidney. 123I‐or 131I‐mIBG accumulation in normal tissues can interfere with its tumor imaging quality and contribute to peripheral toxicities. While mIBG uptake into the neuroblastoma tumors has been shown to be facilitated predominantly by the human norepinephrine transporter (hNET), the molecular mechanisms involved in its uptake into the peripheral tissues have been poorly understood. We hypothesized that the organic cation transporters 1–3 (hOCT1‐3) and the human plasma membrane monoamine transporter (hPMAT) are involved in mIBG transport and accumulation in normal tissues. Thus, the purpose of this project is to characterize the interaction and transport kinetics of mIBG with these transporters and compare them with hNET. This was done by executing uptake assays in human embryonic kidney cells stably expressing hPMAT, hNET, or hOCT1‐3, followed with mIBG quantification by liquid chromatography‐tandem mass spectrometry. Time‐dependent studies showed that mIBG is an excellent substrate of hPMAT, hNET, and hOCT1‐3. Concentration‐dependent studies were fitted to the Michaelis‐Menten equation, resulting in Km values for hPMAT, hNET, and hOCT2 of 42.0 ± 10.7, 35.7 ± 8.91, and 42.9 ± 12.5 μM, respectively, suggesting that mIBG has a similar apparent affinity towards these transporters. Our data demonstrated that mIBG is a novel substrate for hPMAT and hOCTs, providing a molecular basis for these transporters as potential facilitators for mIBG uptake into normal tissues.

Support or Funding Information

This work is supported by NIH Grants GM066233 and T32‐GM007750.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multidrug resistance protein 4 (Mrp4), a drug transporter is a novel genetic determinant for adipogenesis
Authors: Donepudi Ajay C. , Manautou Jose E.
Published: June 2021

Multidrug resistance protein 4 (Mrp4; encoded by Abcc4) is a basolateral; efflux pump that transports several xenobiotic and endogenous molecules such as prostaglandins and cyclic nucleotides. Mrp4 is highly expressed in kidney and bladder, with minimal expression in liver. Recently, our laboratory determined that mice lacking Mrp4 develop hepatic steatosis during compensatory tissue regeneration following partial hepatectomy. These studies indicate that Mrp4 may play a significant role in lipid homeostasis. Recent studies also showed that Mrp4 is expressed in adipose tissue, yet the role of Mrp4 in adipose tissue physiology and lipid metabolism is unknown. To begin addressing this, WT and Mrp4 knockout (Mrp4−/−) mice were subjected to 24‐hr fasting. The mouse embryonic fibroblast cell line 3T3‐L1 was also used to investigate the role of Mrp4 in adipogenesis. The results showed that both male and female Mrp4−/− mice have increased adipose tissue to body weight ratio and adipocyte cell size in both fed and fasted conditions. Additionally, the lack of Mrp4 is associated with increased serum glucose levels and impaired glucose tolerance in mice challenged with the glucose tolerance test. In parallel to increased adipose tissue weights, Mrp4−/− mice also showed increase in leptin gene expression in adipose tissue. In vitro studies using 3T3‐L1 cells showed that pharmacological inhibition of Mrp4 function using either MK571 or C1 increases adipogenesis and expression of adipose tissue markers such as fatty acid binding protein 4 (Fabp4), lipoprotein lipase (Lpl), glucose transporter type 4 (Glut4) and peroxisome proliferator‐activated receptor gamma (Ppar‐γ). In conclusion, our studies show that the absence of Mrp4 or chemical inhibition promotes adipogenesis, in addition to its well‐established role as a drug transporter. The implications of these findings are that alterations in Mrp4 expression and function can now be considered as a potential risk factor in the pathogenesis of metabolic diseases such as fatty liver disease, obesity and diabetes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Blockade of IL‐6 Released from Human Breast Carcinoma MDA‐MB‐231 in Presence of NK‐92 Inhibits an Invasion of Breast Carcinoma
Authors: Lee HwanHee , Cho Hyosun
Published: June 2021

A variety of cytokines are existed in tumor microenvironment, which might affect tumorigenesis and metastasis. Recent studies have demonstrated that interleukin(IL)‐6, a pro‐inflammatory cytokine, is significantly present in breast tumor microenvironment. It is suggested to have a critical role in an epithelial to mesenchymal transmission(EMT) process in breast cancer cells. Natural killer (NK) cells are a large innate lymphocyte that has a cytotoxic effect to cancer cells or virus‐infected cells. However, in breast tumor microenvironment, NK cells are shown to have pro‐tumor activity in presence of various inflammatory cytokines and growth factors. We found that IL‐6 is highly produced in coculture of invasive breast cancer cell MDA‐MB‐231 and natural killer(NK)‐92 cell, but is little produced in coculture of non‐invasive breast cancer cell MCF‐7 and NK cells. Subsequently, we found that the expression of p‐stat3, p‐erk, akt and pi3k proteins is statistically up‐regulated in a coculture of MDA‐MB‐231 and NK‐92 compared to in coculture with MCF‐7. A blockade of IL‐6 by anti‐IL‐6 (1ng/ml) or shRNA in invasive breast cancer cell MDA‐MB‐231 reduced a migratory and invasive capability as well as down‐regulated an expression of p‐stat3 protein. Further we examined the inhibitory effect of apigenin on invasion or migration of invasive breast cancer cell MDA‐MB‐231. We found that a blockade of IL‐6 by apigenin in MDA‐MB‐231 mediates the inhibitory effect on migration and invasion of MDA‐MB‐231.

Support or Funding Information

this research was supported by basic science research program through the national research foundation of korea(nrf) funded by the ministry of education(2016R1A6A1A03007648)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transient Increase in Sympathetic Constriction of Mesenteric Arteries from High Fat Diet‐Fed Dahl Salt‐sensitive Female Rats
Authors: Alula Kibrom Meles , Xu Hui , Galligan James
Published: June 2021

Obesity (BMI ≥ 30) is a risk factor for hypertension. Obesity‐associated hypertension is a risk factor for cardiovascular disease, stroke, type 2 diabetes and kidney failure. Hypertension is less common in premenopausal women compared to age‐matched men. This disparity fades away after the onset of menopause. Increased sympathoexcitation in the neuroeffector junction leads to increase in constriction of blood vessels. Differences in the mechanism/s neurogenic arterial constriction in male and female are not well understood. We hypothesized that sympathetic nerve activity in high fat diet‐induced hypertension is greater in male than female rat mesenteric arteries (MA). Rats were fed either control (C) or high fat (HF) diet beginning at 3 weeks of age, and used at 10, 17 and 24 weeks on diet. Body weight and systolic blood pressure (SBP) were measured weekly. HF males gained more weight than HF females at all time points. SBP was higher in HF rats compared to C rats in both sexes and across all time points. The estrus cycle stage was determined cytologically by vaginal lavage prior to euthanasia. Mesenteric arteries (Inner diameter; male=275–350 μm, female=245–315 μm) were harvested and mounted in a pressure (60 mmHg) myograph and inner diameter was measured continuously. MA contractile responses to electrical field stimulation (30 stimuli at 0.2–30 Hz) and drug applications were measured. Neurogenic constriction of MA from HF females was greater than HF males at 5 and 10 Hz at 10 wks, and at 5–30 Hz at 17 wks. At 24 wks the difference disappears. Constrictions caused by norepinephrine (NE) and ATP were similar in MA from HF male and female rats. Immunostaining for tyrosine hydroxylase (sympathetic nerve marker) and vesicular nucleotide transporter (ATP marker) revealed greater sympathetic nerve intensity in HF male than female rats at 10 and 24 wks. There was also greater intensity in MA from HF males than HF females at 10 and 17 wks. Moreover, HF males have higher NE and ATP nerve density than HF females at 10 and 17 but not for ATP at 24 wks. MA NE levels were similar in male and female rats. Prejunctional α2‐AR and NE transporter functions were not impaired in MA from HF Dahl salt‐sensitive rats. Taken together, despite sex differences in sympathetic nerve density in MA, we detected only transient sex differences in sympathetic neurogenic arterial constrictions. Contrary to our hypothesis, we found that HF females transiently have an upregulation of neurogenic sympathetic activity and this warrants further investigation into the mechanism.

Support or Funding Information

Research supported by PPG NIH Grant P01HL070687

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuroperptides, their interactions and gastric mucosal protection in the rat.
Authors: Gyires Klara , Zádori Zoltán
Published: June 2021

In the last decades several neuropeptides, such as thyreotropin‐releasing hormone (TRH), amylin, neurotensin, ghrelin, melatonin, opioid peptides, substance P (SP), angiotensin II (Ang II) have been shown inhibit the gastric mucosal lesions induced by different ulcerogenic agents given centrally in the dose range of fmol to nmol. The site of action is likely to be the dorsal vagal complex. However, the different neuropeptides can interact with each other at different levels and can affect each other's biological actions. Among others nociceptin ‐ opioid; angiotensin ‐ cannabinoid, opioid; CRF ‐ beta‐endorphin, somatostatin interactions have been demonstrated. Aim of the present study was to elucidate potential interactions between neuropeptides and cannabinoids in gastric mucosal defense.

Methods

Gastric damage was induced by acidified ethanol in the rat (150–170 g, males); the gastric mucosal lesions were detected 60 min after ethanol administration. The dugs were given intracerebroventricularly (i.c.v.) 10 min before ethanol challenge. Gastric mucosal CGRP, somatostatin levels were determined by RIA.

Results

The gastroprotective effect of SP, nociceptin or cannabinoids was reversed by endomorphin2‐antiserum or the opioid receptor antagonist naloxone, while the Ang II‐induced protection was decreased by the CB1 receptor antagonists, AM‐251.

Conclusion

Neuropeptides have prominent role in central regulation of gastric mucosal integrity. In addition, interactions between neuropeptides result in redundancy in centrally‐initiated gastric mucosal defense.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscarinic Receptor‐Mediated Regulation of Epineurial Blood Vessels of the Sciatic Nerve
Authors: Killey Chelsa , Cleary Shane , Orr Julie , Frisbee Jefferson C. , Jackson Dwayne N. , Twynstra Jasna
Published: June 2021

Objective

The objective of this study was to determine the role of muscarinic receptors on the diameter of the epineurial vessels of the sciatic nerve in situ.

Methods

An anesthetized rat model (318.4±7.1 g) was used to expose the sciatic nerve and directly visualize its microvasculature using intravital video microscopy. Rats were tracheotomized and catheters were inserted into the jugular vein for supplemental anesthesia, and the carotid artery for measures of mean arteriolar pressure. Alexa Fluor 488 Isolectin GS‐IB4 conjugate (178.6 μm/ml) was topically applied for 10 minutes to the exposed sciatic nerve to stain the endothelial cells of the epineurial vessels of the nerve. Following a 30‐min equilibration period, acetylcholine (10−4 M, n=8) or atropine (10−5 M, n=6) was topically applied for 4 and 5 minutes, respectively. Images were captured at baseline (immediately prior to drug application) and every minute during the application period. The images were analyzed offline using ImageJ to measure the diameter of the vessels at each time point. All data are expressed as mean ± standard error.

Results

Acetylcholine caused a vasodilation of the epineurial vessels (baseline: 22.9±2.4 μm, acetylcholine: 29.2±2.9 μm, p<0.01). There was no difference in mean arteriolar pressure from baseline (103.8±1.5 mmHg) to maximum vasodilation (102.3±2.7 mmHg). Atropine caused a vasoconstriction of the epineurial vessels (baseline: 26.7±3.2 μm, atropine: 15.5±3.6 μm, p<0.05). There was no difference in mean arteriolar pressure from baseline (104.0±1.9 mmHg) to maximum vasoconstriction (105.2±2.2 mmHg).

Conclusion

Application of acetylcholine, a muscarinic receptor agonist, resulted in a vasodilation while application of atropine, a muscarinic receptor antagonist, caused vasoconstriction. This finding suggests that muscarinic receptors are involved in blood flow regulation of peripheral nerves. Further, since atropine constricted the vessels at baseline conditions, muscarinic receptors may be involved in maintaining baseline diameter.

Support or Funding Information

This study was supported by the SEED Research Grant from The University of Western Ontario.

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