bluuebunny/crossref_metadata_2025 huggingface dataset:
ABC Transporter: Atm1 and the Transport of Heavy Metals
Authors: Ureta Lyle , Hicks Dennis , Yang Janet
Published: June 2021

ATP binding cassette (ABC) active transporters rely on the ability to bind and hydrolyze ATP with the purpose of exporting and importing molecules into and out of cells. In this study, we focus on Atm1, an ABC transporter originally discovered in yeast mitochondria. Atm1 plays a critical role in iron sulfur cluster synthesis, although the exact mechanism is not known. The protein utilized in this research is NaAtm1, from Novosphingobium aromaticivorans, which has been shown to confer heavy metal resistance when overexpressed in E. coli. The overall goal of this research is to confirm that the Atm1 that we are working with does indeed transport heavy metals, and to understand how Atm1 mediates this transport. Toward this end, four mutants of the Atm1 protein were created to dissect the mechanism of transport. The first mutant was made in the Walker B motif and is expected to be ATP hydrolysis deficient. This mutant was created by knocking out the catalysis ability of the protein by replacing a glutamate with a glutamine (E523Q). The second mutant, S526C, will be fluorescently tagged to observe changes in conformation when the protein is bound to nucleotides and/or substrate. The third mutant, K142Q, is designed to uncouple ATP hydrolysis and transport. The last mutant, K400A, is expected to eliminate the ability to bind ATP by changing a lysine to an alanine in the Walker A motif. This collection of mutations will allow us to begin our dissection of the NaAtm1 mechanism.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Avocado Oil Alleviates Renal Damage and decreases NADPH Oxidase Activity, Peroxynitrite Production and Mitochondrial Calcium Uptake in Hypertension Rats
Authors: Olmos‐Orizaba Berenice Eridani , Márquez‐Ramírez Cristian Adrián , Garcia‐Berumen Claudia Isabel , Villagómez Ana Victoria Hurtado , Calderón‐Cortés Elizabeth , Saavedra‐Molina Alfredo , Montoya‐Pérez Rocio
Published: June 2021

Hypertension (HT) has been associated with mitochondrial dysfunction due to both nitrosative and oxidative stresses that are generated by increased activity of NADPH oxidase (Nox), defective calcium transport and enhanced ROS generation by reverse electron transfer (RET) at complex I. These factors contribute to the pathogenesis of endothelial dysfunction and to the progress of hypertensive complications. Previously, we have shown that avocado oil, a rich source of bioactive compounds and antioxidants, decreases blood pressure and ameliorates both mitochondrial dysfunction and oxidative stress in kidney from hypertensive rats. However, it is unknown whether the effects of avocado oil on renal mitochondria are attributable to decreased activity of NADPH oxidase, lowered levels of reactive nitrogen species (RNS) and improved calcium uptake by mitochondria. Thus, we analyzed Nox activity and peroxynitrite (ONOO‐) levels in kidney cytosolic fractions of L‐NAME – induced hypertensive rats as well as mitochondrial calcium uptake and complex I activity. After 60‐days of L‐NAME administration, the hypertensive rats exhibited enhanced Nox activity and ONOO‐ levels, as well as increased complex I activity and excessive calcium uptake. All these alterations were prevented by co‐administration of L‐NAME with avocado oil. Moreover, the kidneys from hypertensive rats shown histological manifestations of renal damage that were also corrected by avocado oil. These data suggest that avocado oil could be beneficial to decrease hypertensive nephropathy progression by improving mitochondrial function, diminishing RNS production and normalizing Nox activity.

Support or Funding Information

This work was supported by a Coordinación de la Investigación Científica‐UMSNH grant (to CCR)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gas7 regulates mitochondrial morphology and physiology
Authors: Bhupana Jagannatham Naidu , Huang Bo‐Tsang , Chang Yu‐Ling , Chao Sue Lin
Published: June 2021

Gas7 belongs to the F‐BAR family of proteins and has been shown to be involved in actin and microtubule dynamics. Gas7 is mainly expressed in mouse brain and testicular tissues. In primary cultured neurons, Gas7 enhances actin polymerization and neurite outgrowth. Mutations in PARKIN and PINK1 genes, which play major roles in mitochondrial quality control and mitochondrial autophagy, are often associated with Parkinson's disease. Here, we report a role of Gas7 in mitochondrial distribution, physiological functions, and enhancement of PINK1‐parkin‐mediated mitochondrial autophagy. In the brain of gas7 knockout mice, the mitochondrial aspect ratio is elevated, suggesting a decrease in the level of mitochondrial fragmentation, resulting in an elongated shape. Furthermore, in primary neuronal cultured cells, we found that mitochondrial distribution was restricted around the nucleus, tubular mitochondrial subtypes increased, indicating decreased mitochondrial division, and increased mitochondrial fusion in Gas7 knockout neurons. Gas7 ectopic‐expression in these primary cultured neurons can restore normal morphology and distribution of mitochondria. Overall, our data suggest that Gas7 is required for normal mitochondrial dynamics and therefore may be important for cerebral neurons to maintain mitochondrial function.

Support or Funding Information

This work was supported by an intramural fund from the Institute of Molecular Biology, Academia Sinica and a theme project of Academia Sinica (2323)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Leptin Delivers β‐cell‐derived C‐peptide to Red Blood Cells
Authors: Meints Lisa , Keshavarz Hamideh , Kauffman Nate , Geiger Morgan , Zinn Kurt , Spence Dana
Published: June 2021

It was recently reported that C‐peptide, the connecting peptide of proinsulin, binds to red blood cells (RBCs) when delivered by albumin. Additionally, treatment of RBCs with C‐peptide, zinc, and albumin results in an increase in ATP release from the RBC and downstream production of endothelium‐derived nitric oxide. Here, we report leptin, an adipocyte‐derived cytokine known for its importance for energy balance and potential glucoregulatory effects, as a potential drug carrier for C‐peptide. Here, we report that C‐peptide increases GLUT1 translocation and ATP release from RBCs, but only in the presence of leptin and zinc. To investigate the nature of the interaction of C‐peptide and leptin with RBCs we have utilized leptin labeled with technetium‐99m (99mTc) to perform saturation binding studies. Samples containing 7% RBCs were treated with different concentrations of labeled leptin and 20 nM C‐peptide. Samples were then incubated, centrifuged, and washed several times prior to quantitative determination of gamma emission from the 99mTc‐labeled leptin bound to the RBCs. Data was correlated back to a standard curve of labeled leptin, and a saturation binding curve was generated. Initial results suggest that total binding of labeled leptin, in the presence of C‐peptide, begins to saturate when 200 picomoles of labeled leptin are added to the RBCs.

Support or Funding Information

National Institutes of Health

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of melatonin on neural reconstruction after acute spinal cord injury through regulation of endoplasmic reticulum stress response and autophagy
Authors: Jin Yunho , Hong Yonggeun
Published: June 2021

It has been reported that autophagy and endoplasmic reticulum (ER) stress response cause improved hindlimb motor function and reduced damage of axons in spinal cord injured animals, respectively. However, the effects of melatonin on neural reconstruction and motor recovery through regulation of ER stress response and autophagy have not been well described. Therefore, the purpose of this study is to elucidate the effects of melatonin treatment on neural reconstruction and motor recovery through regulation of ER stress response and autophagy.

To verify the effect of melatonin injection on post‐SCI alterations regarding neural cells, autophagy, and ER stress response, we analyzed markers at protein level, and morphological changes. At day 3 after SCI, melatonin did not cause behavioral improvement (p<.05). At molecular levels, melatonin suppressed the loss of astrocytes after SCI (p<.05). Beclin‐1 expression was decreased by melatonin treatment (p<.05). In other words, autophagy activation in SCI+MT was lower than that of SCI group, indicating melatonin‐mediated suppression of autophagic cell death. In addition, GRP78 expression was preserved in SCI+MT group with no difference compared to non‐injured groups. Namely, ER stress response was advanced by melatonin. In histological analysis, it was proved that dendritic branches were preserved by melatonin treatment.

In conclusion, exogenous treatment of melatonin may result in neural reconstruction after SCI through regulating autophagy and ER stress response at the injured spinal segments.

Support or Funding Information

NRF‐2017R1A2A2A01067169, KGM4611821, 2016 Creative Research Program of Inje University, Republic of Korea

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Compared to liver, bile duct and skeletal muscle, garlic stimulates only pancreatic insulinogenesis in early streptozotocin‐induced diabetic rats
Authors: Al‐Qattan Khaled K , Al‐Adsani Amani M
Published: June 2021

Background

Streptozotocin (STZ) induces diabetes mellitus (DM: STZ‐DM) type‐1 in rats by pancreatic β‐cells DNA alkylation and consequently reduction in insulin circulating concentration. Garlic aqueous extract (GE) increases insulin circulating concentration in the STZ‐DM rat model.

Objective

This study investigated the insulinogenic mechanisms of GE in the pancreas, liver, bile duct and gastrocnemius skeletal muscle (GSM) of STZ‐DM rats.

Method

The body weight, food and water intake, urine output, fasting blood glucose (FBG), serum insulin (SI) in addition to insulin m‐RNA expression, immunohistochemical localization (IHC) and concentration were investigated before (basal level = BL) and after 1, 4 and 8 weeks of oral treatment in normal rats treated with normal saline (NR‐NS), diabetic rats treated with normal saline (DR‐NS) and diabetic rats treated with GE (DR‐GE).

Results

Compared to NRNS, DR‐NS showed significant typical diabetic behaviors and biophysical symptoms including progressive increases in FBG and reductions in SI, in addition to reductions in pancreatic insulin m‐RNA expression, IHC and concentration. Conversely, and in comparison to DR‐NS, DR‐GE showed significant amelioration of diabetic behaviors and biophysical symptoms, including progressive decreases in FBG and increases in SI, in addition to timely‐progressive increases in pancreatic insulin m‐RNA expression, IHC labeling and concentration. The remaining investigated tissues did not show any advent changes in insulin m‐RNA and IHC in response to GE treatment.

Conclusion

Garlic's short‐term treatment induced hyperinsulinemia observed in the early STZ‐DM type‐1 rat model may result from augmented pancreatic insulinogenesis.

Support or Funding Information

Research Sector ‐ Kuwait University (SL04/14)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lin28a Regulates Pathological Cardiac Hypertrophic Growth through Pck2‐mediated Enhancement of Anabolic Synthesis
Authors: Ma Hong , Yu Shuo , Liu Xiaojing , Zhang Yingao , Fakadej Thomas , Liu Ziqing , Yin Chaoying , Shen Weining , Locasale Jason W , Taylor Joan M , Qian Li , Liu Jiandong
Published: June 2021

During pathological hypertrophy, the heart undergoes extensive metabolic changes prior to the onset of structural remodeling, yet, it remains largely unexplored whether and how this metabolic remodeling could contribute to cardiac hypertrophic growth. Here, we identified the RNA‐binding protein Lin28a as a critical regulator of pathological cardiac hypertrophy and metabolic re‐patterning. Cardiac‐specific deletion of Lin28a attenuated pressure overload‐induced hypertrophic growth, cardiac dysfunction and alterations in cardiac transcriptome. Mechanistically, Lin28a directly bound to mitochondrial phosphoenolpyruvate carboxykinase 2 (Pck2) mRNA and positively modulated its level. Manipulation of Pck2 expression phenocopied hypertrophic growth phenotypes of manipulating Lin28a expression. Epistatic analysis demonstrated that Pck2 mediated, at least in part, the role of Lin28a in cardiac hypertrophic growth. Furthermore, metabolomic analyses highlighted role for Lin28a and Pck2 in promoting cardiac anabolic biosynthesis required for cell growth. Thus, our study revealed a critical role of Lin28a in the regulation of pathological cardiac hypertrophic growth through Pck2‐mediated regulation of cardiac metabolism.

Support or Funding Information

This study was supported by AHA 13SDG17060010, Ellison Medical Foundation (EMF) AGNS‐13 1064‐, and NIH/NHLBI R01HL128331 to L.Q., NIH/NHLBI R00 HL109079, R56HL133081, and American Heart Association (AHA) 15GRNT25530005 to J.L.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cyclic stretching establishes cardiac model expressing ANF and β‐MHC that responds to anti‐oxidizing drug
Authors: Wong Tzyy‐Yue , Tseng Yu‐Kai , Jhong Rong‐Chang , Lee Wen‐Hsien , Cheng Pei‐Wen
Published: June 2021

The road to drug discovery for heart‐related diseases is challenged by advancement in whole‐heart and reliable animal heart models. In vitro model is a solution to replacing the unpredictable in vivo heart models. However, the techniques are time‐ and cost‐consuming. Cardiomyocyte cell line that expands in culture can be trained by mechanical stimulation to express cardiac biomarkers. The use of mechanical stimulation such as cyclic stretching can be more predictable, reliable in terms of cardiac marker expressions. The heart is an organ that is in constant dynamic which requires regulation for reactive oxygen species (ROS) to prevent pathological process. Here, we used cyclic stretching to stimulate cardiomyocytes with mild (5%), and aggressive (25%) strains at 1Hz. The stimulated cardiomyocytes expressed the cardiac characteristic markers Atrial natriuretic factor (ANF), and β‐myosin heavy chain (β‐MHC) after 24h of stretching. The 3H‐1,2‐dithiole‐3‐thione (D3T) has been reported to be a potent inducer of antioxidant genes through activation of the transcription factor Nrf2, and Nrf2 has been reported to play a role in cardiac remodeling.

In this study, we investigated the effect of the D3T on the cardiomyocytes stimulated at mild, or aggressive strain. Results show that the aggressive strain reduced the β‐MHC expression, and the D3T enhanced the β‐MHC expression in cardiomyocytes that were stimulated with aggressive strain.

Support or Funding Information

This research is supported by VGHKS107‐076, VGHKS107‐168, VGHKS107‐175, MOST104‐2320‐B‐0751B‐003‐MY3, and MOST106‐2320‐B‐075B‐001.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plakophilin‐dependent Dsg3 oligomerization and binding properties are involved in desmosomal hyper‐adhesion
Authors: Fuchs Michael Tobias , Foresti Marco , Vielmuth Franzsika , Waschke Jens
Published: June 2021

Under mechanical stress, intercellular adhesion of keratinocytes is critically dependent on desmosomes which, during maturation, acquire a hyper‐adhesive state – at which desmosomal cadherins become independent from Calcium (Ca2+). The desmosomal plaque proteins plakophilins (Pkps) are known to be involved in desmosomal turnover and hyper‐adhesion. However, the molecular mechanism underlying is not elucidated yet. Thus, we here used atomic force microscopy (AFM) in combination with biochemical approaches in murine keratinocytes lacking either Pkp1 or Pkp3 to dissect the underlying mechanism by which Pkps regulate desmosomal cadherin binding properties and desmosomal hyper‐adhesion. Intercellular adhesion was impaired in Pkp3‐ and more severe in Pkp1‐deficient keratinocytes as revealed by keratinocytes dissociation assay. In accordance, Pkps regulated the single molecule binding properties of desmoglein 3 in a manner that Pkp loss led to decreased binding frequency and strength. This was further accompanied by compromised oligomerization of Dsg3. Furthermore, loss of Pkp1 but not Pkp3 causes increased lateral mobility and impaired cluster size of Dsg3. Thus, we questioned whether Pkp‐dependent changes in Dsg clustering and mobility participate in development of a hyper‐adhesive state in those keratinocytes. Interestingly, in keratinocyte dissociation assays wt keratinocytes become partly hyper‐adhesive after 72h whereas Pkp‐loss abolished the development of Ca2+‐independency. Further, Pkp1‐deficiency reduced development of Ca2+‐independent Dsg3 oligomers on the contrary to wt and Pkp3 k.o. keratinocytes. Furthermore, in wt but not in Pkp‐deficient keratinocytes, Dsg3 single molecule binding strength was increased in wt keratinocytes when cells where hyper‐adhesive. Whereas this increase was missing in Pkp‐deficient keratinocytes, which could serve as a molecular explanation for their failure to reach a proper hyper‐adhesive state. In summary, the data provided show that Pkp‐dependent changes in Dsg3 oligomerization as well as binding properties provide a new concept of the molecular mechanism of hyper‐adhesion.

Support or Funding Information

Else‐Kröner‐Fresenius‐Stiftung 2016_AW157

DFG SPP1782

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synonymous Changes in Specific Leucine Codons Impact Morbilliviral Protein Production from Human & Canine Codon Optimized Constructs
Authors: Uhl Elizabeth W , Osborn Michelle L , Michel Frank , Jelesijevic Tomislav , Hogan Robert J
Published: June 2021

Protein expression is increased by optimization of human measles virus (HMV) & canine distemper virus (CDV) nucleoprotein (N) gene to human or canine codon usage bias (CUB) and decreased by suboptimization. Six leucine codons were all replaced by CTG in human CUB optimized N gene constructs and by CTG & CTC in canine CUB optimized N gene constructs. For CUB suboptimized N gene constructs only CTT coded for leucine. The purpose of this experiment was to determine the effects of synonymous leucine codon changes. Protein expression from human & canine CUB optimized constructs in which CTG was replaced with CTT was compared using flow cytometry to that from WT, human & canine CUB optimized versions. Protein expression from CUB suboptimized versions with CTT changed to CTG was also compared to that from unmodified constructs. Human & canine CUB optimized CTT versions of the HMV N gene had significantly decreased protein production, with expression from the CTT canine CUB optimized construct the most suppressed. Replacing CTG with CTT in the human optimized CDV N gene constructs decreased the level of protein expression, while changing CTG to CTT in the canine optimized CDV N gene construct did not significantly change the level of protein expression. Protein expression from the CTT modified suboptimized CDV N gene construct was significantly increased compared to that from the unmodified suboptimized version. These results indicate that synonymous changes in specific leucine codons can significantly impact morbilliviral protein production in ways that vary with the virus and the context in which they occur.

Support or Funding Information

The project depicted was sponsored by the Department of Defense Threat Reduction Agency. The content of the information does not necessarily reflect the position or the policy of the federal government, and no official endorsement should be inferred

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pathogenicity of Mycoplasma hyopneumoniae strain in Naturally Infected Slaughtered Pigs in Nigeria: Immunohistochemical and Ultrastructural Studies
Authors: Olaniyi Moshood , Omotainse Samuel , Ajayi Lawrence , Ojo O , Brown Corrie , Shields John , Ard Mary , Nagy Tamas , Alaka O
Published: June 2021

Enzootic pneumonia caused by Mycoplasma hyopneumoniae (MHYO) remains a serious concern to swine industry in many countries including Nigeria. MHYO strains isolated from different countries and geographical locations are known to vary in pathogenicity in pigs. There is paucity of information on the pathogenicity of MHYO strain affecting pigs in Nigeria. This study investigated the pathogenicity of MHYO strain in naturally infected slaughtered pigs in Nigeria using immunohistochemistry and electron microscopy. Two hundred and sixty four lungs were randomly collected from abattoirs at Abeokuta, Ibadan and Lagos, Southwest Nigeria. A sub‐sample of 104 pneumonic lungs was selected, processed for routine histopathological examination and immunohistochemistry, while 3 lung tissues were selected for ultrastructural studies. The most significant microscopic changes were suppurative broncho‐interstitial pneumonia associated with varying degrees of lymphoid hyperplasia of the bronchus‐associated lymphoid tissue (BALT) and thickening of alveolar septa due to cellular infiltration. Immunohistochemically, MHYO antigens were detected in 86/104 (82.7%) lungs. Bronchial and bronchiolar epithelial cells typically exhibited a granular brown reaction on the surface, in the BALT and luminal cellular exudates of MHYO‐infected lung tissues. Transmission electron microscopy revealed numerous Mycoplasmas in the lumina of respiratory tract, in between degenerated cilia, while a few Mycoplasmas were located within the alveoli. It is concluded that the MHYO strain detected in this study is pathogenic to pigs and capable of inducing pneumonic lesions, and therefore implicated in the pathogenesis. This is the first investigative study on pathogenicity of MYHO strain in slaughtered pigs in Nigeria.

Support or Funding Information

Support from the Federal University of Agriculture, Abeokuta

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary Sugars and bacteria interactions affect cariogenic properties of cavity‐causing bacteria
Authors: Wu Janina , Yang Chan
Published: June 2021

Cavity is becoming more prevalent in our society. One major contributor is the modern diet, specifically the large amounts of sugars in diet. Another key factor is bacteria in our mouth that eat these sugars. Streptococcus mutans is one of the leading bacteria that initiate and promote cavity. They metabolize sugars to build up plaque over time and simultaneously produce acid that erodes tooth surface and causes cavity. We were interested in studying how different sugars interact with S. mutans: how different sugars influence pH changes and affect S. mutans colonization, which would help us recognize what sugar is beneficial or harmful to your teeth.

We used a fruit fly model because fruit flies like sugars and mixing S. mutans with sugars can facilitate S. mutans colonize fruit flies. To make sugary food I mixed 8 different sugar solutions with S. mutans (labeled with green fluorescent protein) and soaked them to small filters. Then we grew fruit flies and separated them into 8 different tubes that were prepared with the sugar‐bacteria soaked filters on top. We then waited for 6 days to let flies eat the food and grow at room temperature. After the incubation, we took 1–2 flies every 2 days and frozen them to image the green color under Fluorescent Microscope, which indicates S. mutans colonization. On the final day, we harvested all of the remaining flies, ground them up, made different solutions, and then streaked various dilutions onto agar plates and incubated them for 2 days to count bacterial colonies, which represents bacterial colonization of flies. Flies fed with five sugars including sucrose showed bacterial colonization by direct imaging and calculating bacterial colony forming units. Flies fed with sucrose exhibited the best colonization, while flies fed with xylitol did not show any colonization. Overall the data indicated that S. mutans utilizes some dietary sugars more efficiently and as a result, can colonize more by eating some sugar than others.

To evaluate the effects of different sugars on the pH of S. mutans cultured in bacterial medium Todd Hewitt Broth, with pH adjusted to 7.5, we added various sugars to the medium and incubated for 30 minutes to monitor the pH changes using a pH meter. With all 8 sugars, the pH dropped significantly after five minutes. The pH did not drop below 4.5 with 6 sugars. But the pH dropped even below 4.5 with two sugars, glucose and sucrose. The pH dropped at an average of 2.91 during the 30 minutes testing period, suggesting sugars and bacteria fuel acid production. These studies reveal different sugars ate by S. mutans can produce different amounts of acid that drives the pH drop of S. mutant culture media, thus interactions between bacteria and sugars are important for the development of cavity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thrombospondin‐1 worsens acetaminophen‐induced acute liver failure in mice through its activation of TGFβ1
Authors: McMillin Matthew , Jefferson Brandi , Grant Stephanie , Frampton Gabriel , Ali Malaika , DeMorrow Sharon
Published: June 2021

Introduction

Acute liver failure results from a significant loss of liver function and has few effective treatments and poor clinical outcomes. Previously we have demonstrated that transforming growth factor beta 1 (TGFβ1) is upregulated during azoxymethane‐induced acute liver failure in mice and this signaling pathway promotes hepatic encephalopathy progression. Recently TGFβ1 has been shown to promote acetaminophen‐induced liver injury by promoting senescence of hepatocytes. TGFβ1 requires interactions with other proteins, such as thrombospondin‐1 (TSP‐1), to become activated and to be capable of binding its receptors. Therefore, we hypothesize that TSP‐1 activates TGFβ1 during acetaminophen‐induced acute liver failure worsening outcomes.

Methods

Male C57Bl/6 mice or TSP‐1 knockout mice were injected with acetaminophen (APAP; 300 and 600 mg/kg) to generate acute liver injury. APAP‐treated mice had serum and liver collected at up to 24 hours post injection. Liver pathology was assessed by H&E staining, TUNEL staining, malondialdehyde assay, glutathione assay, nitrotyrosine expression and transaminase assessments. TGFβ1, TSP‐1, IL‐1β, IL‐6, TNF‐α, SOD1, iNOS and phosphorylated/total JNK expression were assessed by immunoblotting, immunohistochemistry, immunofluorescence, ELISA and/or real‐time PCR assays.

Results

Mice injected with APAP had elevated hepatic TGFβ1 and TSP‐1 levels, which co‐localized primarily to hepatocytes. APAP‐treated mice with genetic knockout of TSP‐1 had reduced hepatic injury and hepatocyte necrosis as evident by improved serum transaminases, restored glutathione levels, reduced phosphorylated JNK signaling and less TUNEL staining compared to wildtype APAP‐treated mice. Expression of oxidative and nitrosative stress markers, as well as proinflammatory cytokines, were reduced in TSP‐1 knockout mice compared to wildtype mice administered APAP.

Conclusions

Strategies employed to reduce TSP‐1 signaling in APAP‐treated mice reduced liver damage and pathology. Therefore, targeting TSP‐1 signaling may be a novel therapeutic target for the management of APAP‐induced acute liver failure.

Support or Funding Information

This study was funded by NIH R01 awards (DK082435 and DK112803) and a VA Merit award (BX002638) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. DeMorrow. This study was also funded by a VA Career Development award (BX003486) from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service to Dr. McMillin.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin enhances presynaptic glutamate release via opioid receptor‐mediated disinhibition
Authors: Ferrario Carrie R. , Santana‐Rodriguez Zuleirys , Oginsky Max F.
Published: June 2021

Recent studies show that insulin modulates the function of brain reward and motivation systems. For example, insulin receptor activation enhances dopamine release in the nucleus accumbens (NAc), reduces excitatory transmission in the VTA, and decreases food intake when infused into the NAc. Excitatory transmission in the NAc plays critical roles in motivation, and diet‐induced obesity alters NAc activity and peripheral sensitivity to insulin. However, insulin's effects on NAc excitatory transmission are unknown. Therefore here, we used whole‐cell patch clamp recordings in adult rat brain slices to determine how insulin affects excitatory transmission onto NAc medium spiny neurons (MSNs) and the mechanisms involved. Importantly, in addition to insulin receptors, insulin‐like growth factor receptors (IGFRs) are also expressed in the NAc and can be activated by moderate to high concentrations of insulin. Thus, a wide range of insulin concentrations were examined here (30–500 nM), and the contribution of insulin receptor activation vs. IGFR activation to insulin's effects was determined using pharmacological approaches. We find that insulin receptor activation increases presynaptic glutamate release via a previously unidentified form of opioid receptor‐mediated disinhibition, whereas activation of IGF receptors by insulin decreases presynaptic glutamate release. Furthermore, diet‐induced obesity (60% high‐fat; Open Source Diets D12492, 8 weeks) resulted in a loss of insulin receptor‐mediated increases and a reduction in NAc insulin receptor surface expression compared to chow fed controls (Lab Diet 5001, 13% fat), while preserving reductions in transmission mediated by IGRFs. These results provide the first insights into how insulin influences excitatory transmission in the adult brain and have broad implications for the regulation of motivation and reward related processes by peripheral hormones.

Support or Funding Information

This work was supported by NIDDK R01DK106188, R01DK115526, and the Brain and Behavior Research Foundation N018940 awards to CRF; MFO was supported by NIDA T32DA007268 and NIDDK FDK112627A. The authors declare no competing financial interests. Studies utilized the Chemistry Core of the Michigan Diabetes Research and Training Center funded by DK020572, and the University of Michigan Animal Phenotyping Core supported by P30 grants DK020572 (MDRC) and DK089503 (MNORC). We thank Drs. Marina Wolf, Larry Reagan, William Birdsong, Travis Brown and Garret Stuber for helpful conversations and Dr. Kenner C. Rice (Drug Design and Synthesis Section, NIDA IRP) for the gift of (+)‐naloxone.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
κ‐Opioid Receptor Activation Induces the Autophagic Machinery and Alters Dendritic Spine Density
Authors: Georgoussi Zafiroula , Karoussiotis Christos
Published: June 2021

It is already evident that opioid receptors modulate the neuronal function by regulating neurite outgrowth and neurotransmission with unknown yet mechanisms (1, 2). Autophagy is a lysosomal degradation pathway which eliminates misfolded proteins and dysfunctional organelles essential for neuronal function and homeostasis (3). Recent observations have shown that G‐protein coupled receptors could mediate the autophagic machinery and that methamphetamine induces autophagy in endothelial cells through the κ‐OR (4). However, it is unknown whether specific κ‐opioid agonists mediate these effects and whether opiodergic alterations in neuronal cells could be controlled through induction of autophagy. Our data indicate that administration of κ‐selective agonists increase the levels of the autophagosome marker LC3‐II (Fig. 1) and the pre‐autophagosomal proteins Beclin 1 and ATG5 respectively in a dose and time dependent manner. These effects are reversed upon exposure of neuroblastoma cells and cortical neurons with the opioid receptor antagonist naloxone, suggesting that it is a κ‐OR‐mediated effect. Pre‐treatment of cells with pertussis toxin blocked the κ‐OR mediated Beclin 1 upregulation and LC3‐II formation, suggesting that Gi/Go proteins are involved in κ‐OR induced autophagy. Futhermore, κ‐opioid agonist exposure of primary neuronal cortical cultures decreased the levels of Neurabin II, a neuronal protein enriched in dendritic spines, and the levels of Synaptophysin, a synaptic terminal protein, suggesting that these proteins are engulfed in the κ‐OR mediated autophagic cargo. Collectively, the present results demonstrate a novel regulatory mechanism via which specifιc κ‐OR agonists affect dendritic spine morphology via induction of autophagy.

Support or Funding Information

This work was supported mainly by the GSRT Excellence II grant, 3722 entitled “Neurite Outrgowth: ALternative G‐ protein coupled Opioid receptor Signaling”‐ “NO‐ALGOS” to Z.G. We acknowledge support of this work also by the project “Target Identification and Development of Novel Approaches for Health and Environmental Applications” (MIS 5002514) which is implemented under the Action for the Strategic Development on the Research and Technological Sectors, funded by the Operational Programme “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co‐financed by Greece and the European Union (European Regional Development Fund). The facilities of the Hellenic Research Infrastructure OPENSCREEN‐GR were used.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Morphine drug discrimination: A behavioral model to assess novel synthetic opioid substances
Authors: Eneanya Chidubem , Walker Teneille , Tella Srihari , Akinfiresoye Luli , Walker Ellen
Published: June 2021

A large number of novel psychoactive substances including opioid‐like compounds have appeared on the illicit market in the past decade. Several substances structurally related to fentanyl are being abused alone and in combination with other drugs (e.g., heroin and/or cocaine), resulting in overdoses at an alarming rate. There is a paucity of information about the pharmacology of these novel synthetic substances. In this study, we evaluated the opioid‐like stimulus effects of crotonyl fentanyl, thiophene fentanyl, benzodioxole fentanyl, and methoxyacetyl fentanyl, four newly identified substances structurally related to fentanyl. We compared the potencies of these new substances to schedule II opioid analgesics such as fentanyl and morphine using a preclinical abuse liability assessment model called drug discrimination. We also studied the susceptibility of the stimulus effects of these test drugs to antagonism by the opioid antagonist naltrexone, a drug used in patients to reverse the overdose effects of heroin and fentanyl. Using a two‐lever operant experimental chamber under a fixed ratio‐10 schedule for food delivery, rats (Sprague Dawley) were trained to discriminate between 3.2 mg/kg morphine and saline, subcutaneously. We evaluated saline and multiple doses of morphine, fentanyl, crotonyl fentanyl, thiophene fentanyl, benzodioxole fentanyl, and methoxyacetyl fentanyl. Crotonyl fentanyl, thiophene fentanyl, and methoxyacetyl fentanyl, similar to morphine and fentanyl, fully substituted for the morphine discriminative stimulus and decreased response rates with slightly differing potencies. Benzodioxole fentanyl failed to produce significant morphine‐like responding or rate‐decreasing effects at any dose although testing was limited by solubility issues. Crotonyl fentanyl, thiophene fentanyl, and methoxyacetyl fentanyl were blocked by naltrexone indicating that mechanisms of action of these three drugs are similar to that of morphine and fentanyl. The opioid‐like discriminative stimulus effects of crotonyl fentanyl, thiophene fentanyl, and methoxyacetyl fentanyl indicate that subjective effects and abuse liability of these three novel substances are likely to be similar to that of morphine and fentanyl.

Support or Funding Information

DJD‐17‐HQ‐P‐0646

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of the Kappa Agonist U69,593 on Naltrexone's Discriminative Stimulus Effects in Subjects Given Chronic, Intermittent Sucrose Access
Authors: Marek Morgan , Schulz Emily , Fruit Nicholas , Vile Oriana , Rothbauer Dylan , Brandt Lucas , Jewett David
Published: June 2021

Naltrexone (NTX) is a nonspecific opioid antagonist that binds to mu‐, kappa‐, and delta‐opioid receptors. Previous research has demonstrated that rats given chronic, intermittent sucrose access have increased endorphin function. We have shown that rats given chronic, intermittent sucrose can discriminate 1.0 mg/kg NTX from saline in a two‐lever, operant choice procedure. We wondered if NTX's discriminative stimulus effects were mediated by kappa‐opioid receptors. To examine this possibility, we tested the effects of the kappa‐opioid agonist U69,593 in our discrimination procedure. Male Sprague‐Dawley rats were given access to 12‐hour access to a 25% sucrose solution and trained to discriminate NTX (1.0 mg/kg) from saline. Once discrimination criteria (80% or greater condition‐appropriate responding for 8 of 10 consecutive sessions) were reached, generalization testing began. We used a cumulative dosing procedure to determine if U69,593 alters the ability of NTX to produce its discriminative stimulus effects. Subjects were pretreated with U69,593 (0.0001 mg/kg – 0.1 mg/kg, s.c.) followed by increasing doses of NTX (0.001 mg.kg – 10.0 mg/kg, s.c.). Fifteen to 30 minutes after the injection, a test session began. Injections continued until response rates were suppressed or 10.0 mg/kg NTX was administered. Reversal tests were also conducted. During these tests, the training dose of NTX was administered and a test session began 15 minutes later. Then a single dose of U69,593 (0.001 mg/kg – 0.1 mg/kg, s.c.) was administered, followed by test sessions 15 to 120 minutes after the injection. U69,593 did not significantly alter the discriminative stimulus effects of NTX. This suggests that kappa‐opioid receptor antagonism is not sufficient to produce NTX's discriminative stimulus effects in rats given chronic, intermittent sucrose access.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Serotonin‐2A Receptor Modulation on Glutamate Levels in the Medial Prefrontal Cortex following Methamphetamine Self‐Administration
Authors: Dolezal Alexandra , McFadden Lisa , Severson Marie
Published: June 2021

Methamphetamine (METH) use can lead to psychosis‐like behaviors. Previous studies have indicated that there is an increase in frontal cortex serotonin (5HT)‐2A receptors as well as observed hallucinogenic‐drug induced behaviors following METH self‐administration. The 5HT‐2A receptor is thought to play a role not only in psychosis‐like behaviors, but also the regulation of glutamate in the frontal cortex. Our research seeks to determine if METH self‐administration will lead to alterations in METH ‐induced glutamate release in the frontal cortex. Further, the role of the 5HT‐2A receptor in these changes will be explored. Male Sprague‐Dawley rats received METH or saline self‐administration 8 hours/day for 7 days. Two days following self‐administration, a microdialysis probe was inserted into the medial prefrontal cortex. Following basal assessment, animals received an injection of the 5HT‐2A antagonist or vehicle followed by a 5 mg/kg injection of METH or saline. Basal and METH ‐induced glutamate levels were assessed. Preliminary results suggest that prior METH self‐administration result in higher glutamate levels in the medial prefrontal cortex following a vehicle injection and a METH challenge. Ongoing efforts will determine if pretreatment with the 5HT‐2A antagonist will reduce these changes in glutamate. Overall, the results of this study will determine the role of the 5HT‐2A receptor in regulating glutamatergic activity in the frontal cortex following METH self‐administration.

Support or Funding Information

This research was supported by the National Institute on Drug Abuse of the National Institutes of Health (DA036012) and provided by the NIDA grant (R25‐DA033674).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EAAT3 endocytosis activated by psychostimulants in diverse populations of neurons
Authors: Underhill Suzanne , Colt Mark , Amara Susan
Published: June 2021

The primary mechanism of action of psychostimulants, including amphetamine (AMPH), has largely been attributed generally to an increase in extracellular dopamine. Dopamine potentiation has been ascribed to several mechanisms including endocytosis of the dopamine transporter, DAT. We have found that this cascade involves entry of AMPH into the cell through DAT, stimulation of an intracellular GPCR for trace amines, TAAR1, and activation of the small GTPase, RhoA. Changes in extracellular glutamate concentrations have also been reported in response to AMPH and we have found that the neuronal glutamate transporter, EAAT3, also undergoes endocytosis via this cascade in dopamine neuron cultures, in acute brain slices and in vivo. EAAT3 is broadly expressed in neuronal populations throughout the brain, suggesting additional actions of AMPH on glutamatergic signaling in cells other than dopamine neurons. AMPH is a transported inhibitor of DAT as well as the norepinephrine transporter, NET, so we explored AMPH‐induced EAAT3 trafficking in NET(+) neurons. By total internal reflection fluorescence (TIRF) microscopy we found that AMPH can cause endocytosis of EAAT3 as well as NET. We developed NET(+) primary cultures derived from E15 murine locus coeruleus. Catecholamine neurotransmitters enter these cells through a desipramine‐sensitive pathway, indicating that this transport is mediated by NET. These cultures also exhibit sodium‐dependent and dihydrokainate‐insensitive tritiated‐glutamate uptake that is mediated by EAAT3. Pretreatment with AMPH decreases both NET and EAAT3 transport capacity in these cultures. Furthermore, through the use of a RhoA inhibitor, we determined that this loss depends on activation of RhoA, as seen previously for AMPH‐mediated internalization of DAT and EAAT3 in midbrain dopamine neuronal cultures. These data indicate that TAAR1 agonists, such as AMPH and dopamine, can affect glutamate signaling through internalization of EAAT3 in noradrenergic as well as dopaminergic neurons.

Support or Funding Information

This work was supported by Intramural Research at the NIH, NIMH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Abuse Potential of Phendimetrazine and its Effects on Cocaine Self‐Administration in Rhesus Monkeys
Authors: Minkiewicz Molly L. , Czoty Paul W. , Blough Bruce E. , Nader Michael A.
Published: June 2021

Cocaine use disorder (CUD) continues to be a major public health and social problem with >1.5 million users resulting in 14,556 deaths in 2017 in the United States and with no FDA‐approved medications available (Volkow and Koob, 2016). Preclinical studies in nonhuman primates have shown that monoamine releasers such as amphetamine can reduce the reinforcing strength of cocaine under several schedules of cocaine self‐administration (SA; Czoty et al. 2010; Negus et al. 2003a, b). However, the high abuse potential of this category of drugs has hindered advancement of their clinical use for treatment of CUD. More recently, preclinical researchers have investigated phendimetrazine (PDM), an FDA‐approved Schedule III anorectic agent, with evidence of efficacy in animal models (Banks and Negus, 2013; Czoty et al. 2016). As a pro‐drug for the monoamine releaser phenmetrazine, PDM has a slow onset and long duration of action. Only two studies (Griffiths et al. 1976; Corwin et al. 1987) have evaluated the reinforcing effects of PDM, with opposite conclusions. The goal of the present study was to compare, in the same subjects, the efficacy of PDM to decrease cocaine SA and the reinforcing strength of PDM relative to cocaine. For 5 consecutive days, three adult male rhesus monkeys responded under a daily fixed‐ratio 20 schedule of food presentation, which, on the fifth day, was followed by a progressive‐ratio (PR) schedule of cocaine (0.003–0.3 mg/kg/injection) presentation (limited hold 30‐min, maximum 4‐hr). PDM was administered daily (1.0–6.0 mg/kg, PO, BID). The primary dependent variable under the PR schedule was the number of injections delivered. Under baseline conditions, monkeys typically earned 10 food reinforcers and the number of cocaine injections was characterized as an inverted U‐shaped function of dose. Under treatment conditions, in two subjects, food‐maintained responding was disrupted at higher PDM doses that also decreased cocaine SA. In the third subject, food reinforcement was not affected and PDM did not show efficacy in decreasing cocaine SA. When PDM (0.1–1.0 mg/kg/injection) was substituted for cocaine, it functioned as a reinforcer in all three subjects; the number of injections observed at peak doses of PDM (0.3 and 1.0 mg/kg/injection) were similar to those observed at the peak dose of cocaine (0.03 mg/kg/injection). These findings suggest that PDM has abuse liability and was ineffective in decreasing cocaine SA. These data do not support the use of PDM as a potential treatment for CUD.

Support or Funding Information

P50 DA06634‐27

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.