bluuebunny/crossref_metadata_2025 huggingface dataset:
ER Stress marker GRP78 is not expressed on ER−/PR−/Her2− human breast cancer cell surface or secreted
Authors: Banerjee Dipak K. , Serrano Negron Jesus E. , Ruiz Andrea Rivera , Baksi Krishna
Published: June 2021

GRP‐78 (a Mr 78kDa calcium dependent glucose binding protein) located in ER lumen. It functions as ER chaperone and translocates proteins for glycosylation at the asparagine residue present in the sequon Asn‐X‐Ser/Thr (i.e., N‐linked glycosylation). This important biochemical event is essential for glycoprotein folding and function. When targeted in ER−/PR−/HER2+ breast tumor in athymic nude mice with the N‐glycosylation inhibitor tunicamycin, the cancer growth was reduced ~55% in three weeks. Paraffin sections of the tumor exhibited reduced N‐glycan level, and reduced angiogenesis but increased GRP‐78 expression in microvasculature as well as in the tumor tissue (J. Biol. Chem. 286, 29127–29138, 2011). This supported the presence of ER stress. We have now evaluated the effect of tunicamycin on the proliferation of metastatic ER−/PR−/HER2− (MDA‐MB‐231) and non‐metastatic ER+ (MCF‐7) human breast cancer cells. Tunicamycin inhibited proliferation of both cells types in a time and dose‐dependent manner. Interestingly, GRP‐78 expression (protein and mRNA) was higher in tunicamycin (1.0 μg/ml) treated MDA‐MB‐231 and in MCF‐7 cells and supported by both Western blotting and qPCR. Since GRP‐78 is an ER stress marker, so we have followed its intracellular localization by immunofluorescence microscopy after treating the cancer cells with tunicamycin as well as after subjecting them to various stress conditions. We have used unfixed cells and stained with either FITC‐conjugated Concanavalin A (Con A), or Texas‐red conjugated wheat germ agglutinin (WGA) or with anti‐GRP‐78 antibody. There were expression of N‐glycans but not GRP‐78. GRP‐78 however became detectable after permeabilization of cells with a brief exposure to ice‐cold methanol. GRP‐78 was not detected in the conditioned media of the cancer cell but a high level of MMP‐1 did. We have therefore, concluded that GRP‐78 is neither expressed on the outer‐leaflet of the (ER−/PR−/HER2−) human breast cancer cells nor it is secreted into the culture media during tunicamycin‐induced ER stress. This is against the current dogma that GRP‐78 expression on the tumor cell surface or its secretion interferes with the cancer therapeutic(s) and make them as tumor promoters and not tumor suppressors. Our study, thus, suggests strongly that anti‐angiogenic and anti‐tumorigenic action of tunicamycin can be modeled to develop next generation cancer therapy, i.e., glycotherapy for treating breast tumor and all other sold tumors.

Support or Funding Information

Supported in part by the university funds (DKB), NIH‐NIDDK STEP‐UP Program (AR) and NIH/NIMHD G12MD007583 (KB)..

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using neurogenic explants to assess the role of the stem cell niche structure fractones in adult neurogenesis and pathology
Authors: Wulff Alina Sophia , Davis Harry , Mercier Frederic
Published: June 2021

Neurogenesis occurs throughout adulthood in restricted zones of the adult brain. Growth factors influence neural stem cell proliferation and differentiation. However, the mechanisms orchestrating growth factors to regulate neurogenesis are not well known. In the adult brain, neural stem cells and their immediate progeny reside in a nurturing stem cell niche environment. These niches are located in the ventricular/sub ventricular zone (VZ/SVZ) of the lateral ventricles. In the SVZ, stem cells are directly contacted by extracellular matrix (ECM) structures that we have named fractones. Using in vivo experiments, we have previously shown that fractones contain heparan sulfate proteoglycans (HSPG) that bind, concentrate and dispatch growth factors to the target stem cells to ultimately regulate stem cell fate. Here, an ex vivo model was devised to expand our ability to study fractones, stem cells, and growth factor interactions in culture in a near‐physiological context. This model consists of brain tissue pieces of the neurogenic zone, termed neurogenic explant. Neurogenic explants survive for months in a simple culture medium. They contain neural stem cells, their immediate progeny, ependymocytes, fractones, all forming the VZ/SVZ, plus the adjacent superficial part of the caudate nucleus. Using mini‐incubator and fluorescence microscopy, we demonstrate that we can visualize fractones, cell proliferation and growth factor binding on the living neurogenic explants. The ease in altering the ECM of explants and assessing the effects on neurogenic stem cells will allow us to understand the role of fractones in neurogenesis. By using explants originating from animal models, we will also be able to understand the relationship between disrupted adult neurogenesis and neural pathology. We anticipate that we can determine from the neurogenic explant model which HSPGs are responsible for the binding of individual growth factors. This will provide insight into the possibility of manipulating HSPG expression in explants to ultimately alter the cell‐fractone interactions responsible for the progression of disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of botulinum toxin type A combined with exercise on the functional recovery after spinal cord injury
Authors: Jin Yunho , Sumsuzzman Dewan Mohammed , Hong Yonggeun
Published: June 2021

Spinal cord injury (SCI) is a result of a traumatic damage of the vertebral column. This injury interrupts the signal conduction from upper motor neuron to lower motor neuron and spasticity may occur. Spasticity is common motor dysfunction induced from upper motor neuron lesions. Botulinum toxin type A (BTA) can inhibit muscle contraction as well as acetylcholine release by without affection to acetylcholine synthesis. Treadmill exercise (Ex) is known to reduce body and fat mass resulting improvement of neuroplasticity. Therefore, the purpose of this study was to evaluate the effect of BTA combined with treadmill exercise after SCI. Experimental animals were divided into three groups; SCI, SCI+BTA, SCI+BTA+Ex. BTA was injected into the gastrocnemius of SCI+BTA and SCI+BTA+Ex rats. And, SCI+BTA+Ex rats were assigned to treadmill exercise. After 14 postoperative day, all animals were sacrificed to perform further analyses. SCI+BTA+Ex group showed the greatest decrease of body weight and gastrocnemius mass. Also, BTA‐injected groups underwent significantly decreased muscle degradation compared to non‐injected group. Similarly, BTA treated muscle showed increased levels of autophagy markers. Especially, both beclin‐1 and LC3 were increased in the gastrocnemius of SCI+BTA+Ex group. Interestingly, this group showed the highest motor functioning scores. In addition, by light microscopy, several vacuoles were observed in the muscle fibers of SCI group rats, indicating delayed functional recovery. In this study, BTA is thought to have slight spasticity‐alleviating effect, and this effect is extended by combination of treadmill exercise.

Support or Funding Information

Funds: NRF‐2017R1A2A2A01067169, KGM4611714

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histopathological effect of some medicinal plants on kidney and liver functions in rats.
Authors: ADEYEMO ADENIKE CHRISTY , ADENIYI ADEDAYO FRANCIS
Published: June 2021

Three medicinal plants which are Aframomum melegueta, Ricinus communis and Helianthus annuus are botanicals used in traditional medicine and they are known to possess insecticidal properties. This study aimed to determine the toxic effect these plant materials leave powders on kidney and liver function which was evaluated through histological examination of tissues from both organs from rats fed with the formulated diets. Forty two (42) male Wistar rats were randomly divided into seven groups (six each) and were fed daily with 1 and 10% of the three plant materials included in their diet for 30 days while the control group fed with basal diet without supplementation. At the end of 30 days, the rats were sacrificed to remove the kidney and liver for toxicity evaluation.

Support or Funding Information

NONE

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of non‐esterified fatty acids on the expression and secretion of angiopoietin‐like protein 4 and fibroblast growth factor 21 in calf hepatocytes cultured in vitro
Authors: ZHU Xiaoyan , WANG Jianguo , LIU Lei
Published: June 2021

Introduction

Negative energy balance (NEB) is considered as the pathological basis of energy metabolic disorders, such as fatty liver and ketosis, in periparturient dairy cows. An elevated serum non‐esterified fatty acids (NEFA) concentration is one of the most important indicators of NEB. Angiopoietin‐like protein 4 (ANGPTL4) and fibroblast growth factor 21 (FGF21) have been identified as hepatokines, which involved in regulation of metabolic adaptations. In dairy cows, most circulating ANGPTL4 and FGF21 originate from the liver. However, the direct effects of NEFA on the expression and secretion of ANGPTL4 and FGF21 in bovine hepatocytes are not entirely clear. The purpose of the present study was to investigate the effects of different NEFA concentrations on the expression and secretion of these two hepatokines in calf hepatocytes cultured in vitro.

Methods

Bovine hepatocytes were cultured using the collagenase perfusion method. After culture for 72 h, the hepatocytes were treated with NEFA at final concentrations of 0 (control), 0.6, 1.2, 1.8 and 2.4 mmol/L. After 24 h of continuous culture, the mRNA and protein expression levels of ANGPTL4 and FGF21 in the hepatocytes were determined by real‐time PCR and Western blot, respectively. ANGPTL4 and FGF21 secretions in the supernatant were determined by enzyme‐linked immunosorbent assay (ELISA).

Results

The results showed similar levels of ANGPTL4 expression and secretion following treatment with 0.6 and 1.2 mmol/L NEFA, which were higher than those observed for the controls (P<0.05). ANGPTL4 expression and secretion were also similar at 1.8 and 2.4 mmol/L NEFA and significantly higher than those observed for controls (P<0.01). The expression and secretion of FGF21 at 0.6 mmol/L NEFA‐treated hepatocytes was higher than that of the control group (P<0.05). The FGF21 expression and secretion were similar at 1.2, 1.8 and 2.4 mmol/L NEFA‐treated hepatocytes and significantly higher than those observed for controls (P<0.01).

Conclusion

The results of this study indicate that high concentrations of NEFA significantly increase the expression and secretion of ANGPTL4 and FGF21 in bovine hepatocytes cultured in vitro in a dose‐dependent manner. Specifically, NEFA promotes the expression and secretion of ANGPTL4 and FGF21, which may worsen fat mobilization under NEB conditions and thereby lead to the pathological processes of energy metabolism disorders in dairy cows in the peripartum period. However, further studies are needed to elucidate the underlying mechanisms.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Pilot Study to Examine the Perspectives of Pharmacy Students towards Adding a Nutrition Course in Pharmacy Degree Curriculum
Authors: Syed‐Abdul Majid Mufaqam , Kabir Syed Sadath , Soni Dhwani Satishkumar , Barnes Jeremey T. , Faber Tony A. , Timlin Maureen T.
Published: June 2021

Today's pharmacists are likely to encounter questions about nutritional products sold in the pharmacy. This is due, in part, to the increased number of pharmacies in grocery stores and the availability of pharmacists. However, many pharmacists report that they lack nutrition knowledge and believe the best time to educate pharmacists about nutrition is during pharmacy school.

PURPOSE

The purpose of this study was to determine if pharmacy students realize the importance of nutrition education during pharmacy school and if a background in nutrition education or pharmacy‐related experience influences student's responses.

METHODS

Both male and female students from nine different pharmacy schools from southern part of India [IND, n=225, age: 18–34y, male: n=74 (32.9%), female: 151 (67.1%)] and two pharmacy schools (Alabama and Illinois) from the United States [USA, n=95, age: 18–45y, male: n=23 (24.2%), female: 72 (75.8%)] participated in this study.

RESULTS

The response rate from IND was 97.0% (seven students did not complete survey) and USA was 100% (no incomplete surveys). When the students were asked about their nutrition education, 77.3% of students from IND and 55.8% students from the USA reported never taking a nutrition course. Students who took a nutrition course had to take it as an elective. While 37.8% students from IND and 93.7% students from USA had pharmacy‐related experience, 6.7% of students from IND and 3.2% students from USA said they were very confident in handling nutrition‐related questions. Regardless of previous nutrition education and pharmacy‐related experience, 88.0% of students from IND and 70.5% of students from the USA, believed that nutrition education is important and should be incorporated in the pharmacy school curriculum. Among those who took a nutrition course, 81.8% of students from IND and 82.9% from USA believed that a nutrition course should be incorporated into the pharmacy school curriculum. When pharmacy‐related experience was considered, 92.9% from IND and 73.3% from USA also believed that a nutrition course should be incorporated into the pharmacy school curriculum.

CONCLUSION

Results from this study suggest that a nutrition course should be incorporated into the pharmacy school curriculum, regardless of past nutrition education or pharmacy‐related experience. A limitation of this study was that only students from the southern part of the India and two pharmacy schools in the United States were surveyed. Additional data is needed from other pharmacy schools in order to generalize the results of this study and influence curriculum changes for pharmacy schools.

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 redesigned pharmacology series increases students' satisfaction and is associated with improved performance in therapeutics
Authors: Brandl Katharina , Schneid Stephen , Tsunoda Shirley , Awdishu Linda
Published: June 2021

Objective

In this study we describe the redesign, implementation and evaluation of a three‐quarter pharmacology course within a Pharm.D. curriculum.

Methods

This retrospective study examined the pre and post implementation of a redesigned three‐course pharmacology curriculum for second‐year pharmacy students. Course evaluations and examination scores were analyzed from four different cohorts of students (Class of 2014 to Class of 2017).

Qualitative analysis of the course evaluations identified key areas of weakness to help the new course director implement changes to the entire pharmacology curriculum starting with the Class of 2014. Descriptive statistics, qualitative analysis, and correlation analysis were used to evaluate outcomes of the new course.

Results

Qualitative analysis of 155 second‐year pharmacy student evaluations from the Class of 2014 identified lack of integration and coordination within the three‐course pharmacology sequence. Poor exam quality and the absence of active teaching methods were the most frequently described shortcomings of the pharmacology curriculum. In response to this analysis, exam question flaws were eliminated and active teaching modalities were introduced. Also, the new director took over more lectures to reduce the number of different guest lecturers teaching in the course. Immediately following these changes, the course evaluations dramatically improved and continued to improve over the following three years (One‐Way ANOVA with Bonferroni post‐test, p< 0.05). Significant correlations were identified between course evaluations and the number of lecturers taught by the new course director as well as the number of active teaching methods (Pearson Correlation coefficient, p<0.05). Students' performance in the years following the redesign correlated better with their performance in the third‐year Therapeutics course providing evidence for improved exam validity (Pearson Correlation coefficient, p<0.05).

Conclusion

A rigorous redesign of a large pharmacology course resulted in increased students' satisfaction and an improved association with exam performance in the Therapeutics course. This study highlights the importance of a well‐designed pharmacology course for continued success in core courses in the PharmD curriculum.

Support or Funding Information

N/A

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development and Preclinical Testing of a Vaccine for 3,4‐Methylenedioxypyrovalerone ((R,S)‐MDPV) Substance Use Disorders
Authors: McClenahan Samantha Jo , Kormos Chad , Gunnell Melinda , Hambuchen Michael , Lamb Pamela , Carroll Ivy , Lewin Anita , Owens Samuel Michael
Published: June 2021

Synthetic cathinones like 3,4‐methylenedioxypyrovalerone ((R,S)‐MDPV) are medically dangerous psychoactive drugs which at high doses produce psychosis, cardiovascular toxicity, and death. The goal for these studies was to develop and test a vaccine for treating ((R,S)‐MDPV) substance use disorders. A racemic MDPV conjugate vaccine was developed from a hapten structure capable of generating antibodies that bind with high affinity to (R,S)‐MDPV and other potent synthetic cathinones. We hypothesized that treatment with an optimized MDPV vaccine could produce high affinity antibodies against (R,S)‐MDPV capable of mitigating the pharmacological effects of (R,S)‐MDPV. An (R,S)‐MDPV‐like hapten was conjugated to different carrier proteins, mixed with Sigma Adjuvant System, and used to immunize male Sprague Dawley rats (n=5/group). The immunochemical characteristics of the rats' immune response was then evaluated. The KD value as determined by radioimmunoassay for (R,S)‐MDPV binding was 2.5 ± 1.9 nM, which demonstrated production of high affinity antibodies. The KD value for the individual (R)‐ and (S)‐MDPV binding was 0.71 ± 0.8 nM and 2.8 ± 2.3 nM, respectively. These similar KD values for the MDPV enantiomers indicates antibodies for both enantiomers contribute to the high binding of (R,S)‐MDPV. The rat antisera also showed similar high affinity binding of (R,S)‐α‐pyrrolidinovalerophenone (α‐PVP). No significant cross reactivity was detected with over‐the‐counter medications like (+)‐pseudoephedrine, neurotransmitters (dopamine, norepinephrine, epinephrine, and serotonin), or non‐cathinone drugs of abuse (cocaine, morphine, phencyclidine, (+)‐methamphetamine, (+)‐MDMA, and (−)‐MDMA). Moreover, the vaccination protocol produced high serum antibody titers, determined by rapid equilibrium dialysis, from 5 weeks after the start of immunizations until termination of the rats 3 months later. Finally, locomotor studies of control and vaccinated rats were conducted to test the vaccine's efficacy using a series of increasing doses of (R,S)‐MDPV (0.3–5.6 mg/kg, sc, 2 days apart). The immunized rats displayed significantly (p<0.05) lower MDPV‐induced locomotor activity and a significantly shorter duration of action compared to controls. In conclusion, active vaccination with an (R,S)‐MDPV conjugate vaccine produced very high affinity antibodies against (R,S)‐MDPV; and the vaccinated rats showed significantly reduced (R,S)‐MDPV‐induced locomotor effects compared to controls.

Support or Funding Information

Funded by NIDA grants DA039195 and T32 GM106999

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 natural product composition targeting oxidative stress induced mitochondrial dysfunction in Alzheimer's Disease
Authors: Bhaskar Manju , Chintamaneni Meena
Published: June 2021

Mitochondria regulates cellular metabolism and apoptosis. Oxidative stress induced mitochondrial dysfunction, appears to play a role in the pathogenesis of Alzheimer's disease. Natural antioxidants have been known to play an important role in alleviating the deleterious effects induced by free radicals by blocking the initiation and propagation of oxidizing chain reactions. Hence, the study hypothesizes the effect of natural product composition (combinatorial extract of Eclipta alba, Morinda pubescens and Withania somnifera) on restoring the mitochondrial functional capacity and mitochondrial enzyme levels. The individual and combinatorial methanolic extracts of dried whole plants of Eclipta alba (MEEA), dried fruits of Morinda pubescens (MEMP) and dried roots of Withania somnifera (MEWS) and MECE were prepared. The extracts were screened for in vitro antioxidant activity by DPPH assay. A rodent behavioural model to assess the effect of the extracts on short term memory was performed subsequent to scopolamine hydrobromide induced amnesia and oxidative stress. The brain homogenates from these rodents were subjected to screening in vivo antioxidaint activity using a battery of assays. The effect of extracts on restoring mitochondrial functional capacity (using MTT assay) and the levels of key mitochondrial enzymes succinate dehydrogenase and isocitrate dehydrogenase; was performed using synaptosomal fractions prepared from rodent brain samples.

MEEA, MEMP, MEWS and MECE compared with ascorbic acid exhibited 50% scavenging effect at 69.34 μg/ml, 72.19 μg/ml, 65.84 μg/ml, 45.09μg/ml and 57.64 μg/ml, respectively by DPPH assay. Pretreatment with MEEA, MEMP and MEWS at the doses 50,100 and 200 mg/kg and MECE at 50 and 100mg/kg for 8 days followed by scopolamine hydrobromide treatment significantly (p<0.001) improved the antioxidant defense system, the mitochondrial functional capacity and mitochondrial enzyme levels.

The natural product composition strengthened the endogenous antioxidant defense system, restored the mitochondrial functional capacity and enzyme levels. Hence, it has a potential neuroprotective role in Alzheimer's disease which needs to be investigated further in a clinical setting.

Support or Funding Information

NMIMS University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a Novel Radioligand for Characterization of the Mas Receptor for Angiotensin 1–7
Authors: Speth Robert Charles , Pang Hong Wang , Neagra Christopher T. , Bergoine Samantha , Linares Andrea , Stoyell‐Conti Filipe F.
Published: June 2021

It is well established that Mas (NP_002368) is the receptor for angiotensin 1–7 (Ang 1–7), however, to date, no published studies have characterized Mas receptor radioligand binding in tissue membrane preparations. Our laboratory has repeatedly failed to reliably demonstrate high affinity, saturable binding of 125I‐Ang 1–7 in liver, kidney, brain and testes membrane preparations from both rats and mice. While the presence of a single iodine‐125 molecule on the tyrosine of Ang II does not impair its binding to the AT1 and AT2 receptors, it is not known whether the presence of an iodine‐125 molecule on the tyrosine of Ang 1–7 is equally innocuous. To begin to address this question, we prepared a novel analog of Ang 1–7: Tyr0‐Ang 1–7, to provide an alternate site for radioiodination of Ang 1–7. Tyr0‐Ang 1–7 was radioiodinated using the chloramine T procedure of Hunter and Greenwood (1962) with a 7 times excess of peptide to iodine to minimize di‐iodination of the peptide. Two radioiodinated peaks, presumed to be mono125I‐Y0‐Ang 1–7 and mono125I‐Y4‐Ang 1–7, were resolved from the uniodinated peptide peak by HPLC (C18 reverse phase column, 14.5% acetonitrile: triethylamine phosphate, pH 3.0 isocratic mobile phase). Saturation and competition radioligand binding assays were run for each peak using rat liver membrane preparations in the presence and absence of 10 μM Ang 1–7. While the identity of the early and late 125I‐Y0‐ Ang 1–7 peaks has not yet been established, preliminary studies indicate that both 125I‐Y0‐Ang 1–7 peaks displayed high affinity, saturable binding to rat liver membranes: early peak: KD = 7.7±2.3 nM, BMAX = 3.3±0.72 fmol/mg wet weight; and late peak KD = 11±2, BMAX = 3.6±0.4 fmol/mg wet weight. Competition binding assays run using the late peak 125I‐Y0‐Ang 1–7 revealed IC50's of 32±15 nM for Ang 1–7 and 15 ± 9.6 nM for Y0‐Ang 1–7. Comparison of the ability of a variety of Ang peptides to compete for late peak 125I‐Y0‐Ang 1–7 binding at a 10 μM concentration gave a rank order of Ang 1–7>Ang 3–7>Ang IV>Ang III>Ang 2–7>Ang II>Ang I. These results suggest that addition of a tyrosine at the amino terminus of Ang 1–7 may increase its binding affinity for Mas and that the presence of an iodine‐125 on either Y0 or Y4 of Ang 1–7 does not preclude high affinity saturable binding to Mas.

Support or Funding Information

This work was supported by the Cardiovascular Neuroscience Fund, Nova Southeastern University and the Peptide Radioiodination Shared Resource, Georgetown University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of Caveolin‐3 (Cav‐3) Promotes G‐protein‐regulated Matrix Metalloprotease 14 (MMP14) Activation in the Aged Heart
Authors: Haushalter Kristofer J. , Overland Aaron C. , Patel Hemal H. , Insel Paul A.
Published: June 2021

Age‐associated cardiovascular disease significantly correlates with morbidity and mortality worldwide; therefore, investigation of cellular signaling pathways in the heart that change with age may help identify novel therapeutic strategies for mitigating the progression of cardiovascular pathologies. Our lab has uncovered a membrane‐delimited receptor transactivation pathway in the heart, wherein G‐protein coupled receptors (GPCRs) signal via heterotrimeric G‐proteins to activate membrane‐tethered matrix metalloprotease 14 (MMP14/MT1‐MMP) and consequently induce “shedding” of membrane‐bound receptor ligands (J Biol Chem 290: 9941‐7). Specifically, G‐protein‐regulated MMP14 releases heparin‐binding EGF‐like growth factor (HB‐EGF), which then triggers activation of epidermal growth factor receptor (EGFR) signaling. We hypothesized that the components of this mechanism are compartmentalized within caveolar lipid raft domains (i.e. cholesterol‐rich membranes containing caveolin‐1 (Cav‐1) and caveolin‐3 (Cav‐3) isoforms), wherein co‐localization with caveolin proteins serves to repress MMP14 enzymatic activity. Furthermore, we hypothesized that age‐dependent loss of Cav‐3 in the heart increases MMP14 activity and thereby alters the signaling pathway to contribute to deleterious cardiac aging phenotypes. We thus sought to compare changes in G‐protein‐regulated MMP14 activity and subsequent activation of HB‐EGF/EGFR signaling in membrane fractions isolated from cardiac myocytes (CMs) and cardiac fibroblasts (CFs) of young (2–3 month) and aged (18–21 month) mice, with or without transgenic CM‐specific overexpression of Cav‐3 (i.e., Cav‐3 OE), to determine if preserved Cav‐3 expression prevents or lessens harmful age‐associated membrane signaling. We observed approximately a two‐fold increase of MMP14 activity in membranes from CMs of aged wild‐type (WT) mice compared to young controls (p< 0.05). Immunoblot analysis of buoyant fractions isolated by differential centrifugation of cardiac tissue in a discontinuous sucrose density gradient confirmed that MMP14, G‐proteins, HB‐EGF, and EGFR co‐localize with caveolin protein isoforms in lipid rafts. Work in progress will test if co‐localization of these proteins is reduced in aged wild‐type (WT) animals but not in Cav‐3 OE animals, and if MMP14 and Cav‐3 co‐immunoprecipitate (co‐IP) within lipid raft domains and thus form a functional signaling module that is disrupted in aged WT subjects but remains intact in Cav‐3 OE mice. Taken together, the results suggest that GPCR transactivation of EGFR and regulation by signaling components in caveolar lipid rafts may be a feature of membrane signaling that is altered in the aged heart.

Support or Funding Information

Supported by NIH grants R21AG053568 and R21AG52914.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Soluble Epoxide Hydrolase (sEH)/Fatty Acid Amide Hydrolase (FAAH) Inhibitors with Improved Potency Towards Rodent Species
Authors: Kodani Sean David , Hwang Sung Hee , Morisseau Christophe , Hammock Bruce Dupree
Published: June 2021

Soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH) are therapeutic targets that are independently known to regulate pain. Concurrent inhibition of these two targets is known to be synergistic at reducing pain; thus, we have sought to design dual inhibitors that leverage this synergy in a potent small molecule. Previous efforts have led to very potent and selective inhibitors that target human species but are poorly active on rodent enzymes. In the current work, we attempted an alternative approach towards inhibitor design that was intended to be more potent on rodent enzyme. The approach modified the FAAH inhibitor URB597 (mouse IC50 = 11 nM) to include sEH pharmacophores leading to two new inhibitors: C‐14 (mouse FAAH IC50 = 0.27 nM, mouse sEH IC50 = 68 nM) and C‐20 (mouse FAAH IC50 = 4.4 nM, mouse sEH IC50 = 1.3 nM). C‐20 is 320‐fold and 15‐fold more potent on mouse FAAH and sEH, respectively, than our previous series of dual sEH/FAAH inhibitors. Relative to URB597, these inhibitors had a higher selectivity ratio towards other serine hydrolase; however, caution is required in their application to prevent confounding results. Future studies will test their therapeutic potential in rodent models of pain and other diseases.

Support or Funding Information

This work was supported by the National Institute of Environmental Health Sciences grant R01 ES002710 and the National Institute of Environmental Health Sciences Superfund Research Program grant P42 ES004699. SDK was supported by a NIGMS‐funded Pharmacology Training Program (T32GM099608).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Vitro and In Vivo Metabolism of Soluble Epoxide Hydrolase Inhibitor 1‐ Trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) Urea (TPPU)
Authors: Wan Debin , Yang Jun , McReynolds Cindy B. , Wagner Karen M. , Barnych Bogdan , Morisseau Christophe , Sun Jia , Hwang Sung Hee , Hammock Bruce D.
Published: June 2021

Epoxy fatty acids (EpFAs) are highly potent lipid mediators, formed by cytochrome P450 (CYP450) from polyunsaturated fatty acids. In living organisms, they will be rapidly metabolized by soluble epoxide hydrolase (sEH) to these 1,2‐diol metabolites. A still growing body of evidence suggests that the stabilization of EpFAs by inhibition of sEH has effect of anti‐inflammatory, analgestic, antihypertension and so on. Therefore, sEH inhibitors of high potency are designed for targeting sEH for disease treatment. Among them, 1‐trifluoromethoxyphenyl‐3‐ (1‐propionylpiperidine‐4‐yl)urea (TPPU) of high potency (IC50 of HsEH, 0.6 nM) is the most promising drug. However, the drug metabolism of TPPU has not been studied. Nowadays, drug metabolism has become essential consideration for the safe and efficacious use of drug in human. Therefore, we in this study reported the comprehensive metabolic profiling of TPPU by in vitro and in vivo studies. A developed sensitive liquid chromatography–mass spectrometry (LC‐MS) method was used for identify all these phase I and phase II metabolites of TPPU. Our results indicated three major metabolic pathways of TPPU from phase I metabolism, and one from phase II metabolism.

Support or Funding Information

NIEHS grant R01ES002710 and NIEHS Superfund Research Program P42 ES004699 (to B.D.H.), the West Coast Metabolomics Center at UC Davis (NIH/NIDDKU24DK097154) (to B.D.H.) and NIH grant R01 AG051618 (to J. O.)

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 Dual‐Inhibitor of Soluble Epoxide Hydrolase and p38 Kinase Alleviating Tau Hyperphosphorylation and Amyloid Neurotoxicity for Potential Treatment of Neuroinflammation in Alzheimer's Disease
Authors: Liang Zhibin , Morisseau Christophe , Hwang Sung Hee , Hammock Bruce D. , Li Qing X.
Published: June 2021

Alzheimer's disease (AD) is the most common dementia that cannot be prevented, cured or even slowed. Neuroinflammation associated with tau hyperphosphorylation and amyloid toxicity is a major hallmark in AD. Soluble epoxide hydrolase (sEH) and p38 kinase are therapeutic targets involving in inflammatory signaling. Development of selective inhibitors to modulate both sEH and p38 kinase is an attractive strategy for AD. We herein studied a potent and blood‐brain barrier permeable sEH inhibitor, 1‐(1‐propanoylpiperidin‐4‐yl)‐3‐[4‐(trifluoromethoxy)phenyl] urea (TPPU) (half maximum inhibition concentration, IC50, 0.64 nM), on neuroprotection in a human AD neuronal model. TPPU effectively alleviates tau hyperphosphorylation and amyloid neurotoxicity in vitro with a half maximum effective concentration (EC50) value of 49 nM. In vitro kinase selectivity profiling indicated that TPPU is a specific p38β kinase inhibitor with an IC50 value of 0.27 μM. The results suggested that TPPU shows a synergistic effect of neuropharmacology through a dual inhibition of sEH and p38. Additional knowledge of TPPU on the neuroinflammatory protection against tau and amyloid pathologies warrants further investigation.

Support or Funding Information

This work was supported in part by the NIH National Institute on Minority Health and Health Disparities grant 8G12MD007601.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Advancing Soluble Epoxide Hydrolase Inhibitors for the Treatment of Osteoarthritis in Companion Animals.
Authors: McReynolds Cindy , Hwang Sung Hee , Yang Jun , Wagner Karen , Schmidt William K. , Hammock Bruce
Published: June 2021

Soluble epoxide hydrolase inhibitors (sEHI) stabilize biologically active lipid metabolites responsible for regulating homeostatic biological processes. Over the past 20 years, researchers at the University of California at Davis synthesized a large chemical library of potent sEHIs to elucidate the role of this enzyme in biological systems. These chemical inhibitors helped identify analgesic effects of inhibiting the sEH enzyme. Further characterization of safety, enzyme potency and efficacy in mice, rats and dogs identified compound, EC1728, as a lead compound for translation into clinical development for the treatment of painful conditions, such as osteoarthritis.

Data will be presented to provide an overview of the screening results that led to the selection of EC1728 for use in companion animals, as well as the results of the initial efficacy study in dogs. To assess efficacy, EC1728 was tested in a naturally occurring arthritis model. Aged beagles (8 per group) with naturally occurring arthritis confirmed by radiographic score were used in this study. Two additional animals were included in a satellite group to measure pharmacokinetic parameters. Dogs were treated once daily with an oral dose of sEHI for five days. Pain measurements were based on an observational questionnaire administered by a blinded clinician that assessed both pain and function. After five days, EC1728 significantly reduced cumulative pain at a dose of 5 mg/kg compared to vehicle controls. Pharmacokinetic evaluation showed concentrations exceeding the enzyme potency in both plasma and synovial fluid. These results provide proof of concept that EC1728 is efficacious in treating osteoarthritis in dogs.

EC1728 is a non‐NSAID, non‐opioid compound targeting a novel mechanism of action. Considering these positive efficacy results and good safety profile, as well as the selective potency of inhibiting the sEH enzyme, EC1728 is being developed for use as a safer alternative for the treatment of osteoarthritis in companion 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.
Kinetics of Letrozole Metabolism by Cytochrome P450 3A4: Modulation and Fitting to Non‐Michaelis Menten Models
Authors: Colson Ryan Patrick , Heers Hayley , Chan Jeannine , Harrelson John
Published: June 2021

Variable blood plasma concentration is an issue for many drugs, including letrozole, which can contribute to toxicity or sub‐effective dosing levels. There is evidence that letrozole is primarily metabolized by CYP2A6 however, CYP2A6 genetic variation only explains a portion (about 30%) of the variability in letrozole plasma concentrations. We hypothesized that CYP3A4 may play a larger role in letrozole metabolism than the current evidence suggests due to the influence of multiple ligand binding and the observation of non‐Michaelis‐Menten kinetic plots. The kinetics of letrozole metabolism by CYP3A4 were analyzed in the presence and absence of ketoconazole, a CYP3A4 modulator, and fitted to multiple ligand binding functions as well as the Michaelis‐Menten model. Further, the time‐dependent inhibition parameters of CYP2A6‐mediated letrozole metabolism were measured in the presence of the CYP2A6‐specific inactivator, trans‐cinnamic aldehyde (tCA). While the Eadie‐Hofstee plot for CYP3A4‐mediated letrozole metabolism was ambiguous, curve fitting showed the Michaelis‐Menten model appropriately characterized metabolism in the absence of ketoconazole (KM: 142+/−23.0 μM, Vmax: 13.0+/−1.44 pmol/nmol/min), suggesting a single ligand bound to the enzyme. A biphasic model most closely modeled letrozole metabolism in the presence of ketoconazole (KM1: 4.71+/−4.55 μM KM2: 513+/−164 μM, Vmax1:0.525+/−0.392 Vmax2: 30.9+/−7.60 pmol/nmol/min) and this was also reflected in the Eadie‐Hofstee plot, which was clearly biphasic; this supports a mechanism involving multiple‐ligand binding. TDI analysis of tCA revealed less effective inhibition of CYP2A6‐mediated letrozole metabolism by tCA (KI: 136 μM, kinact: 0.019 min−1) as compared to CYP2A6‐mediated coumarin metabolism. Together, these results suggest that CYP3A4 may contribute to variability in letrozole metabolism especially in the presence of an effector molecule (i.e., ketoconazole) since the value of KM1 is much closer to the letrozole plasma concentrations observed clinically than the KM value observed in the absence of an effector.

Support or Funding Information

Funding from National Institute on Drug Abuse

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tumor Suppressor Alpha‐arrestin ARRDC3 Controls GPCR Signaling and Breast Cancer Invasion
Authors: Pan Wen An , Arakaki Aleena , Trejo JoAnn
Published: June 2021

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with poor prognosis and lacks druggable targets. The development of chemotherapy resistance substantially accounts for the high recurrence and death of TNBC patients. Therefore, understanding how TNBC acquires chemoresistance will provide an opportunity to identify new target therapies. G protein‐coupled receptors (GPCR) are the largest family of FDA‐approved drugs but remain overlooked in cancer therapy. Substantial evidence indicates that dysregulated GPCR signaling drives cancer initiation and progression, this is particularly relevant for protease‐activated receptor‐1 (PAR1). However, how PAR1 signaling is dysregulated and confers chemoresistance in aggressive cancers is poorly understood. Arrestin‐domain containing protein 3 (ARRDC3) is a newly identified tumor suppressor in breast cancer and its expression is lost or suppressed in the basal‐like breast cancers, which constitutes the majority of TNBC. Whether and how ARRDC3 play a role in PAR1 signaling and impacts chemoresistance of breast cancer is unclear. The study objective is to address the link between the ARRDC3 and PAR1 signaling and breast cancer chemoresistance. PAR1 is a promising drug target for breast cancer and its expression is correlated with high tumor grade and poor prognosis in breast cancer patients. PAR1 is irreversibly activated by thrombin and its overexpression is sufficient to promote neoplastic transformation and essential for breast cancer metastasis in vitro and in vivo. In invasive breast carcinomas, PAR1 signals constitutively due to a defect in lysosomal trafficking and promotes invasiveness. We hypothesize that ARRDC3 represses PAR1 signaling and chemoresistance in TNBC cells. In this study, we used a lentiviral based ARRDC3 doxycycline‐inducible (pSLIK‐ARRDC3) system in TNBC lines to examine the role and mechanism by which ARRDC3 and PAR1 signaling contribute to chemoresistance. Here we report that ARRDC3 and PAR1 expression are inversely correlated in luminal versus basal‐like invasive breast carcinoma and in patient tissue samples. Activation of PAR1 promotes chemoresistance, while ARRDC3 expression re‐sensitizes cells response to chemotherapy agents. Mechanistically, ARRDC3 specifically represses the magnitude of endosomal PAR1‐induced p38 mitogen‐activated protein kinases (MAPK) signaling but has no effect on extracellular signal‐regulated kinase‐1,2 (ERK1,2) in pSLIK‐ARRDC3 MDA‐MB‐231 cells. In conclusion, our study suggests that PAR1 may be a therapeutic target for attenuation of chemoresistance in ARRDC3‐deficient TNBC patients.

Support or Funding Information

This work is funded by NIH R01 GM090689‐05 and UC Tobacco‐related Disease Research Program High Impact Pilot Award 26IP‐0050

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Angiotensin II in Cardiovascular Stress Responses in Rats Exposed to Glucocorticoids in Utero
Authors: Madhavpeddi L , Hammond B. D. , Royal C. R. , Handa R. J. , Hale T. M.
Published: June 2021

It is well known that even transient prenatal insults can impact cardiovascular function in adulthood. We have hypothesized that adult cardiovascular disease may have its origins in utero as a result of exposure to elevated levels of glucocorticoids. In support of this, we have shown that when pregnant rat dams are treated with the glucocorticoid, dexamethasone (DEX), for the last 4 days of gestation, female‐specific changes resulting in autonomic dysfunction and enhanced pressor and tachycardic responses to stress are detected in their adult offspring. Given the known role of angiotensin II in influencing the autonomic nervous system, the present study investigated the impact of angiotensin receptor antagonism on the cardiovascular stress responses in adult male and female rats that were exposed to DEX in utero. Pregnant dams were administered DEX (0.4 mg/kg per day, s.c.) or vehicle on gestation days 18–21. This resulted in a significant reduction in birthweight in DEX‐exposed males and females. At 2–3 months of age, arterial pressure was assessed via radiotelemetry. In order to assess whether prenatal DEX alters stress‐induced hypertensive and tachycardic responses, rats were placed in a restraint tube for 20 minutes, followed by a 2hr recovery period. Restraint‐stress testing was performed on diestrus in females. Restraint stress was then repeated following a 5‐day treatment with the angiotensin type 1 receptor antagonist, losartan (30 mg/kg per day, i.p). In female offspring, prenatal exposure to DEX resulted in enhanced elevations in systolic and diastolic pressure as well as heart rate in response to restraint stress relative to vehicle‐exposed rats. Treatment with losartan reduced the pressor and tachycardic responses to restraint, only in female rats that were prenatally exposed to DEX. In male rats, neither in utero exposure to DEX, nor losartan treatment altered the pressor or tachycardic response to stress in adult rats. These findings suggest that prenatal programming of cardiovascular disease due to exposure to glucocorticoids may be mediated in part due to long‐term changes in the renin angiotensin system, and this effect may be sex‐specific.

Support or Funding Information

ABRC ADHS14‐082990

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anchored Gs‐coupled purinergic receptor regulation of L‐type CaV1.2 and vascular tone in diabetic hyperglycemia
Authors: Prada Maria Paz , Syed Arsalan U. , Buonarati Olivia R. , Ghosh Debapriya , Nystoriak Matthew A. , Reddy Gopireddy Raghu , Sasse Kent C. , Ward Sean M. , Xiang Y. Kevin , Santana Luis F. , Nieves‐Cintrón Madeline , Hell Johannes W. , Navedo Manuel F.
Published: June 2021

Enhanced arterial myocyte contractility in response to elevated extracellular glucose (hyperglycemia) has been associated with protein kinase A (PKA) dependent phosphorylation of the CaV1.2 pore‐forming subunit (α1C) at serine 1928 (Ser1928), but how PKA is activated is unknown. Considering that glucose stimulates extracellular nucleotide release, we hypothesized that a Gs‐coupled purinergic receptor is a key upstream activator of PKA‐mediated L‐type Ca2+ channel (LTCC) potentiation and vasoconstriction by elevated glucose. Using human and murine arteries and arterial myocytes, we found protein expression of predicted molecular weight and sarcolemmal distribution for the only Gs‐coupled P2Y receptor P2Y11, as well as nanometer proximity of this receptor to CaV1.2 and PKA. A FRET‐based reporter confirmed that both elevated glucose and a specific P2Y11 activator stimulate cAMP production, and this was blocked in the presence of a specific P2Y11 inhibitor. Consistent with our hypothesis, the ectonucleotidase apyrase and a P2Y11 inhibitor, but not a Gq‐coupled P2Y6 inhibitor, prevented increased Ser1928 phosphorylation and LTCC activity in human and murine arterial myocytes and augmented vasoconstriction in murine arteries by elevated glucose. Similar results were observed by knocking down the expected P2Y11 protein. Comparable to glucose effects, a P2Y11 activator potentiated Ser1928 phosphorylation, LTCC activity, and vasoconstriction. Moreover, enhanced Ser1928 phosphorylation and LTCC activity upon chronic elevated glucose exposure were prevented in cells pretreated with the P2Y11 inhibitor. Altogether, our findings suggest a key role for a Gs‐coupled P2Y receptor that fits the profile of P2Y11 in regulation of LTCCs and vascular tone during diabetic hyperglycemia, with important clinical and therapeutic implications for diabetic hypertension.

Support or Funding Information

This work is supported by NIH R01HL098200 and R01HL121059.

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