bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploring the structural basis of the Cross‐Talk between GlcNAcylation and Phosphorylation Using Physical Models
Authors: Shania Sharon , Schwabe Michael , Garcia Nina Marie , Sonnenschein Halie , Bell Ellis
Published: June 2021

The focus of this project was to explore Structure Function relationships in the enzyme UDP‐GlcNAc O transferase protein and design and build a physical model that illustrates key functional features of the protein. In particular our focus is on the Cross‐Talk between O‐GlcNAcylation and Phosphorylation that plays a critical role in overall regulation. Using the pdb file 4gyy.pdb from the paper “Crystal structure of human O‐GlcNAc Transferase with UDP‐5SGlcNAc and a peptide substrate” The structure illustrates the binding sites for both UDPGlcNAc and the peptide YPGGSTPVSSANMM with the requisite PV prior to the GlcNAcylatable S. Recent work by Leney et al (Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):E7255–E7261) has shown that the preceding T can be phosphorylated and that the sequence TPVS is common in proteins thought to undergo GlcNAc‐Phosphorylation cross talk. We have also used computational models to create and built models of the peptide phosphorylated at the N‐3 Threonine. These models clearly illustrate that the phosphate group clashes with the U in UDP‐GlcNAC. Since the kinetic mechanism of OGT is ordered Bi‐Bi with UDPGlcNAc as the obligate first substrate, the phosphorylated peptide can no longer bind in the peptide substrate pocket explaining why excludes GlcNAcylation. The models described here can be used to illustrate three different aspects contained in the ASBMB 8 Core Concepts of Macromolecular Structure and Function: #3. Structure and function are related, #4. Macromolecular interactions, and #6. The biological activity of macromolecules is often regulated. In addition construction and use of the models illustrates the use of core concept #8, A variety of experimental and computational approaches can be used to observe and quantitatively measure the structure, dynamics and function of biological macromolecule.

Support or Funding Information

Funded in part by NSF‐DUE 1725940 for the CREST Project.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circulating Caspase‐3 p17 Fragment as a Novel Marker for Cardiac Apoptosis During Cardioplegia
Authors: Lorinsky Michael K. , Kim MJ , Gold Courtney , Lahey Stephen , Fusco Daniel , Rosinski David , Pawlak Dorota , Liang Bruce
Published: June 2021

Myocardial cellular necrosis occurs with acute myocardial infarction and can be identified through the measurement of serum biomarkers, such as troponin I and the MB isozyme of creatine kinase (CK‐MB). However, both ischemia and reperfusion of cardiac myocytes in mouse models has been shown to result in cell death via apoptosis, in addition to necrosis. Apoptosis of cardiac myocytes during myocardial ischemia in humans has less, but nonetheless supporting evidence. Caspase‐3 is an end‐effecter of cardiac myocyte apoptosis, which yields a measurable p17 fragment in the serum after activation of the apoptotic cascade. Here we show the presence of elevated p17 levels in patients that undergo cardioplegia for cardiac surgery, which is a clinically controlled ischemia‐reperfusion condition. Serum levels of p17 were measured along with the traditional cardiac necrosis biomarkers CK‐MB and cardiac troponin I. Blood was drawn before, during, and after cardioplegia in three locations—peripheral venous, aorta, and coronary sinus. Compared to biomarker levels in healthy subjects, the levels of p17 were statistically significantly elevated before, during, and after cardioplegia, in conjunction with similar elevations of the traditional necrotic biomarkers. In addition, the ratio of caspase‐3 in the coronary sinus to that in aorta after reperfusion was greater than 1.0—a finding that was also found with the traditional cardiac biomarkers, indicating a cardiac origin of these biomarkers. These findings support that apoptosis of cardiac myocytes is the primary source of the elevated p17 after reperfusion of hypoxic cardiac myocytes during cardioplegia and that, in addition to necrosis, cardiac apoptosis occurs during cardioplegia and likely contributes to cardiac injury in this commonly performed operation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Regulation of mTORC1 Activity in The Plasma Membrane
Authors: Zhong Yanghao , Zhou Xin , Zhang Jin
Published: June 2021

The mechanistic/mammalian target of rapamycin (mTOR) complex 1 (mTORC1) plays a central role in cells by sensing a variety of signal molecules and integrating those signals to control pivotal cellular functions, including protein synthesis, cell growth and autophagy. Dysregulation of mTORC1 signaling pathway can cause many severe diseases such as cancer and type‐II diabetes. Emerging studies have shown that mTORC1 is regulated in a location‐ and signal‐specific manner, however, the mechanism underlying mTORC1 activities in different cellular compartments is poorly understood. By using a novel mTORC1 kinase activity reporter (TORCAR) based on fluorescence resonance energy transfer (FRET), our study aims to elucidate how mTORC1 activity is regulated in the plasma membrane compartment in response to different stimulations. We have shown that the growth factor signal but not the amino acid signal can induce mTORC1 activity in the plasma membrane compartment. Interestingly, we found that platelet derived growth factor (PDGF) induced mTORC1 activity showed a two‐phase pattern. Depleting calcium removed the fast responding component while keeping the slow one intact. Our preliminary results indicate calcium‐dependent regulation of mTORC1 activity at the plasma membrane. This result suggests signaling pathways including G‐protein coupled receptor (GPCR), especially Gq subclass, might contribute to the regulation of mTORC1 activity in the plasma membrane.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LOW‐LEVEL LASER THERAPY REDUCED BLOOD PRESSURE RESPONSE TO MAXIMUM EXERCISE IN POLICE OFFICERS
Authors: De Moura Jose Roberto , Alves Cleber Rene , Lemos Jose Ribeiro , Jordão Camila Paixão , Vicente Wanda Rafaela , Dalboni Maria Aparecida , Trombetta Ivani Credidio
Published: June 2021

Police officers (PO) in Brazil are exposed to a stressor environment, being wide‐open to higher cardiovascular risk. Arterial stiffness and endothelial dysfunction and impairment blood pressure response to exercise are markers of cardiovascular risk. Previous experimental studies have shown that low‐level laser therapy (LLLT) promotes increase in flow‐mediated dilation (FMD). Thus, we hypotetized that LLLT would improves endothelial function and decrease blood pressure in response to maximum exercise in police officers. We studied PO, submitted to LLLT (n=11, 39.4±2 years; 91.9±5 kg) for 6 months, and a matched group for age and weight that remained without any therapy was studied as a control group (C, n=7, 38.2±3 years; 83.8±3 kg). LLLT was applied twice weekly in the sublingually, with a maximum of three days between one application and another. Endothelial function was evaluated by brachial artery FMD. Systolic and diastolic blood pressure (BP) were analyzed during cardiopulmonary exercise test. After intervention, LLLT increased FMD, whereas C did not change (Δ of increase= 19.5±2 and 1.5±5 %, respectively, P=0.002). Interestingly, BP response to maximum exercise decreased only in PO submitted to LLLT (pre vs. post, SBP=198±6 vs. 177±4, P=0.02; DBP= 106±2 vs. 90±1 mmHg; respectively, P<0.001) and did not change in C (p=0.19). In conclusion, our data demonstrated that LLLT could significantly increase the vascular reactivity also in humans, what seems to explain, at least in part, the improved BP response to exercise. In police officers, who are exposed to high levels of stress, LLLT would be an alternative therapeutic to prevent cardiovascular risk.

Support or Funding Information

CAPES ‐ Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the antioxidant enzyme‐mimetic activity and neuroprotective effects of cerium oxide nanoparticles stabilized with varying ratios of citric acid and EDTA
Authors: Estevez Ana Y. , Boateng Yvonne , Lipps Jennifer , Stadler Brad , Erlichman Joseph S.
Published: June 2021

Cerium oxide (CeO2) nanoparticles (nanoceria or CeNPs) are potent antioxidants that are being explored as a potential therapeutic for diseases featuring oxidative stress. However, both beneficial and toxic effects of CeNPs have been reported. We hypothesize that the method of synthesis as well as the choice of stabilizers coating the particles can lead to disparate biological effects. In the present study we tested the antioxidant enzyme‐mimetic properties of a series of custom‐synthesized CeNPs that differed in the citric acid (CA) and EDTA ratio (CA:EDTA) used to stabilize them. We first spun each type of CeNP at 100,000 RPM (434,513 xg) in order to separate it into supernatant (SUP) and pellet (PEL) fractions. We then used commercially available enzymatic assay kits to assess the catalase‐ and superoxide dismutase (SOD)‐mimetic activity of each fraction. The CeNPs stabilized with only CA (CA:EDTA ratio of 100:0) exhibited significantly higher catalase‐mimetic activity in both the SUP and PEL fractions compared to the other samples. Varying the CA:EDTA ratio had opposing effects in the SOD‐mimetic activity of the SUP and PEL fractions. For example, CeNPs stabilized with only CA (CA:EDTA ratio of 100:0 ) displayed most of the SOD‐mimetic activity in the PEL. In contrast, CeNPs stabilized with only EDTA (CA:EDTA ratio of 0:100) showed highest level of SOD‐mimetic activity in the SUP. Intermediate ratios (CA:EDTA of 40:60) showed intermediate levels of SOD‐mimetic activity in the SUP and PEL fractions. We then tested the effects of these CeNPs in a mouse hippocampal brain slice model of ischemia where cell death was assessed using the fluorescence indicator SYTOX green. Interestingly, no significant neuroprotection was observed in brain slices treated with CeNPs stabilized with the extreme ratios of CA:EDTA (100:0 and 0:100). However, slices treated with CeNPs stabilized with intermediate ratios of CA:EDTA (50:50, 40:60, 30:70) showed significantly reduced ischemic cell death. When the SUP and PEL fractions of the CA:EDTA 50:50 sample were tested separately, only the slices treated with the pellet fraction showed significantly reduced cell death. These results demonstrate that CeNPs varying in the ratio of CA:EDTA used for stabilization display different enzyme‐mimetic activity. More importantly, our data suggest that CeNPs synthesized with intermediate CA:EDTA stabilization ratios demonstrate the best neuroprotection profile in ischemic brain slices and that the neuroprotective activity resides in the PEL fraction of the CeNPs.

Support or Funding Information

This work was supported by St. Lawrence University and Cerion Advanced Materials.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intestinal D‐allulose transport is likely mediated by glucose transporter type 5 (GLUT5)
Authors: Kishida Kunihiro , Iida Tetsuo , Yamada Takako , Ferraris Ronaldo P. , Toyoda Yukiyasu
Published: June 2021

D‐allulose is found in a wide variety of food products in limited amounts, and therefore is referred to as a rare sugar. It is an epimer of fructose that is 70% as sweet as sucrose and has been demonstrated to have antihyperglycemic, antihyperlipidemic, and antiobesity effects by various studies including humans. The mechanism underlying intestinal absorption of D‐allulose is unknown, hence we tested the hypothesis that intestinal D‐allulose transport is mediated by glucose transporter type 5 (GLUT5). Adult rats were initially fed for a week a high‐fructose (control: high‐glucose) diet to induce GLUT5 expression and then gavaged with 1g/kg BW D‐allulose alone, D‐allulose with 1g/kg BW glucose or D‐allulose with 1g/kg BW fructose. Plasma D‐allulose concentrations were then compared to examine whether D‐allulose shares transporters with fructose and/or glucose and whether its absorption is affected by luminal glucose or fructose. When D‐allulose was gavaged alone, the fructose‐fed rats showed significantly higher plasma D‐allulose levels at 30, 60, 90 min and conversely lower (P = 0.05) level at 120 min after administration than those of the glucose‐fed rats. This suggests that more D‐allulose was rapidly absorbed via highly expressed GLUT5 in the fructose‐fed rats. When D‐allulose was gavaged with fructose, appearance of D‐allulose in the plasma was delayed compared to when D‐allulose alone was administrated, indicating that fructose inhibited D‐allulose transport into plasma. Moreover, the levels of plasma fructose peaked earlier than those of D‐allulose, indicating that luminal fructose is more rapidly absorbed than D‐allulose. When D‐allulose was gavaged with glucose, higher plasma D‐allulose concentrations at 30 min after administration were observed compared to when D‐allulose was gavaged with fructose in both glucose‐ and fructose‐fed rats. These results suggest that intestinal D‐allulose transport is likely mediated by GLUT5 and its affinity for GLUT5 is lower than that of fructose.

Support or Funding Information

This work was partly supported by Matsutani Chemical Industry Co., Ltd.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Cellular Mechanisms of DNA Replication Restart in Escherichia coli
Authors: McKenzie Aidan , Wessel Sarah , Keck James
Published: June 2021

In E. coli, replisomes duplicate only a fraction of the chromosome before being ejected from the DNA due to collisions with damaged DNA or nucleoprotein complexes. Premature replisome ejection leaves an abandoned replication fork that must be reinitiated by DNA replication restart proteins (PriA, PriB, PriC, and DnaT in E. coli). PriC can mediate helicase loading without the help of other replication restart proteins in vitro and it directly interacts with the DnaB/DnaC (helicase/loader) complex. Interestingly, ΔpriC strains appear wild‐type but have synthetic lethal relationships with genes not implicated in DNA replication restart. Our poster describes recent structural efforts to probe the PriC/DnaB/DnaC complex and a transposon‐sequencing approach to identify genes linked to priC and other DNA replication restart genes in E. coli.

Support or Funding Information

NIH R01GM098885.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthesis and production of granatane alkaloids in hairy root cultures of Punica granatum
Authors: Glockzin Kyle , D'Auria John
Published: June 2021

Punica granatum, the common pomegranate plant, contains compounds known as granatane alkaloids. These compounds possess medicinal properties that have positive effects on cardiovascular disease and reducing high blood pressure(1). Granatane alkaloids include compounds such as pelletierine, isopelletierine, pseudopelletierine, and N‐methylpelletierine. Granatane alkaloid biosynthesis is believed to occur in the root bark. In order to study the metabolism of these compounds, hairy root cultures were established by inoculating roots of seedlings with Agrobacterium rhizogenes strain MSU440(2). The bacterium harbored a pK2gw7 vector containing an eGFP (enhanced green fluorescent protein) sequence. Our ultimate goal is to use next generation sequencing on biochemically and transcriptionally active granatane producing tissues for gene discovery purposes. This relies on a confirmation of the hairy root culture as a tenable model system. In one of the hypothetical granatane producing pathways, the first five membered ring would originate from the decarboxylation of the amino acid lysine, which would form the compound cadaverine (1,5‐pentadiamine). The suspected enzyme for this step is PgLDC (Punica granatum lysine decarboxylase). We aim to isolate and characterize this enzyme with an established hairy root culture system. To date, several candidate genes have been identified.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elicitation of HIV‐specific Antibodies Targeting The Carbohydrate‐Occluded Neutralization Epitopes Through Rational Protein Design
Authors: Zhu Cheng , Dukhovlinova Elena , Council Olivia , Benhabbour S. Rahima , Ping Lihua , Potter E. Lake , Kincer Laura P. , Swanstrom Ronald , Dokholyan Nikolay V.
Published: June 2021

Background

The HIV‐1 viral surface Env protein encodes a dense array of carbohydrates. Many of these N‐linked surface‐covering carbohydrates appear at highly conserved positions, although most HIV‐1 isolates are missing one or more glycosylation sites, which potentially reveal underlying surface structures. We have grouped the Env surface that underlies these carbohydrates into a set of Carbohydrate‐Occluded Neutralization Epitopes (CONEs). We have examined the utility of small protein mimics of the CONEs to serve as potential immunogens.

Methods

We selected four distinct CONEs as targets (three‐stranded β‐sheet, α‐helix, and two separate loops) and identified compatible motifs in the PDB for each epitope‐scaffold design. We then grafted the exposed residues of each CONE onto the respective scaffolds followed by computational redesign to increase thermodynamic stability. The chimeric proteins were expressed in E. coli and purified then used in structural analysis and rabbit immunization.

Results

We determined the structures of two designed proteins C2S5 and C4S3 as mimetics of the α2‐helix and the C loop of Env. Crystal structures were solved to resolutions of 1.2 Å and 2.0 Å, respectively. Comparison with the SOSIP trimer structure (PDB 4TYP) revealed a high degree of mimicry: within the epitope region the overall RMSDs between the designs and Env structure are 0.64 Å (C2S5) and 0.36 Å (C4S3), suggesting high‐accuracy structural design and our accurate presentation of the CONE residues on heterologous protein scaffolds.

In addition to the primary scaffolds we also made alternative scaffolds to examine the specificity of the corresponding antibodies. We generated antibodies to the primary immunogens and detected cross‐reactivity to the alternative scaffolds by ELISA, suggesting the antibodies react to the HIV‐specific segment of our immunogens.

We next evaluated the interactions between the polyclonal antibodies and gp120 (fully‐glycosylated and under‐glycosylated) through western blot. The rabbit sera reacted specifically to the under‐glycosylated gp120. Importantly, we observed antibody‐mediated neutralization towards tier 2 viruses with under‐glycosylated Env.

Conclusions

We have been able to design structural mimetics of surface features of the HIV‐1 Env protein that are typically but not always occluded by carbohydrate, and to use these mimetics to raise HIV‐specific antibodies. These small and thermally stable immunogens represent a starting point for eliciting a new class of antibodies that in combination could neutralize most isolates.

Support or Funding Information

NIH Grant R01AI102732
Solvent Extraction and Antibacterial Analysis of Chinese Traditional Herbs
Authors: San Angelo Michael , Isovitsch Ralph , Valenzuela Hector
Published: June 2021

The increased development of antibiotic resistant bacteria has created a need for new antibiotics. To obtain new antibiotics by solvent extraction, studies are currently utilizing Chinese traditional herbal medicines to acquire antibacterial compounds. Eight Chinese traditional medicinal herbs were obtained from the Yunnan Province of China: Semiaquileqia adoxoides, Albizia julibrissin bark, Dendrobium nobile stem, Panax notoginseng stem, Lygodium japonicum spore, Prunellae vulgaris, Lycium ruthenicum, and Lepidium meyenii. These specific herbs have been primarily used as anti‐inflammatories throughout the history of Chinese traditional medicine. Using the knowledge that inflammation is often a sign of infection, we obtained these specific herbs that could potentially contain antibacterial components. To determine potential antibacterial properties of these herbs, the disc‐diffusion method was conducted using 10 mg/mL extracts within water, ethanol, cyclohexane, ethyl acetate, and chloroform. After dissolving the extracts within their respective solvents screening was performed against the following gram‐positive, gram‐negative, and gram‐variable bacteria: E. coli, M. luteus, S. aureus, P. aeruginosa, P. mirabilis, S. typhimurium, and B. subtilis. All eight of the herbs utilized, proved to have some form of antibacterial properties ranging from 0.5 mm to 6 mm zone of inhibition. The Dendrobium nobile stem chloroform extract had the most significant antibacterial resistance with a 6 mm zone of inhibition after three triplicate trials containing three 10 mg/mL discs per trial. Amoxicillin (10 μg/disc) and penicillin (10 μg/disc) were used as a reference to compare the zone of inhibition obtained from the disc‐diffusion assays in addition to the solvents used for extraction. Gas chromatography analysis proved that the chloroform extract of Dendrobium nobile stem had a total of four components with the following retention times: 15.427, 20.388, 21.142, and 23.279 minutes. Mass Spectroscopy data showed a molecular ion with the m/z ratio of 503.2 within the Denrobium nobile bark extracts that had significant antibacterial resistance against M. luteus. In addition the following molecular ions were observed: 338.1 m/z and 429.2 m/z.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Responses Produced by the Central Microinjection of Salvinorin A and B in conscious Rats
Authors: Franklin Cynthia , Anatrella Elizabeth , Bombaywala Pooja , Kaur Satwinder , Lucas Evan , Henderson Richard , Rangel Yolanda , Gottlieb Helmut
Published: June 2021

Intracerebroventricular (ICV) injection of the kappa opioid agonists produces a marked diuresis and antinatriuresis in rats. Salvinorin A and B have also been proposed to evoke renal effects, however these drugs are very hydrophobic and requires the use of detergent like vehicles (e.g., DMSO). We observed that DMSO alone is able to evoke changes to cardiovascular and renal function, thus not an ideal diluent. As such, the purpose of this study is to examine the diuretic effects of two drugs with affinity for the kappa opioid receptor, Salvinorin A and B dissolved in acetonitrile vehicle. This study is unique since past studies utilized drug dissolving vehicles (e.g., DMSO) that produced significant cardiovascular and renal effects. As such, we are utilizing a new approach to determine the renal effects produced by these kappa opioid receptor agonists.

Methods

Arterial blood pressure (ABP), heart rate (HR) and urine output was recorded in conscious rats. Catheters were placed in both a femoral vein, for drug delivery and infusion of isotonic saline (55 ml/min), and in the urinary bladder for urine collection. Urine was sampled during two 10 min control periods and six 10 min periods beginning 10 min after injection of drug or vehicle into the lateral ventricle.

Results

Salvinorin A increased urine excretion without changing HR, mean ABP, or urinary sodium excretion. Injections of acetonitrile (9:1 dilution) alone or Salvinorin B produced no physiological effects in the measured parameters.

Conclusion

The ability of Salvinorin A to increase urine outflow without affecting sodium excretion raises the possibility that Salvinorin A preferentially targets kappa opioid receptors at brain sites that facilitate water excretion, but not sodium. Furthermore, Salvinorin B did not appear to be as efficacious as Salvinorin A in producing diuresis. This is different when compared to synthetic kappa opioid agonists, which produce water diuresis.

Support or Funding Information

FSOP Intramural Grant

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 a Pre‐Clinical Model of Prostate Cancer on Left Ventricular Function in Sedentary and Exercise Trained Rats
Authors: Baumfalk Dryden R. , Opoku‐Acheampong Alexander B. , Caldwell Jacob T. , Ade Carl J. , Musch Timothy I. , Behnke Bradley J.
Published: June 2021

Background

Prostate cancer is the most common type of non‐skin cancer found in men making it a primary area of concern. Preliminary evidence suggests prostate cancer independent of treatment has atrophic effects on whole heart and left ventricular (LV) mass which are associated with reduced endurance exercise capacity in rats, highlighting a major area of concern. Using a pre‐clinical orthotropic model of prostate cancer, echocardiography was used to test the hypothesis that prostate cancer would impact LV function. We also investigated whether moderate exercise training would attenuate any detriments seen in LV function with prostate cancer.

Methods

Dunning R‐3327 AT‐1 prostate cancer cells (1×104) were cultured and harvested for injection in 0.1 ml of physiological saline solution in the ventral lobe of 3–4 month‐old male Copenhagen rats (n=19). These animals were randomized into 2 groups, Tumor Bearing Exercise (TBEX) n=10 or Tumor Bearing Sedentary (TBS) n=9. After ~1‐week recovery, TBEX animals began exercise on a treadmill at 15 m/min with a 15° incline for 60 min/day for ~30 days. Twenty additional cancer free controls were randomized into exercise control (EXC) (n=10) or Sedentary control (SEDC). Pre‐surgery (V1), and ~30 days post exercise commencement (V3) animals underwent echocardiographic evaluation using the parasternal short axis view to examine ventricle dimensions via a commercially available system (Vivid S6, GE Health Care).

Results

There were no significant differences in tumor mass between groups (TBEX 8.62±1.7g, TBS 6.6±1.3g p=0.29), or body weights (TBEX 299±9g, TBS 284±6g p=0.22) and (EXC 351±5g, SEDC 341±7g). There were no significant differences for V1 for any measures of LV function for all 4 groups. From V1–V3 EXC had significant increases in SV (103±5 ul, 126±7 ul, p=0.003) while SEDC had no significant difference (114±3 ul, 111±10 ul, p=0.78). SV for TBEX and TBS did not significantly change from V1–V3, but at V3 TBEX had significantly larger SV (TBEX=116±6 ul, TBS= 99±5 ul, p=0.022). Strain and strain rate were not significantly different from V1–V3 for all groups, however the strain (p=0.001) and strain rate (p=0.005) were both significantly different between control groups.

Conclusion

Information gleaned from the current investigation suggests prostate cancer itself does not significantly reduce left ventricular function in sedentary animals. However, increases in left ventricular function found in normal healthy exercise trained animals was reduced in tumor bearing animals performing the same exercise regime.

Support or Funding Information

American Cancer Society RSG 14‐150‐1‐CCE (BJB)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Can Daytime Measures of Respiratory Sinus Arrhythmia and Breathing Stability Serve as Biomarkers for OSA?
Authors: DeLucia Claire M. , Barreda Santiago , Vranish Jennifer R. , Bailey E. Fiona
Published: June 2021

Obstructive sleep apnea (OSA) is by definition a disorder of nighttime breathing however, there is good evidence that impairments in cognitive and cardiovascular function persist in the day and that adults with OSA exhibit daytime CO2 retention and hypoxemia. Although a diagnosis of OSA requires in‐home or in‐laboratory overnight polysomnography, these types of tests are expensive and, due to health insurance quotas, may be limited in availability. Here, we outline simple methods for assessing daytime respiratory sinus arrhythmia (RSA) and respiratory stability that show promise as potential diagnostic tools to identify adults at risk for OSA. We recorded heart rate (finger oxy‐photoplethysmography) and respiration (inductance plethysmography) in adults with obstructive sleep apnea (N=6) and age and BMI matched healthy adults (N=8). Subjects completed five minutes of eupneic breathing, five minutes of paced breathing (7.5 breaths/minute) and after equilibration, five minutes of hyperoxic, hypercapnia (5% FICO2). Using de‐identified data, we obtained estimates of RSA amplitude, RSA stability, and respiratory stability for each subject in each condition and performed a regression analysis to determine the relationship between each of these variables and subjects' apnea hypopnea index (0–25 AHI). Our preliminary results indicate that daytime measures of respiratory stability and RSA can differentiate healthy adults from adults diagnosed with OSA (p<0.006). Importantly, the analysis requires minimal instrumentation and short sampling periods (~5 minutes). Summary schematics arising from the analysis may prove useful as preliminary clinical/diagnostic biomarkers of RSA and cardio‐respiratory coupling in adults at risk for OSA.

Support or Funding Information

This work was supported by start‐up monies (Department of Physiology, University of Arizona) awarded to EFB and by The Finley and Florence Brown Predoctoral Fellowship (Sarver Heart Center, University of Arizona) awarded to JRV.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of An Anticancer Peptide Rescuing p53 from Degradation by COP1
Authors: Gao Meng , Huang Yongqi
Published: June 2021

A 28 amino‐acid peptide derived from azurin (amino acids 50–77, p28) preferentially enters human cancer cells and induces apoptosis or growth inhibition. Previous studies have revealed that p28 exerts its anticancer activity mainly by interacting with the p53 DNA binding domain (DBD) and interfering in the p53 degradation signaling pathway. However, the interacting mechanisms between p28 and p53‐DBD are missing and how p28 inhibits p53‐DBD ubiquitination remains largely unknown. In this work, we characterized the interacting mechanisms between p28 and p53‐DBD by a combination of NMR, X‐ray crystallography, ITC and computer simulations. Interactions between p28 and p53‐DBD were mainly mediated by hydrophobic contacts. Structural models of the p28/p53‐DBD complex obtained with NMR datadriven docking indicated that p28 adopted an α‐helix upon binding to p53‐DBD. Val59, Val60, Thr61, Tyr72 and Leu73 of p28 made extensive interactions with the L1 loop and S7–S8 loop of p53‐DBD. Site‐directed mutagenesis and ITC characterization showed that single alanine substitution of the above mentioned hydrophobic residues reduced the binding affinity by more than one order of magnitude. GST pull‐down assays showed that the interactions between p53‐ DBD and the E3 ligase COP1 were blocked by p28. Since alanine substitution of key hydrophobic residues reduced the binding affinity of p28 with p53‐DBD, alanine mutated p28 were less efficient in blocking the p53‐DBD/COP1 interaction. Determination of the p28/p53‐ DBD complex structure is in progress, which will allow us to rationally design p28 derived peptides exhibiting higher affinity with p53‐DBD and stronger anticancer activity in the future.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (Grant number: 21603121, Y.H.) and Hubei University of Technology (M. G. and Y.H.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein Disulfide Isomerase Disrupts and Detoxifies Aggregates of Amyloid Beta Peptide
Authors: Lehew Haley , Evangelista Baggio , Cilenti Lucia , Teter Ken
Published: June 2021

Aggregated fibrils of amyloid beta (Aβ) peptide induce the neurotoxicity characteristic of Alzheimer's Disease. The inhibition of Aβ aggregation could therefore represent a therapeutic target for Alzheimer's Disease. Protein disulfide isomerase (PDI) is an oxidoreductase and chaperone that has been shown to effectively inhibit the formation of Aβ fibrils when present at the onset of aggregation. However, the potential disruption of ongoing Aβ fibrillization by PDI has not been demonstrated. We examined the disruptive effect of PDI on Aβ aggregation using a Thioflavin‐T fluorescence assay to monitor Aβ fibrillization. PDI prevented the aggregation of monomeric Aβ at a sub‐stoichiometric 1:10 molar ratio of PDI:Aβ. Fibrillization was partially inhibited by a 1:50 molar ratio of PDI:Aβ and was not affected by a 1:100 molar ratio of PDI:Aβ. As such, a 1:10 molar ratio of PDI:Aβ was used for all subsequent experiments. We next found that PDI could also disrupt Aβ fibrillization when added 2, 4, or 10 h after the initiation of aggregation. Cell culture studies using an MTS cell viability assay further demonstrated that PDI can protect neuronal cells from extracellular aggregates of Aβ when added 2 h after the initiation of aggregation. These findings demonstrate PDI is able to disrupt Aβ aggregation at sub‐stoichiometric molar ratios and support the potential use of PDI as a novel therapy for Alzheimer's Disease.

Support or Funding Information

Florida Department of Health, Ed and Ethel Moore Alzheimer's Disease Research Program Grant 8AZ12

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 ‘Complex’ Problem of Purine Metabolism
Authors: French Jarrod B
Published: June 2021

The interior of a eukaryotic cell is an extremely crowded environment containing thousands of different types of biomolecules. Within this dense morass, however, are differentiated micro‐environments, sometimes called mesoscale assemblies or biomolecular condensates, which are transiently assembled to respond to cellular stimuli. These protein systems provide a highly tunable and specific level of temporal and spatial control over processes such as metabolism and signaling. One such system has evolved to manage the needs of purine metabolism in eukaryotic cells. The purinosome is a newly discovered protein assembly that is involved in regulating de novo purine biosynthesis. This dynamic complex is composed of all six members of the pathway and several known accessory factors. Recent imaging and complementary biochemical analyses revealed that the purinosome structurally and functionally associates with mitochondria. This mutualistic interaction provides energy for purine biosynthesis while creating the necessary precursors for ATP production. The implications of this observation, additional observations about purinosome trafficking, and our lab's progress towards understanding the mechanisms of assembly will be presented.

Support or Funding Information

This work is funded by the National Institute of General Medical Sciences of the NIH under Award Number R35GM124898.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Δ‐2 Hexadecenal Generated from S1P by Nuclear S1P Lyase Is a Regulator of HDAC1/2 Activity and Histone Acetylation in Lung Epithelial Cells
Authors: Ebenezer David L. , Fu Panfeng , Mangio Lizar A. , Berdyshev Evgeny , Schumacher Fabian , Kleuser Burkhard , Van Veldhoven Paul P. , Natarajan Viswanathan
Published: June 2021

Sphingosine‐1‐phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to Δ‐2 hexadecenal (Δ2HD) and ethanolamine‐phosphate by S1P lyase (S1PL) in mammalian cells. While the role of S1P as a key regulator of cellular functions in normal and pathological conditions is well established, the physiological role of fatty aldehyde released from S1P by S1PL is still unclear. We have recently demonstrated activation of nuclear SPHK2 and generation of S1P and Δ‐2 Hexadecenal in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa (PA). Here, we have investigated the nuclear localization of S1PL and the role of Δ2HD as a modulator of nuclear HDAC activity and histone acetylation.

Nuclear extracts, free from cytosolic and ER contamination, prepared by detergent fractionation method, were probed for purity by Western blotting using organelle‐specific markers Lamin B, ERp72, and GAPDH. Nuclear fractions derived from primary lung bronchial epithelial cells and alveolar epithelial MLE‐12 cell line exhibited S1PL activity, as measured by a fluorogenic substrate assay and mass spectrometric analysis and immunostained for S1PL by Western blotting. Activation of lung epithelial cells with heat‐inactivated Pseudomonas aeruginosa (PA) stimulated Δ2HD generation in the nucleus, which was S1PL dependent. Blocking S1PL activity with an inhibitor, compound C1 (5μM), in MLE‐12 cells inhibited PA‐induced nuclear Δ2HD production and H3 and H4 histone acetylation. In vitro, addition of exogenous Δ2HD (100–10000 nM) to lung epithelial cell nuclear preparation inhibited HDAC 1/2 activity, and increased acetylation of Histone H3 and H4. Overexpressing wild‐type Sgpl1 gene into mouse embryonic fibroblasts (MEFs) deficient in Sgpl1 decreased basal and PA‐induced nuclear HDAC activity, underlining the role of S1P degradative products in modulating HDAC activity. Furthermore, in Sgpl1 −/−MEFs, PA‐induced HDAC activity was higher compared to Sgpl1+/+MEFs treated with PA. Additionally, incubation of Δ2HD with HDAC1 generated 5 different amino acid adducts as detected by LC‐MS/MS; the predominant adduct being Δ2HD with lysine residues of HDAC1.

These data show nuclear localization of S1PL in lung epithelial cells and indicate a crucial epigenetic role for Δ2HD, released from S1P by S1PL in the nucleus, in regulating HDAC1/2 activity and histone acetylation. Thus, nuclear S1PL and generation Δ2HD may have a physiological and pathophysiological relevance to lung inflammatory injury.

Support or Funding Information

This work was supported by NIH grant HL 098050 to VN.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unique enzyme specificity of three human phospholipases A2 toward phospholipids containing sn‐2 omega‐3 and omega‐6 fatty acids
Authors: Hayashi Daiki , Mouchlis Varnavs D. , Dennis Edward A
Published: June 2021

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyzes the sn‐2 acyl‐chain of membrane phospholipids producing lyso‐phospholipids and free fatty acids. The products of PLA2 have various functions, especially omega‐3 and omega‐6 fatty acids which are precursor of various eicosanoids and related metabolites involved in numerous pro‐inflammatory and pro‐resolving cellular responses. The PLA2 superfamily is composed of six different types of enzymes, each with distinct structural features. Among them, the cytosolic PLA2 (cPLA2), calcium‐independent PLA2 (iPLA2), and secreted PLA2 (sPLA2) are well studied. cPLA2 is the main provider of free arachidonic acid (AA), an omega‐6 fatty acid involved in the inflammatory cascade. Several knockout mice studies demonstrated the contribution of iPLA2 to the metabolism of the omega‐3 fatty acid docosahexaenoic acid (DHA) in brain (J. Lipid Res. 2010, 51, 3166–3173). sPLA2 is known for its antibacterial action and involvement in atherosclerosis. Previous studies in our laboratory determined the substrate specificity of all three PLA2s toward a variety of membrane phospholipids (J. Am. Chem. Society 2018 140, 3285–3291). However, the specificity of these enzymes toward phospholipids containing omega‐3 and omega‐6 fatty acids at the sn‐2 position has been unclear. In this study, we have employed a lipidomics‐based LC‐MS/MS assay, which measures the lysophospholipid product to measure the activity of PLA2, and to investigate the sn‐2 acyl‐chain specificity in various phospholipids. We have also developed a lipidomics‐based GC‐MS assay which allow us to measure free fatty acids in mixtures of phospholipid substrates. Although cPLA2 showed relatively high activity toward the omega‐3 eicosapentaenoic acid (EPA), it prefers AA. iPLA2 showed a distinct preference for EPA over AA and a much lesser activity toward phospholipids containing DHA. Unlike the other two PLA2s, sPLA2 showed a remarkable preference for DHA among the omega‐3 and omega‐6 fatty acids. Importantly, similar sn‐2 acyl‐chain specificity was obtained in mixtures of phospholipids. This study investigated the preference of three human PLA2 enzymes for the sn‐2 omega‐3 and omega‐6 fatty acids for the first time. Our findings will help us to understand the difference of function of PLA2 enzymes at the cellular level and their implications in inflammatory diseases.

Support or Funding Information

Supported by NIH grant GM20501

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perilipin 5 and Rab 18 Occupy Similar Cellular Pools
Authors: Hughes Erin K , Tansey John T
Published: June 2021

Type II diabetes mellitus and non‐alcoholic fatty liver disease are two examples of metabolic diseases that are contributing to the emerging health crisis in the west. Key to understanding these diseases and their progression is an understanding of neutral lipid metabolism. Perilipins are a class of five conserved proteins that are key regulators of lipid metabolism and are potentially involved in lipid trafficking within cells. The most recently discovered member of the family is perilipin 5, which is expressed most strongly in tissues that are highly oxidative such as heart, oxidative muscle, and fasting liver. Perilipin 5 is predominantly located on the surface of lipid storage droplets, but has also been found in the cytoplasm, nucleus, and the endoplasmic reticulum and mitochondria. We hypothesized that perilipin 5 moves between these cellular pools via interactions with other proteins. Furthermore, we hypothesized that interactions with Rabs were necessary for this trafficking to occur. CHO cells ectopically expressing perilipin 5 and endogenously expressing Rab18 were assayed using western blotting. These proteins were found in the same cellular pools. Immunoprecipitations are being used to assay for interactions between perilipin 5, Rab18, and Rab32. Immunofluorescence microscopy indicates that perilipin 5 and Rabs colocalize to the same cellular pools. Collectively these data will provide a more detailed picture of perilipin 5 in the cell.

Support or Funding Information

Otterbein University Student Research Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatocyte‐Specific Deletion of eNOS Impairs Mitochondrial Function and Exacerbates Hepatic Steatosis
Authors: Cunningham Rory P. , Moore Mary P. , Meers Grace M. , Nguyen Nhu , Boccardi Luigi , Jepkemoi Vivien , Takahashi Takamune , Rector R. Scott
Published: June 2021

Introduction

Whole body loss of endothelial nitric oxide synthase (eNOS) worsens hepatic mitochondrial function and exacerbates nonalcoholic fatty liver disease (NAFLD) development and progression. However, the precise role of eNOS in hepatocytes in the contribution to NAFLD has not been established. Here, we examine the effects of in vivo hepatocyte‐specific ablation of eNOS on hepatic steatosis and NAFLD susceptibility.

Methods

Hepatocyte‐specific eNOS knockout (eNOShep−/−) mice were generated by crossing homozygous eNOS floxed (eNOSfl/fl) mice on a C57/BL6J background with albumin‐Cre recombinase transgenic mice. At 10 weeks of age, male eNOSfl/fl and eNOShep−/− mice (n = 12–14) were fed a control diet (10% fat) for 16 weeks.

Results

Body weight, body fat, and food intake did not differ between eNOSfl/fl and eNOShep−/− mice. eNOShep−/− mice developed elevated histological hepatic steatosis and increased fasting serum insulin (~30%) compared to eNOSfl/fl mice (p<0.05), along with trends for elevations in serum liver enzymes AST and ALT (p<0.17). Elevated hepatic steatosis in eNOShep−/− was related to significant reductions (−50%) in hepatic mitochondrial fatty acid oxidation and ADP‐stimulated and maximal uncoupled hepatic mitochondrial respiration (p<0.05). However, hepatic citrate synthase activity, a marker of mitochondrial mass, was significantly elevated in eNOShep−/− mice compared to eNOSfl/fl mice (p<0.05), while protein content of oxidative phosphorylation complexes and peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) did not differ between groups. Furthermore, eNOShep−/− mice presented with significant reductions (p<0.05) in key proteins involved in mitophagy and autophagy including PTEN‐induced kinase 1 (PINK1) and phosphorylated Unc‐51 Like Autophagy Activating Kinase 1 (pUlk1/Ulk1) and tended to have significant reductions in BCL2/adenovirus E1B 19 kDa protein‐interacting protein 3 (BNIP3) compared to eNOSfl/fl mice.

Conclusion

Here we demonstrate for the first time that hepatocyte‐specific deletion of eNOS impairs hepatic mitochondrial function, reduces markers of autophagy/mitophagy, and exacerbates hepatic steatosis development. Further studies are necessary to determine the mechanisms by which deletion of eNOS regulates mitochondrial homeostasis.

Support or Funding Information

VA Merit Grant I01BX003271‐01 (R.S.R.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transfer RNA post‐transcriptional modifications of Bacillus subtilis
Authors: Psihountas Christina , Jora Manasses , Limbach Patrick A
Published: June 2021

Transfer Ribonucleic Acids (tRNAs) have more posttranscriptional modifications to nucleosides than other species of RNA.1 It has been found that when bacteria are analyzed at different stages of a growth curve, changes in these nucleoside modification identities or amounts exist.2 Similarly, nucleoside modification profiles are dependent on growth media.3 Here we are examining whether domestication of Bacillus subtilis lead to differences in nucleoside modification profiles for tRNAs. We have compared tRNA modifications from two different strains: (i) a domesticated lab strain 168, the most widely studied strain, and (ii) strain NCIB 3610, a Marburg strain isolate. NCIB 3610 is reported in literature to be “wilder” than strain 168.4 We are using strain 168 as the B. subtilis model against which we can evaluate how domestication may impact the types and quantities of tRNA modifications. Our experimental approach is to use liquid chromatography tandem mass spectrometry (LC‐MS/MS). Once nucleosides are identified and quantified, we will then compare those results to the genetic variations arising between the domesticated and undomesticated strains to understand how such modification changes may arise.

Support or Funding Information

The National Science Foundation (CHE 1507357)

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