bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

BackgroundThe 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.MethodsWe 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.ResultsWe 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.ConclusionsWe 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 InformationNIH 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.MethodsArterial 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.ResultsSalvinorin 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.ConclusionThe 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 InformationFSOP Intramural GrantThis 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

BackgroundProstate 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.MethodsDunning 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).ResultsThere 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.ConclusionInformation 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 InformationAmerican 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 InformationThis 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 InformationThis 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 InformationFlorida Department of Health, Ed and Ethel Moore Alzheimer's Disease Research Program Grant 8AZ12This 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 InformationThis 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 InformationThis 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.