bluuebunny/crossref_metadata_2025 huggingface dataset:
Design of Novel Lectins by Computer‐Guided Directed Evolution
Authors: Sharma Prerna , Kazan I. Can , Ozkan Sefika Banu , Ghirlanda Giovanna
Published: June 2021

Changes in the level and type of glycosylation are implicated in mediating countless physiological and disease processes, yet the glycome is poorly defined. To overcome this barrier, we integrate state of the art computational methods with guided directed evolution for the design of selective glycan binding reagents based on a natural lectin, cyanovirin.

Cyanovirin‐N (CV‐N) is a small (11 kDa) lectin that displays potent antiviral activity by binding to the oligomannosides of gp120. The solution structure shows two quasi‐symmetric domains, defined as A (residues 1‐38/90‐101) and B (residues 39–89), which are connected on each side by a short helical linker. Each domain contains a carbohydrate binding pocket, which binds selectively Mana(1®2)Mana termini in branched high‐mannose oligosaccharides. We assessed the binding affinity of WT CV‐N through ITC titrations with dimannose revealing a high‐affinity binding site (Kd=15.3 mM) and a low‐affinity site (Kd=0.400 mM). To increase the binding affinity for glycosylated gp120, we exploited avidity effects by designing a dimeric CV‐N in which a CV‐N sequence is spliced into another. The construct contains four glycan binding domains in two sets (A, A′, B, B′), and binds dimannose with Kd=24.5 mM and 0.95 mM respectively. Four indistinguishable binding events, with Kd=1.1 mM, are observed with Man9.

We recently developed computational tools to predicted protein‐glycan interactions accurately by using multiple sequence alignment (MSA) and flexibility analysis to identify co‐evolved positions, and coupling this analysis to our flexible docking tool (BP‐Dock). These tools were validated by assessing the energetic contribution of polar residues within the binding pocket, docking dimannose to single‐point CVN mutant models. We found that the E41A/G and T57A mutations led to a significant decrease in binding energy scores due to rearrangements of the hydrogen‐bond network that reverberated throughout the binding cavity. N42A decreased binding by affecting the integrity of the local protein structure. In contrast, N53S resulted in a high binding energy score. Experimental characterization of the five mutants by NMR and ITC confirmed the binding affinity pattern predicted computationally.

Computational analysis also yielded a set of mutations at several positions beyond the immediate binding pocket that impact glycan recognition. These predictions were validated by directed evolution in yeast display; screening resulted in mutants with increased affinity and selectivity for dimannose. We are currently using the same library to identify mutants with high affinity and selectivity towards glycan biomarkers.

Support or Funding Information

This work is supported by NIH‐IR21AI129895 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.
Use of Bioorthogonal N‐Acetylcysteamine (SNAc) Analogues and Peptidoglycan O‐Acetyltransferase B (PatB) to Label Peptidoglycan
Authors: Lazor Klare M. , Wang Yiben , DeMeester Kristen E. , Liang Hai , Heiss Tyler K. , Grimes Catherine L.
Published: June 2021

In Gram (−) bacteria, peptidoglycan O‐acetyltransferase B (PatB) is responsible for the O‐acetylation of peptidoglycan to prevent degradation by lysozyme and other lytic transglycosylases generated by its host. In this work, PatB was exploited as a tool to incorporate bioorthogonal functionality on the carbohydrate backbone of the peptidoglycan by using a variety of donors, either p‐nitrophenol (pNP) or N‐acetylcysteamine (SNAc). Using tri‐N‐acetylglucosamine (GlcNAc)3 as a peptidoglycan mimic, a variety of functionalities were installed, including an alkyne and azide handle, on this mimic. Further studies in vitro showed that these modifications have a protective effect on isolated peptidoglycan from B. subtilis, E. coli, P. putida, and V. parahaemolyticus against lysozyme degradation. Intrigued by these results, an alkyne handle was subsequently installed on whole bacterial cells of B. subtilis using the SNAc alkyne derivative and the carbohydrate backbone was fluorescently labeled via click chemistry. Altering the peptidoglycan post‐synthetically with these derivatives will aid in the isolation and identification of immune stimulatory fragments from bacteria, as well as in the development of antibiotics and immune modulators.

Support or Funding Information

The Delaware COBRE and INBRE programs supported this project with a grant from the NIGMS (5 P30GM110758‐02, P20GM104316‐01A1 and P20GM103446) from the NIH.

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 Role of Sphingomyelin Synthase on the Healthspan of Organisms Via Modulating Neurotransmission
Authors: Chuh Steven Sung Hwan , Wentz Curtis , Chan Jason , Dries Daniel
Published: June 2021

Individuals are living longer due to medical interventions; however, simply increasing lifespan without proportionally increasing healthspan will result in longer periods of decrepitude. Healthspan is defined as the length of time before the onset of morbidity. One metric of healthspan that decreases as organisms age is neuronal function. To determine signaling molecules that may mediate healthy neuronal function, we examined neuromuscular function in the model organism C. elegans. In particular, we systematically analyzed a class of lipids called sphingolipids. Sphingolipids mediate for a variety of cellular signaling functions such as cell proliferation and apoptosis, but also involved in neurotransmission by interacting with neurotransmitter receptors and promoting neurotransmitter release. Sphingolipids are interconverted by metabolic enzymes that balance the levels of lipids within cells. We examined changes in neuromuscular function with age of C. elegans knockouts in several sphingolipid converting enzymes, including sphingomyelin (SM) synthases (sms‐1, sms‐3), ceramidase (asah‐2) and ceramide synthase double mutant (lagr‐1, hyl‐1). Sphingomyelin synthase converts ceramide to sphingomyelin. Additionally, ceramide synthase converts sphingosine to ceramide. We experimented with these mutants because both sphingomyelin and ceramide play important roles in the production of neurotransmitter receptors. To examine neuromuscular changes with age, we examined worm trashing behavior in 5, 10, and 15 day old worms. We found that mutants lacking sms‐1/SM synthase had decreased thrashing in comparison to wildtype worms at all ages. As stated, SM affects neurotransmission and development, but it is still unclear at which age window SM levels will have the most prominent effect. Therefore, we aim to further observe the role of SM during development and adulthood. We will expose wildtype and mutant worms with a pharmacological agent, D609 which inhibits sphingomyelin synthases. We will treat D609 specifically during development or adult time frames. Lastly, we wanted to further investigate this phenomena by investigating changes in aspects of synaptic vesicle cycling such as exocytosis and endocytosis. To do this, we will examine changes in the synapses of the neuromuscular junction (NMJ) by analyzing changes in GFP‐tagged synaptic markers with age and when exposed to the D609 drug treatments. These results will help identify how sphingomyelin processing enzymes might affect neurotransmission that may have a cascading effect on health span in aging animals.

Support or Funding Information

ASBMB

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Investigation into the Antibacterial Properties of Citrus and Plant Essential Oils
Authors: Axup Antonetta , Sostarecz Audra Goach
Published: June 2021

Langmuir Monolayers of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and E.coli lipid extract were used to investigate the antibacterial properties of essential oils, in particular sweet orange oil. DPPC served as the control membrane while DPPG served as the gram positive bacteria model and E.coli lipid extract was the gram negative bacteria model. The Langmuir Monolayer technique is useful for forming model cell membranes and allows for the analysis of the interactions between phospholipids and their molecular organization in the presence of another molecule. The main chemical in sweet orange oil, limonene, was found to disrupt phospholipid‐phospholipid interactions in all models except for DPPG simply because PG headgroups hydrogen bond with each other and there is no space for the hydrophobic limonene molecule. The largest disruption was observed for the E.coli extract lipids due to the unsaturated tails in these lipids resulting in less tight packing. Monolayers were seen in all three membrane systems to be destabilized as evidenced by the decrease in collapse pressure of the monolayer film but the E.coli lipid extract monolayer was the most affected in terms of fluidity as seen by the compression modulus. The antibacterial effects of orange oil on these systems is currently being compared to that of peppermint oil.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stainless Imaging To Identify The Biochemical changes during Bleomycin‐Induced Pulmonary Fibrosis By Fourier Transform Infrared Technique
Authors: Suryadevara Vidyani , Nazeer Shaiju Suhara , Sreedhar Hari , Natarajan Viswanathan , Walsh Micheal
Published: June 2021

Idiopathic pulmonary fibrosis (IPF) is a chronic and ultimately fatal pulmonary disease due to scarring of the lung tissue. While biopsies are the gold standard for diagnosis of IPF, there is definitive need for increased efforts towards identifying new molecular markers associated with the disease for efficient diagnosis. Fourier Transform Infrared imaging (FT‐IR) combines imaging at a spatial resolution that approaches light microscopy coupled with biochemical sensitivity of spectroscopy. In this study, bleomycin induced pulmonary fibrosis in rodents, a pre‐clinical model of IPF was used to detect the biochemical changes that occur during different stages of fibrosis. Based on the time point after bleomycin challenge, the animals were grouped as 1. Control; 2. Day 7 (Inflammation); 3. Day 14 (Onset of fibrosis); 4. Day 21 (fibrosis); 5. Day 28 (recovery phase). FT‐IR images were acquired from the lung tissues in each group in reflectance mode using an Agilent Cary 600 Series FT‐IR spectrometer. The spectral profile across the entire lung was obtained and pre‐processed for further analysis using multivariate data analysis. Non‐hierarchical k‐means cluster analysis of the spectrum resulted in delineation of the entire lung tissue based on the biochemical changes into different clusters that reflects the histological changes in the tissue. Further, specific changes in the biochemical composition of the fibrotic and non‐fibrotic regions in the lung during various stages of fibrosis was quantified by the changes in the spectral ratios of the biomolecules of interest. PCA‐LDA analysis could score the probability of occurrence of fibrosis based on the spectral changes. FT‐IR imaging will be a potentially powerful clinical tool, giving new insights into the biochemistry of tissue in a label‐free and non‐perturbing manner.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resolving the structure of metallochaperone AztD
Authors: Fullam Stephanie , Neupane Durga , Yukl Erik
Published: June 2021

Zinc is an essential nutrient, and proteins mediating zinc homeostasis are important virulence factors among pathogenic bacteria, making them attractive targets for the development of novel antibiotics. Of particular interest are the ATP binding cassette (ABC) transporters, which utilize extracellular solute binding proteins (SBPs) to bind zinc with high affinity and specificity for delivery into the cell via integral membrane permeases. We are studying the zinc import machinery of Paracoccus denitrificans as a model for highly homologous systems in pathogenic bacteria including the carbapenem resistant enterobacteraceae (CRE) species Klebsiella pneumoniae and Citrobacter koseri. Specifically, these species encode an unusual zinc ABC transporter operon aztABCD that encodes a periplasmic metallochaperone (AztD) that transfers zinc specifically to the SBP (AztC). The structure for PdAztC has been solved and features a long, protruding loop containing three His residues. Deletion of the loop or mutation of any of the His residues to Ala completely abrogated zinc transfer from AztD but had no impact on metal binding from solution. From this data, we hypothesized that the loop must be required to access the zinc binding site in AztD, chelate the metal and return it to the AztC binding site. However, without structural information on the AztD chaperone the details of this mechanism remained unclear. We have recently solved high‐resolution crystal structures of AztD from both P. denitrificans and C. koseri using single wavelength anomalous dispersion (SAD) on a lead derivative crystal. This was necessary as there were no homologous of AztD in the protein databank. The protein is a 7‐bladed beta propeller, with zinc bound to three conserved His residues in the central pore. A second zinc binding site was also identified, consistent with solution data indicating that AztD can bind up to three zinc ions. A detailed mechanism for zinc transfer from the primary AztD binding site to AztC is proposed based on the existing solution data, new structures, accessibility of the AztD zinc binding site and electrostatic complementarity between AztD and AztC surfaces. Given the importance of dynamic zinc transfer for bacterial zinc homeostasis and the role of this process in pathogenesis, high‐resolution structural data on zinc binding and transport proteins may be very useful in the development of novel antibiotics for resistant bacteria such as C. koseri.

Support or Funding Information

National Institutes of Health under award number 1SC2GM111170‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Atherogenic diet induced cardiovascular disease and bone loss in ApoE knockout mice
Authors: Cheng Wei‐Yuan , Lan Ying‐Wei , Chen Hsiao‐Ling , Chen Chuan‐Mu
Published: June 2021

Cardiovascular diseases and bone loss are chronic degenerative diseases which have been considered to be independent pathway. Hyperlipidemia is the dominant risk of cardiovascular disease that caused atherosclerosis, seriously, it will cause vascular calcification. In recent years, growing evidences indicated that the hyperlipidemia also be a risk factor of bone loss which leads to an increased risk of bone fracture. In clinical studies, aortic calcification often exists in patients with osteoporosis, independently of age. Apolipoprotein E (ApoE) is a class of proteins mediated cholesterol metabolism in the body, which plays an important role in cardiovascular disease. The aim of this research is using the ApoE knockout mice to investigate the correlation of cardiovascular disease and bone loss under atherogenic diet treatment. In this study, B6:129P2‐Apoetm1Unc/J knockout mice were used as experimental animal that exhibiting lipid metabolism dysfunction. Mice were divided into two groups (n=6) and placed on either a chow diet or a atherogenic diet for 13 weeks. C57BL/6 mice at the same age were used as a wild‐type normal control. The analysis data were collected including blood chemistry, pathological section, and micro‐computed tomography (μ‐CT). In the blood biochemical examinations, result showed that total cholesterol (TC), oxidized low‐density lipoprotein (Ox‐LDL), calcium content, and carboxyl‐terminal cross‐linking telopeptides of type I collagen (CTX‐1) were significantly increased in atherogenic diet‐induced ApoE KO mice group; however, procollagen type 1 amino‐terminal propeptide (P1NP) was significantly decreased compared to chow diet‐fed ApoE KO mice group. Histopathological results revealed that the severe atherosclerosis and developing vascular calcification in the atherogenic diet‐induced ApoE KO mice group. In the analyses of femurs trabecular bone parameters, bone volume/tissue volume (BV/TV), trabecular separation (Tb.Sp), trabecular thickness (Tb.Th), trabecular number (Tb.N) and trabecular bone mineral density (Tb.BMD) were obviously lost in the atherogenic diet‐induced ApoE KO mice group. In conclusion, we have successfully used the atherogenic diet to induce cardiovascular disease and bone loss in ApoE knockout mice. This study provides a platform to further investigate common pathophysiologic mechanism between cardiovascular disease and bone loss. It also provides a drug screening animal model test the prevention or therapy at both conditions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deficiency of Lysosomal Ceramide Hydrolysis Contributes to Enhanced Exosome Release and Calcification in Coronary Artery Myocytes
Authors: Yuan Xinxu , Bhat Owais M. , Lohner Hannah , Zhang Qinghua , Zhang Yang , Boini Krishna , Li Pin‐Lan
Published: June 2021

Ceramide has been shown to contribute to vascular calcification under different pathological conditions. However, the precise mechanism mediating this process remains unknown. The present study investigated the role of lysosomal ceramide metabolism via acid ceramidase (AC) in the regulation of exosome excretion in coronary artery myocytes (CAMs) and the possible implication in the development of calcification. In cultured CAMs from smooth muscle (SM)‐specific Asah1 (mouse code of AC gene) knock‐out mice (Asah1fl/fl/SMcre), we observed that deletion of Asah1 gene increased lysosomal ceramide level compared to their control littermates, as detected by immunostaining of ceramide co‐localized with lysosome marker lamp‐1. Treatment of CAMs from wild type mice produced similar effects to reduce lysosomal ceramide given its effect to inhibit AC activity. Alizarin Red staining showed that more remarkable calcium deposition or calcification by 1.9 folds occurred in cultured CAMs from Asah1fl/fl/SMcre mice compared to cells from their control littermates, if these cells were treated with 3 mM NaH2PO3 and atherogenic stimulator, 7‐ket. Correspondingly, CAMs from Asah1fl/fl/SMcre mice were found to have significant phenotype transition as shown by Western blot analysis, increased expression of synthetic and osteogenic markers (more vimentin and osteopontin) and decreased contractile phenotypic marker (less SM22α), and this synthetic or osteogenic phenotype was enhanced by 7‐ket treatment. Using super‐resolution imaging with Structured Illumination Microscopy (SR‐SIM), we found that 7‐ket markedly reduced colocalization of Rab‐7a (multivesicular body (MVB) marker) with lamp‐1 in 7‐ket‐treated WT CAMs by 3 folds or CAMs from Asah1fl/fl/SMcre mice by 2.5 folds, suggesting inhibition of MVB‐lysosome encountering or fusion during inhibition of AC expression and activity. Using nanoparticle tracking analysis, 7‐ket‐induced excretion of exosomes (80–140 nm in size) was found to be significantly increased in CAMs of Asah1fl/fl/SMcre mice by 2.1 folds compared to CAMs from their control littermates. These results suggest that AC‐mediated ceramide hydrolysis in lysosomes critically controls MVB fate and exosome release and that insufficient AC expression and activity may lead to enhancement of exosome release, contributing to calcium deposition or calcification in CAMs or arterial media.

Support or Funding Information

(supported by NIH grants HL057244, HL075316 and HL122937).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Disease‐associated mutation in PTH reveals molecular mechanisms in endosomal GPCR signaling
Authors: Jean‐Alphonse Frederic , Sutkeviciute Ieva , Xiao Kunhong , Vilardaga Jean‐Pierre
Published: June 2021

Parathyroid hormone (PTH) and its related peptide (PTHrP) activate PTH receptor (PTHR) signaling, but only PTH sustains GS–mediated cAMP production after PTHR internalization into early endosomes. The physiological relevance of this unexpected behavior for PTHR is unclear. Here we present evidence that endosomal PTHR signaling through cAMP also has disease relevance. We demonstrated that R25C mutation recently identified in PTH as a new cause of hypocalcemia in humans affects receptor binding at endosomal pH resulting in rapid ligand dissociation and recycling of PTHR preventing sustained endosomal cAMP signaling. Using hydrogen–deuterium exchange coupled with mass spectrometry (HDXMS) to map structural changes of PTHR upon PTH binding, we identified acidic clusters in the receptor's first extracellular loop as key determinants for Arg25 binding and endosomal cAMP signaling. Determination of the dynamic of protein‐protein interaction using FRET approaches revealed that PTH R25C has a lower potency to form and stabilize the functional complex between β‐Arrestin, Gβγ and PTHR required for endosomal cAMP generation. Overall, these new findings indicate that PTH‐induced endosomal cAMP signaling is likely a primary signaling component regulating calcium homeostasis.

Support or Funding Information

NIH R01‐DK102495 and R01‐DK087688 National Research Foundation of Korea NRF‐2013R1A1A1A05005629

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Live‐cell super‐resolution imaging of PKA activity reveals microdomain organization and regulation
Authors: MO Gary , Zhang Jin
Published: June 2021

Compartmentalized biochemical activities are crucial to many signaling processes in the cell. However, it has been difficult to visualize dynamic protein activities in living cells at the super‐resolution required to see their sub‐diffraction‐limit spatial regulation. Recently, we introduced a novel class of fluorescent biosensors that can detect biochemical activities in living cells at a resolution up to three‐fold better than the diffraction limit. Harnessing a specific binding‐induced change in protein fluorescence dynamics called Fluorescent fLuctuation INcrease by Contact (FLINC), these biosensors translate kinase activities or protein‐protein interactions into changes in fluorescence fluctuations. These fluctuations can then be quantified through Stochastic Optical Fluctuation Imaging (SOFI), permitting us to follow the protein activity dynamics, over tens of minutes, at super‐resolution. Utilizing one such biosensor for Protein Kinase A (PKA), we resolved minute activity microdomains on the plasma membrane of living cells, and discovered the role of clustered anchoring proteins in organizing these active PKA microdomains. We further demonstrate a new version of FLINC – REmoved Dronpa FLINC (red‐FLINC) – and use it to contextualize the PKA activity microdomains with the location of an additional protein. With red‐FLINC, live cell super‐resolution activity imaging requires only a single channel of the visible spectrum, freeing the others for imaging other proteins, or perturbing their activities with optogenetics. Using this technology. we determined the spatial relationship between membrane PKA activity microdomains and important participants in the PKA pathway such as AKAP79 and L‐type calcium channel. Our studies suggest that biochemical activities of the cell are spatially organized into an activity architecture.

Support or Funding Information

NIH DP1 CA174423, R35 CA197622 and R01 DK073368

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing the Activity of Synthetic Opioid AH‐7921 and U‐47700 Analogs in Cloned Human Mu‐Opioid Receptor Expressing Fibrosarcoma HT‐1080 Cells
Authors: Hsu Tom , Mallareddy Jayapal , Yoshida Kayla , Lee Tim , Kemboi Edna , Park Emily , Krstenansky John , Zambon Alexander
Published: June 2021

While important for their antinociceptive properties in medicine, opioids are commonly abused in America due to easily accessible opiate prescription pain medication. Classic opioids can induce adverse side effects such as gastrointestinal dysfunction and fatal respiratory depression, the latter of which is a key underlying factor contributing to the rising rates of lethality of opioid abuse. New synthetic opioids are emerging on the black market, many of which have not been characterized for structure activity relationships at human mu‐opiate receptors (hOPRM1). The rise of new synthetic opioids complicates the opioid crisis in America as they often escape the traditional illicit drug classifications established by law enforcement and may be more potent and lethal than conventional molecules. In this study, we developed a stable amino terminal HA‐tagged hOPRM1 expressing human fibrosarcoma (HT1080) cell line for determining the pharmacology of a panel of opioids analogs derived from synthetic opioids AH‐7921 and U‐47700. Expression of hOPRM1 was verified by Western blot and immunocytochemistry. Assessing the morphine response in this cell line revealed that morphine EC50 (39.3 nM) was comparable to published findings and were naloxone reversible, thereby validating this cell line. The analogs were assessed for their ability to suppress forskolin‐induced cyclic AMP (cAMP) levels in a hOPRM1‐dependent manner revealing a subset of structurally‐related analogs showing functional activity. The preliminary results found in this study indicate that some of the synthetic opioid analogs are likely to have abuse potential based on their ability to bind to and activate the human OPRM1 receptor.

Support or Funding Information

This project was supported by Award No. 2016‐R2‐CX‐0059, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication/program/exhibition are those of the author(s) and do not necessarily reflect those of the Department of Justice.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alpha‐2c Adrenergic Receptor Promotes the Malignant Phenotype of Colon Cancer Cells
Authors: Njeim Rachel , Eid Ali
Published: June 2021

Cancer remains one of the leading causes of death worldwide, with colorectal cancer (CRC) being the third most common type. Accumulating evidence shows that stress plays a pivotal role in the malignant phenotype of CRC, by promoting hallmarks of CRC including cellular proliferation, migration, invasion, and angiogenesis. During stressful situations, the sympathetic nervous system is activated, causing an elevation in levels of catecholamines, including epinephrine which elicits its effects mainly by activating adrenoceptors. Interestingly, however, the role of alpha2C adrenergic receptor (a2c‐AR) in CRC malignancy remains obscure. Here, we proposed that activation of a2c‐AR potentiates the malignant phenotype of human CRC cells. Treatment of human colon cancer cell lines, SW480 and HT‐29, with increasing concentrations of a2‐AR specific agonist (UK 14,304; 10, 30 or 100 nM) for different time points (24, 48, or 72 hours) did not cause a significant change in proliferation. However, using monolayer scratch assay, a significant increase in migration was observed after treatment with UK 14,304 (100 nM) for 8 and 12 hours in both cell lines. This was further supported by a transwell migration assay where UK 14,304 increased cell migration. Pretreatment with a specific a2c‐AR antagonist (JP 1302; 100 nM) abolished the UK 14,304‐induced cell migration. This increase in migratory capacity was concomitant with increased Rho activation evident by translocation of Rho from cytoplasm to the membrane as well as via a Rhotekin‐binding activation assay. Moreover, treatment with UK 14,304 evoked actin reorganization, an element known to be a prerequisite for cell migration. The induced Rho activation and actin cytoskeletal rearrangement could be mediated by increased oxidative stress since treatment with UK 14,304 increased ROS production. Importantly, ROS is known to induce migration of these cells. Furthermore, adhesion of colon cancer cells to TNF‐alpha‐activated HUVECs was promoted by UK 14,304. This UK14,304‐induced adhesion was followed by a dramatic increase in transendothelial migration of SW480 through HUVECs, clearly indicating that a2c‐AR activation may promote intra‐ or extravasation. Because breaking the endothelial cell barrier requires matrix digestion, we measured the levels of matrix metalloproteinases (MMPs). Importantly, stimulation of a2c‐ARs by UK14,304 increased the production of MMP2 and MMP9, and JP 1302 abolished this UK14,304‐induced production. Taken together, our data suggests that a2c‐AR potentiates the migratory and invasive capacities and hence the malignant phenotype of human colon cancer cells. Therefore, a2c‐AR may represent a novel target in the treatment of CRC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering the Role of SLX4IP in Telomere Maintenance Mechanisms
Authors: Whited Tawna , Robinson Nathaniel , Grabowska Magdalena , Schiemann William , Taylor Derek
Published: June 2021

Telomeres are protective caps found at chromosomal ends that act to prevent attrition of the genome and aberrant activation of DNA repair mechanisms. In somatic cells, telomeric DNA progressively shortens with rounds of replication until a critically short length is reached triggering the cell to undergo senescence to prevent loss of genomic information. However, some cells express telomerase to elongate telomeres and avoid reaching this critical length, thus instilling additional replicative capacity. Notably, 85% of all cancer cells upregulate telomerase and the remaining 15% primarily utilize the alternative lengthening of telomeres (ALT) pathway which relies upon homologous recombination. There is evidence to suggest that cancer cells have the ability to switch to the ALT pathway, should telomerase activity be compromised, but the molecular switch regulating this process is unknown. A recently identified protein, SLX4IP, appears to play a critical role in telomere homeostasis and the telomerase‐to‐‐ALT conversion. In order to identify the basic function of SLX4IP in telomere homeostasis, the interactome of SLX4IP must be interrogated to identify additional proteins involved under specific conditions. For example, identifying differences between the interactomes in telomerase‐positive and ALT‐positive cells will provide insight regarding SLX4IP in telomere maintenance mechanisms. Initially, immunoprecipitation experiments were carried out to identify discrepancies in the proportion of known SLX4IP binding partners among telomerase‐positive and ALT‐positive cell lines. As expected, SLX4IP interacts with TRF2, a telomeric binding protein, and SLX4, an ALT‐associated nuclease scaffold protein, but with varying degrees among the cell types tested. To further dissect the interactome of SLX4IP, wild type SLX4IP, an N‐terminal mutant being interrogated for structural studies, and a C‐terminal mutant which represents a deletion mutant identified in acute lymphoblastic leukemia patients exhibiting poor prognosis were stably overexpressed in telomerase‐positive and ALT‐positive cell lines followed by subsequent immunoprecipitation and proteomic evaluation. This evaluation will provide valuable information as to which regions of SLX4IP are responsible for orchestrating particular interactions in each cell type. With this knowledge, a greater understanding of SLX4IP's role in telomere homeostasis and the telomerase‐to‐ALT conversion can be elucidated.

Support or Funding Information

This work was supported in part by an NIH T32 (GM008803) 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.
A Hydrogen Sulfide Donor, JK1, Preserves Endothelial Function and Improves Exercise Capacity in Pressure Overload ‐ Induced Heart Failure
Authors: Li Zhen , Carnal Jean , Kang Jianming , Xian Ming , Lefer David J.
Published: June 2021

Background and Hypothesis

Hydrogen sulfide (H2S) protects against heart failure by ameliorating oxidative stress, improving mitochondrial function, and attenuating apoptosis. At present there is a lack of data demonstrating the protective actions of H2S in the late stages of heart failure. And the effects of H2S on exercise intolerance, the clinical hallmark of heart failure, has not been evaluated in preclinical models. In current study, we tested the efficacy of delayed therapy with a novel H2S donor, JK1, on cardiac function, endothelial function, and exercise capacity in a murine model of pressure overload‐induced heart failure.

Methods

Male C57/BL6J mice at 10 weeks (wks) of age were subjected to transverse aortic constriction (TAC) for 18 weeks. JK1 (200 μg/kg/d, n=20) or the H2S deficient control compound (200 μg/kg/d, n=18) was administered via i.p. injection twice per day starting at 10 wks post TAC, in which time all mice had EF lower than 45%. Echocardiography was performed at baseline and tri‐weekly to assess LV structure and function. LV hemodynamics, cardiac fibrosis, vascular reactivity (isolated thoracic aorta), and exercise capacity were measured at the endpoint, 18 wks post TAC.

Results

Delayed treatment with JK1 initiated at 10 wks post TAC reduced LV dilation (p < 0.01) and attenuated eccentric hypertrophy (p < 0.01). However, no significant difference was noted in LV ejection fraction and cardiac fibrosis. JK1 treated mice displayed lower LV end‐diastolic pressure (LVEDP 17.8 ± 1.8 mmHg vs. 22.9 ± 1.23 mmHg, p < 0.05) and improved relaxation time constant (Tau 8 ± 0.27 ms vs. 10.05 ± 0.58 ms, p < 0.01). In addition, aortic vasorelaxation in response to acetylcholine treatment was markedly improved in mice received JK1 treatment (Max. Relaxation 51.8 ± 4.2% vs. 31.5 ± 3.6%, p < 0.01). More importantly, mice received JK1 treatment exhibited improved exercise capacity (Distance 312 ± 13.4 m vs. 89 ± 6.3 m, p < 0.05) as compared to those received control.

Conclusion

These results demonstrate that delayed administration of a novel H2S donor, JK1, improves exercise capacity in severe heart failure following TAC. JK1 treatment initiated at 10 weeks post TAC did not improve systolic function or reduce cardiac fibrosis, suggesting that the beneficial effects of 10‐wks‐delayed H2S therapy in pressure overload heart failure are a result of the improved vascular function and LV hemodynamics.

Support or Funding Information
AMP‐activated protein kinase activation by traditional herbal medicines and their active compounds ameliorates oxidative stress as mediated with liver kinase B1 and farnesoid X receptor
Authors: Baek Su Youn , Lee Eun Hye , Kim Young Woo
Published: June 2021

Background

Oxidative stress is a disorder in the oxidant‐antioxidant balance potentially leading to cellular damage in human. In the liver, oxidative stress stimulated by iron accumulation can induce a variety of chronic liver diseases, including liver fibrosis, cirrhosis and cancer. Oxidative stress resulting from iron accumulation may induce the release of arachidonic acid, and excessive amounts of arachidonic acid can cause high levels of cellular and mitochondrial ROS that reduce the functioning of mitochondrial respiration in the liver.

Methods

This study was designed to examine the effects of traditional herbal medicines and their active compounds against severe oxidative stress and mitochondrial impairment and its cellular mechanisms of action in vivo and in vitro.

Results

In cell experiments, the medicinal herbs such as Fructus Ligustri lucidi and Glycyrrihiza Radix effectively protected hepatocyte from arachidonic acid + iron‐induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of reactive oxygen species, increases in mitochondrial membrane permeability and altered expression of apoptosis‐related proteins, all of which are similar with aminoimidazole‐carboxamide ribonucletide treatment, as a positive control. In mice, The administration of the drug candidates also protected against oxidative stress and liver injury induced by carbon tetrachloride injection, as indicated by decreases in histological liver damage and serum alanine amino transferase level. Moreover, active compounds from the herbs also induced the activation of AMP‐activated protein kinase, which was mediated by its upstream kinase liver kinase B1 and farnesoid X receptor.

Conclusions

Our results confirmed that beneficial herbs have the ability to protect hepatocytes and liver against oxidative injury through regulation of the AMP‐activated protein kinase signaling pathway. This work was supported by the National Research Foundation of Korea grant funded by the Korea government [MSIP] (No. 2017R1D1A3B03027847) and (2015K1A3A1A59069800). All authors disclose no conflict of interests.

Support or Funding Information

This work was supported by the National Research Foundation of Korea grant funded by the Korea government [MSIP] (No. 2017R1D1A3B03027847) and (2015K1A3A1A59069800). All authors disclose no conflict of interests.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of Endothelial Nitric Oxide Production by the Sigma Receptor Agonist Afobazole
Authors: Motawe Zeinab Y. , Katnik Christopher P. , Trujillo Andrea N. , Cuevas Javier , Breslin Jerome W.
Published: June 2021

Afobazole is a sigma‐1 receptor agonist that has been shown to enhance survival in a rat mid‐cerebral artery occlusion stroke model. The mechanism has been previously attributed to an inhibition of ischemia‐induced calcium dysregulation that lowers glial cell loss and infarct volume. A recently published finding is that afobazole can stimulate nitric oxide (NO) production from lymphatic endothelial cells. In the current study, we investigated the ability of afobazole to relax arterioles from the brain and potential interactions between the sigma‐1 receptor and endothelial NO synthase (eNOS). Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. Afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM. The ability of afobazole to elicit NO production from cultured human brain microvascular endothelial cells (HBMEC) was assessed using 4‐Amino‐5‐Methylamino‐2′,7′‐Difluorofluorescein Diacetate (DAF‐FM) loading and fluorescence microscopy. Phosphorylation of eNOS on Ser‐1177 in the absence or presence of afobazole was evaluated by Western blotting. Lastly, intracellular localization of eNOS and the sigma‐1 receptor were evaluated by immunofluorescence and laser confocal microscopy. The results show that 200 and 250 μM afobazole elicited significant dilation of parenchymal arterioles. Afobazole also elicited elevated NO production from HBMEC in association with elevated phosphorylation of eNOS on Ser‐177. Lastly, eNOS and the sigma‐1 receptor were found partially colocalized in what appears to be the Golgi complex in confocal image stacks of HBMEC. This study shows for the first time a novel mechanism by which afobazole can relax brain arterioles via eNOS‐mediated NO production. This mechanism is likely associated with previously reported improvements of outcomes following stroke induced brain injury.

Support or Funding Information

Supported by NIH grant R01GM120774.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hyperglycemia Enhances Constriction of Retinal Venules to Endothelin‐1 via Activation of the Reverse‐Mode Sodium‐Calcium Exchanger
Authors: Chen Yen‐Lin , Xu Wenjuan , Rosa Robert H. , Kuo Lih , Hein Travis W.
Published: June 2021

The ocular level of vasoconstrictor endothelin‐1 (ET‐1) is elevated in diabetic retinopathy, which is associated with hyperglycemia and increased retinal vascular fluid filtration, a process heavily influenced by postcapillary pressure and venous constriction. However, the impact and mechanistic action of ET‐1 on venular vasomotor activity in the retina remain unclear. We addressed these issues by examining the contributions of ET‐1 receptor subtypes, extracellular calcium, and reverse‐mode sodium‐calcium exchanger (NCX) to ET‐1‐induced constriction of retinal venules during exposure to euglycemia and hyperglycemia. Retinal venules (<150 μm maximum diameter) were isolated from streptozocin‐induced diabetic pigs (2‐wk; chronic hyperglycemia: 456 ± 26 mg/dl) or age‐matched control pigs (euglycemia: 98 ± 3 mg/dl) and then cannulated and pressurized to 5 cmH2O for vasoreactivity study. For acute hyperglycemia study, vessels from euglycemic pigs were exposed intraluminally to high glucose (25 mM ≈ 450 mg/dl) for 2 hr, and normal glucose (5 mM ≈ 90 mg/dl) served as the control. Porcine retinal venules developed stable basal tone (constricted to about 90% of maximum diameter) and constricted to ET‐1 in a concentration‐dependent manner. This vasoconstriction was significantly increased after exposure to acute or chronic hyperglycemia and was abolished in the presence of ETA receptor antagonist BQ123 or extravascular calcium‐free solution, but not ETB receptor antagonist BQ788. The reverse‐mode NCX inhibitors SEA0400 and KB‐R7943 prevented the enhanced constriction of retinal venules to ET‐1 during chronic hyperglycemia, but they did not alter the ET‐1‐induced constriction of retinal venules during acute or chronic euglycemia. Furthermore, the mRNA levels of ETA receptors, ETB receptors, and NCX isoforms (NCX1, NCX2, and NCX3) in retinal venules were not influenced by chronic hyperglycemia. In conclusion, ET‐1 elicits ETA receptor‐dependent constriction of porcine retinal venules related to extracellular calcium entry. Although the calcium entry mode of NCX does not appear to contribute to venular constriction to ET‐1 during euglycemia, hyperglycemia enhances this vasoconstriction by activating the reverse‐mode NCX independent of changes in NCX and ET‐1 receptor mRNA expression levels. The ability of hyperglycemia to augment venular constriction to ET‐1 may elevate postcapillary pressure and promote retinal tissue edema during diabetic retinopathy. Blockade of reverse‐mode NCX in retinal venules may provide a new therapeutic strategy to mitigate this pathologic process.

Support or Funding Information

NIH Grants R01EY023335 and R01EY024624, Kruse Chair Endowment Fund, and Retina Research Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Association between Cerebrovascular Reactivity and Intravascular Cellular Activation in Postmenopausal Women Following Use of Menopausal Hormone Treatments
Authors: Pearson Andrew G. , Jayachandran Muthuvel , Lahr Brian D. , Joyner Michael J. , Miller Virginia M. , Barnes Jill N.
Published: June 2021

Cerebrovascular reactivity to hypercapnia, an estimate of microvascular responses in the brain, is increased during menopausal hormone treatment (MHT) in postmenopausal women. It is unclear whether this effect of MHT continues beyond the treatment period. We hypothesized that prior MHT use would not affect cerebrovascular reactivity 3 years after treatment. In addition, we have previously shown that cerebrovascular reactivity is associated with vascular activation in postmenopausal women with elevated cardiovascular risk. We hypothesized that cerebrovascular reactivity is not associated with markers of intravascular cellular activation in postmenopausal women with low cardiovascular risk who are within 10 years of menopause. Cerebrovascular reactivity to hypercapnia was measured in 58 postmenopausal women from the Kronos Early Estrogen Prevention Study at Mayo Clinic 3 years after the cessation of MHT [oral conjugated equine estrogen (oCEE), n=15; transdermal 17β‐estradiol (tE2, n=22); or placebo (PLA, n=23)]. Cerebrovascular reactivity was estimated from the middle cerebral artery flow velocity (MCAv) response to hypercapnia. Mean arterial blood pressure (MAP) was measured continuously throughout the protocol. Fasting blood was analyzed for a set of 14 activation markers of platelet and endothelial cells, inflammation and thrombogenicity. Principal components (PC) analysis was used to reduce these measures to fewer dimensions on which the associations of intravascular cellular activation with cerebrovascular reactivity could be based. Women were 60±3 years of age with a body mass index of 28±4 Kg/m2. MAP (99±14 mmHg in PLA, 97±7 mmHg in tE2, 98±9 mmHg in oCEE; p>0.90) and MCAv (60±14 cm/s in PLA, 68±20 cm/s in tE2, 71±18cm/s in oCEE; p>0.16) did not differ among groups. Cerebrovascular reactivity did not differ among women based on previous MHT randomization (1.2±0.7 cm/s/mmHg in PLA, 1.4±1.0 cm/s/mmHg in tE2 and 1.7±1.0 cm/s/mmHg in oCEE; p>0.17). The 14 markers of intravascular cellular activation were reduced to five PC that did not differ based on prior MHT assignment. There was no overall association of cellular activation PC with cerebrovascular reactivity. Effects of MHT on cerebral microvascular reactivity do not extend beyond the time of menopausal hormone treatment. Intravascular cellular activation does not alter cerebrovascular reactivity to hypercapnia in postmenopausal women with low cardiovascular risk profiles 3 years after the cessation of MHT.

Support or Funding Information

NIH grant P50 AG44170

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