bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteolytic Digestion Reveals Novel Insights into the Kv1.5 Structure‐Function Relationship
Authors: Hogan‐Cann Andrew , Zhang Shetuan 
Published: June 2021

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Kv1.5 channel mediated ultra‐rapidly activating delayed rectifier potassium current (IKur) is important for atrial repolarization. Since cardiac IKur is expressed solely in the atria, therapies that target Kv1.5 represent promising strategies for treating AF. Pathologies linked to AF, such as ischemia and hypoxia, have been reported to activate various proteases. It has been shown that cell‐surface Kv1.5 channels are sensitive to cleavage by extracellular proteases, such as proteinase K (PK). In this study, we investigated the consequences of proteolytic digestion on Kv1.5 channels. Our data demonstrate that extracellularly applied PK cleaves mature (75 kDa) Kv1.5 channels at a single locus in the S1‐S2 linker, yielding two digestion products of 42 and 33 kDa, corresponding to the N‐ and C‐terminal fragments, respectively. Surprisingly, whole‐cell patch clamp analysis showed that IKv1.5 was unaltered by the PK cut. Plasma membrane protein isolation via biotinylation indicated that both the N‐ and C‐terminal fragments following PK digestion retain cell‐surface expression over several hours. Co‐immunoprecipitation studies after PK cleavage indicate that the two fragments of the channel do not associate. The time‐dependent decline in the Kv1.5 fragment lacking the N‐terminus and the S1 domain paralleled the amplitude of recorded Kv1.5 currents. Our results suggest that Kv1.5 remains functional without the NH2‐S1 portion of the channel. The resistance of IKv1.5 to proteolytic cleavage may represent an inherent protective mechanism for Kv1.5 function. These findings extend our understanding of the Kv1.5 channel structure‐function relationship.Supported by CIHR & the Heart and Stroke Foundation
Cordycepin Inhibits LPS‐Induced Inflammatory Responses by Modulating NOD‐Like Receptor Protein 3 Inflammasome Activation
Authors: Yang Jing , Li Yunzhou , Zhang Luyong , Zhou Huiping 
Published: June 2021

The inflammasome is a multi‐protein complexthat induces maturation of inflammatory cytokines interleukin (IL)‐1β and IL‐18 through activation of caspase‐1. Activation of inflammasome contributes to host defense during diverse bacterial infections. But prolonged activation of inflammasome can be deleterious due to tissue damage associated with chronic inflammatory responses. Cordycepin, a natural compound and adenosine analogue derived from Cordyceps militaris, has been shown to have a great potential for therapeutic use against a number of inflammatory diseases. However, the molecular mechanisms by which cordycepin regulates inflammatory responses have not been clearly elucidated. The aim of this study is to examine the effects of cordycepin on LPS‐induced inflammatory response in macrophages and further investigate the underlying molecular mechanisms.MethodsCultured mouse RAW263.4 macrophages and human THP‐1‐derived macrophages were used in this study. The mRNA and protein levels of caspase‐1, IL‐1β, IL‐6, TNF‐α, MCP‐1, and COX‐2 were detected by real time RT‐PCR, ELISA and Western blot, respectively. The activation of NLRP3 inflammasome and ERK was determined by Western blot analysis. Results: Cordycepin significantly inhibited LPS‐induced expression of proinflammatory cytokines (IL‐1β, IL‐6, TNF‐α, MCP‐1) and COX‐2. LPS‐mediated NLRP3 and ERK activation was inhibited by cordycepin.ConclusionCordycepin prevents LPS‐induced inflammatory response through influencing NLRP3 inflammasome activation and inhibiting the ERK pathway.
InflammatoryCaspase‐1 Activation and Vegfr‐2 Signaling Regulate Each Other in Lipid Stimulated Endothelial Cells
Authors: Lopez‐Pastrana Jahaira , Ferrer Lucas , Cueto Ramon , Choi Eric , Wang Hong , Yang Xiao‐Feng 
Published: June 2021

BackgroundVascular inflammation is reported to be an antiangiogenic state, however, the underlying mechanism remains unknown. More specifically, the interplays between danger‐associated molecular pattern (DAMP) sensing in endothelial cell (EC), EC activation and EC growth are poorly defined. We tested a novel hypothesis that the caspase‐1 activation and VEGFR‐2 pathway regulate each other in inflammatory lipid stimulated EC.Methods and ResultsWe assayed caspase‐1 activity in human aortic endothelial cells (HAECs) stimulated with lysophosphatidylcholine (LPC). Using flow cytometry we analyzed the EC size, death, caspase‐1 activation and VEGFR‐2 pathway in LPC stimulated EC. We made the following findings: LPC increases caspase‐1 activity in larger size EC first that is associated with EC activation; and then in smaller size EC, which is associated with inflammatory cell death as judged by 7‐AAD staining. LPC decreases VEGFR‐2 expression in EC, whereas the inhibition of caspase‐1 increases the expression of VEGFR‐2 after LPC treatment in EC, which leads to an improvement in tube formation. The caspase‐1 deficient mice have an increase in the blood flow after hind limb ischemia presumably via enhancing angiogenesis.ConclusionOur results indicate for the first time that caspase‐1 inhibition increases VEGFR‐2 signaling pathway after lipid stimuli in EC. The interplays of inflammatory caspase‐1activation and VEGFR‐2 signaling pathway could be a novel therapeutic target for vascular inflammation and angiogenesis.
SUMO‐binding Sites in Mouse Nrf2: Impact on the Transcriptional Activity of Nrf2
Authors: Walters Treniqka , McIntosh Deneshia , Arinze Ifeanyi 
Published: June 2021

The transcription factor Nrf2 induces transcription by binding to the antioxidant response element(s) in target gene promoters, enabling oxidatively stressed cells to mount a response to oxidative stress in order to restore redox homeostasis. Recently, we showed that Nrf2 undergoes posttranslational modification by sumoylation after it is activated [Malloy et al. (2013) JBC 288, 14569‐14583], but the SUMO‐binding sites were not identified. Here, we have used three SUMO site‐predicting algorithms (SeeSUMO, SUMOsp and SUMOplot) to locate putative SUMO‐binding sites in mouse Nrf2. Three of the fifteen predicted sumoylation sites identified by these algorithms have high probability scores on all three prediction algorithms. Of these sites, only one (524LKDE527) matched the classical definition (ΨKXD/E) of a canonical SUMO‐binding site. Using site‐directed mutagenesis and in vitro sumoylation assays we show that this site is a bona fide SUMO‐binding site. Three other lysine residues (K53, K110, and K535) were also identified as SUMO‐acceptor residues. To test whether sumoylation of Nrf2 influenced transcription Nrf2‐dependent gene transcription, we mutated (K→R) some of the SUMO‐acceptor lysines. We found that mutations at K525, K535, and K591 resulted in impaired Nrf2‐dependent gene transcription, as measured by the luciferase reporter gene assay. To understand how sumoylation influenced transcription we assessed, using the Re‐ChIP (Active Motif, Carlsbad,CA) assay, whether sumoylation impacted the ability of Nrf2 to interact with its binding partners, specifically MafG, at the ARE.Supported by NIH grants SC1CA143985 and 5T32 GM07628‐34 and by CTSA award No. UL1TR000445.
Selective and Bioavailable Inhibitors of Collagen Prolyl 4‐Hydroxylase
Authors: Vasta James , Andersen Kristen , Raines Ronald 
Published: June 2021

Collagen is the most abundant protein in animals. A variety of diseases are associated with the overproduction of collagen including cancer metastasis. The stability of collagen relies on post‐translational modifications that occur throughout the secretory pathway. One of the most important modifications is the hydroxylation of collagen strands by collagen prolyl 4‐hydroxylases (CP4Hs). Catalysis by CP4Hs converts proline residues to hydroxyproline residues, which are essential for the conformational stability of mature collagen strands. Importantly, CP4Hs are validated targets for the treatment of metastatic breast cancer, a significant unmet clinical need. Nonetheless, a therapy based upon this strategy has yet to emerge, primarily due to the difficulty in designing selective inhibitors for members of this enzyme family. Herein, we use a chemical approach based upon the classic 2,2′‐bipyridine (bipy) scaffold to probe the active site of human CP4H1 and understand the determinants of selectivity for this enzyme. Then, we redesign the bipy scaffold to enhance its pharmacological properties and generate potent biheteroaryl inhibitors that are selective for human CP4H1. Evaluations of these inhibitors in cell culture suggest that they are bioavailable and inhibit collagen synthesis at concentrations that do not disrupt iron homeostasis or cause general cytotoxicity. These results suggest the potential use of these inhibitors as leads for the development of antimetastatic therapeutics.This work was supported by grant R01 AR044276 (NIH).
Exposure to Phosphodiesterase Inhibitors Impairs Physiological Processes in Juvenile Nematodes
Authors: Schuster Kevin , Danquah Wiseborn , Williamson Valerie , Cote Rick 
Published: June 2021

Meloidogyne spp. (root‐knot nematodes) are the most damaging plant‐parasitic nematodes to commercial agriculture. Currently used nematicides are of limited effectiveness as well as toxic to other organisms. The present study examines whether disruption of cyclic nucleotide signaling pathways using phosphodiesterase (PDE) inhibitors could serve as the basis for novel nematicides with greater efficacy and reduced environmental impact. To explore this, we carried out an evolutionary analysis of nematode phosphodiesterases (PDEs) and found that nematodes contain only 6 of the 11 members of the vertebrate PDE superfamily. Evolutionary trace analysis reveals nematode‐specific amino acid residues in the drug binding pocket of several nematode PDEs, suggesting the feasibility of developing compounds that selectively inhibit individual nematode PDEs. We also examined the physiological effects of PDE inhibitors on both C. elegans and M. hapla. We found that IBMX (non‐selective), cilostazol and milrinone (PDE3‐selective), rolipram (PDE4‐selective), and papaverine (PDE10 inhibitor) can reduce C. elegans dauer locomotion velocity by 30‐50%. IBMX, cilostazol, and rolipram also reduce by 50‐70% the chemotactic response of C. elegans to salt. Treatment of juvenile M. hapla with cilostazol or rolipram reduced by 蠅50% their ability to invade plant roots. We conclude that inhibitors that target individual PDE families present in nematodes can disrupt locomotion, chemosensation, and/or processes required to invade plant roots, and thus may have nematicidal applications.This work was supported by the New Hampshire Agricultural Experiment Station (USDA).
Gemcitabine is a Competitive Inhibitor of Cytidine‐5′‐triphosphate Synthase (CTPS) that Induces Enzyme Filament Polymerization
Authors: McCluskey Gregory , Bearne Stephen 
Published: June 2021

CTP plays a central role in nucleotide and lipid biosynthesis and is synthesized de novo by the catalytic action of CTP synthase (CTPS). CTPS is an important modulator of lymphocyte proliferation and has been identified as a potential therapeutic target for the treatment of acute lymphoblastic leukemia. The cytidine analogue gemcitabine is used to treat various cancers by inhibiting DNA polymerase, and also depleting intracellular CTP levels. While depletion of CTP levels suggested that gemcitabine inhibits CTPS activity in vivo, in vitro characterization of the inhibition is lacking. We demonstrate that gemcitabine is a potent competitive inhibitor of CTPS, binding with an affinity that exceeds that exhibited for UTP by 100‐fold. Site‐directed mutagenesis (E149D) studies reveal that a conserved glutamate (E149) is necessary for gemcitabine recognition, and this residue is also required for catalyzing the conversion of UTP to CTP. CTPS polymerization is an additional mode of inhibition where filament formation apparently prevents conformational changes conducive to enzymatic activity. Using electron microscopy, we detected CTPS filaments in gemcitabine‐treated samples of wild‐type enzyme, but not in E149D CTPS which cannot bind gemcitabine. These studies highlight the critical role that E149 plays in catalysis and the conformational changes of the enzyme.Supported by the Canadian Institutes for Health Research
Isoform specificity and phosphorylation site specificity for 14‐3‐3 protein binding to tyrosine hydroxylase studied by fluorescence anisotropy and chemical cross‐linking
Authors: Daubner Susan Colette , Vogt Ellysa , Cruz Eduardo , Rodriguez Augustin 
Published: June 2021

14‐3‐3 proteins are chaperone proteins that bind phosphorylated proteins and affect their activity or stability. Tyrosine hydroxylase (TyrH) is reported to be activated by 14‐3‐3. TyrH is phosphorylated at serine19 by calmodulin‐dependent protein kinase and by cAMP‐dependent protein kinase (PKA) in vivo. A variant of TyrH was constructed to enable phosphorylation of both serines in vitro by PKA, plus a TyrH variant to limit phosphorylation by PKA to serine19. Therefore TyrH phosphorylated at only serine19, only serine40, at both, or neither, has been studied. Binding between 14‐3‐3 and TyrH using chemical cross‐linking and fluorescence anisotropy will be reported. The TyrH forms variants were phosphorylated and cross‐linked to two of the 14‐3‐3 isoforms, eta and zeta via dimethylpimelimidate protein chemistry. Further, fluorescence polarization anisotropy of dansylated 14‐3‐3zeta has been measured in the presence and absence of these TyrH phosphorylated forms.Anisotropy of dansylated 14‐3‐3zeta is increased upon the addition of TyrH even if unphosphorylated, but it increases more if the enzyme is phosphorylated at serine40, and even more if phosphorylated at serine19. The greatest increase in anisotropy is seen if TyrH is phosphorylated at both positions.The chemical cross‐linking of the same forms of TyrH with 14‐3‐3 suggests that double phosphorylation results in a tighter complex than single phosphorylation at serine19. Cross‐linking between doubly‐phosphorylated TyrH and 14‐3‐3eta is tighter than with 14‐3‐3zeta. These results clearly suggest a 14‐3‐3 isoform specificity for TyrH binding.
Spatiotemporal Analysis of the Role that Protein Kinase A Phosphorylation Plays In Gap Junction Assembly
Authors: Sosinsky Gina , Cavin Gabriel , Solan Joell , Kunkel Maya , Newton Alexandra , Lampe Paul , Cone Angela 
Published: June 2021

Gap junction (GJ) trafficking and intercellular communication are regulated through interactions of the GJ protein, connexin, with several kinases culminating in phosphorylation on specific serines or tyrosines in the connexin C‐terminus. Connexin43 (Cx43) is an important, ubiquitous and highly phosphorylated isoform present in many vertebrate organs. Activation of Protein Kinase A (PKA) causes increased trafficking of Cx43 to the plasma membrane hypothesized to occur by direct phosphorylation on S364/S365. Here, we imaged Cx43 tagged with a PKA phosphorylation FRET biosensor (Cx43‐AKAR3). A strong Cx43‐AKAR3 FRET ratio signal was found in the Golgi apparatus and not in the GJs. Phosphorylation in the Golgi increased after stimulation with forskolin and IBMX, potent stimulators of PKA activity. However, we found no overlap of the PKA FRET ratio signal with antibody labeling for phospho‐S365 before or after PKA stimulation. Inhibition of PKA with H89 decreased but does not abrogate the effect of forskolin/IBMX stimulation. However, treatment with this inhibitor after stimulation did not result in rapid dephosphorylation. Previously published work indicated that direct PKA phosphorylation of Cx43 is low, especially as compared with PKC or MAPK. Thus, PKA activation and localization was not consistent with direct S365 phosphorylation. Most likely, PKA phosphorylates another kinase that increases Cx43 trafficking to the plasma membrane and GJ assembly.Funding: GM072881 (GS), GM043154 & DK054441 (AN), GM055632 (PD) & GM103412 (NCMIR)
Investigation of Cysteamine as a Potential Antioxidant Supplement in TRX1 Yeast Mutants
Authors: Hendershot Allison , Mordan‐McCombs Sarah 
Published: June 2021

In 1957, the free radical theory of aging proposed that cellular aging and death are the result of accumulating oxygen‐derived free radicals within the cell. These free radicals, also referred to as reactive oxidative species (ROS), accrue as a result of mitochondrial metabolism and can damage DNA, lipids, and proteins. Studies suggest that ROS‐related damage is associated with diseases such as Alzheimer's and cancer. Cells combat the detrimental effects of ROS by producing endogenous antioxidants, such as the thiol antioxidant glutathione and its associated enzymes, which reduce free radicals to a more stable state. This research seeks to determine the relationship between endogenous and dietary antioxidants, using Saccharomyces cerevisiae as a model system. Here we investigate the role of the TRX1 protein and its involvement in the thioredoxin and glutaredoxin pathways, and the ability of the exogenous antioxidant, cysteamine, to interact with disrupted antioxidant signaling. We hypothesized that, due to the structural similarities between cysteamine and glutathione, cysteamine may be able to act as a surrogate in the glutaredoxin and thioredoxin pathways in the absence of functional glutathione. ΔTRX1 yeast strains were treated with cysteamine, and analyzed for growth, gene expression, and protein expression changes under oxidative stress from hydrogen peroxide. Results indicate alterations in endogenous antioxidant pathways in gene and protein expression levels with deletion of the TRX1 gene and subsequent treatment with cysteamine.This work is supported by the Biology Department at Franklin College and by a grant from the Indiana Academy of Science.
Temporal Selectivity of Melanocortin‐4 Receptor Agonist to Modulate Signaling by Increased Intracellular cAMP
Authors: Cooney Kimberly , Molden Brent , West Kirk , Baldini Giulia 
Published: June 2021

Melanocortin‐4 Receptor (MC4R) is a G‐protein coupled receptor expressed in the brain where it functions to regulate food intake and energy expenditure. MC4R signals in response to its natural agonist, α‐MSH, by increasing intracellular cAMP. It is not known whether synthetic MC4R agonists exhibit functional selectivity to modulate the cAMP signal. Effects by α‐MSH and two synthetic agonists, Melanotan II (MT‐II) and THIQ, were monitored using Neuro‐2A cells expressing both endogenous MC4R and exogenous tagged HA‐MC4R‐GFP, which can be visualized directly by fluorescence microscopy. By using Fluorescence Resonance Energy Transfer (FRET) we determined that α‐MSH induces a cAMP signal that disappears rapidly, within 15 min after agonist withdrawal. Conversely, MT‐II and THIQ induce a more prolonged cAMP signal, lasting for at least 40 min after agonist withdrawal. Internalization rate of ligand‐free MC4R and MC4R in a complex with a‐MSH and MT‐II, monitored by Fluorescence Recovery after Photobleaching (FRAP) occurred with the same half‐life of ~2.5 min. Together, these data indicate that MT‐II can induce a cAMP signal even after being internalized. Exposure to a‐MSH for 4 hours increased the distribution of MC4R to the intracellular localization more efficiently than MT‐II and THIQ, however desensitization of the receptor was the same for all agonists. We conclude that MC4R agonists have temporal selectivity to modulate the duration of the cAMP signal after agonist withdrawal, but not to change the desensitization properties of the receptor.NIH Grants R01‐DK080424 (to G. B.) and Intramural Funding Support from the UAMS College of Medicine Research Council
Serial Iron, Zinc and Copper Status in Adults with Pulmonary Tuberculosis in the Country of Georgia
Authors: Frediani Jennifer , Tukvadze Nestani , Sanikidze Ekaterina , Kipiani Maia , Hebbar Gautam , Millson Erin , Ramakrishnan Usha , Tangpricha Vin , Blumberg Henry , Ziegler Thomas 
Published: June 2021

ObjectiveTo determine serial dietary intake and serum concentrations of immune‐related micronutrients in patients with TB disease.Methods: Estimated mean daily intake of iron (Fe), zinc (Zn), and copper (Cu) was determined in 192 adults with pulmonary TB within 7 days of diagnosis and at 16 weeks using a validated instrument and NDS‐R. Serum levels of Fe, Zn and Cu were determined by inductively coupled argon plasma spectrometry at baseline and week 16.Results: Mean subject age was 34 y and 64% were male. Serum Fe levels were below normal in 25% at baseline (24±20 umol/L), and rose significantly by week 16 [to 26±15 umol/L (P< 0.01)]. Mean dietary Fe intake at baseline and week 16 was adequate. Serum Zn levels were below normal in 35% at baseline (11.6±2.8 umol/L) and 26% at week 16 (12.2±3.2 umol/L). Mean dietary Zn intake at baseline was 11.7±5.0 mg/day (121% of RDA)] and decreased at wk 16 (to 10±3.5 mg/day;P=0.03). Serum Cu levels were normal at baseline (26.5±0.6 umol/L) and fell significantly by week 16 to 20.5±0.5 umol/L (P<0.01). Mean dietary Cu intake at baseline and week 16 was adequate.ConclusionA significant proportion of adults with newly diagnosed TB exhibited low Fe and Zn levels, while Cu levels decreased over time. Changes during treatment may reflect diet intake, decreased inflammation and endogenous anti‐microbial processes.
Vitamin D Correlation with Testosterone Concentration in US Army Special Operations Personnel
Authors: Wentz Laurel , Berry‐Cabán Cristóbal , Eldred Jerad , Wu Qiang 
Published: June 2021

Vitamin D has been positively correlated with testosterone in older men, but these hormonal relationships have not been examined in younger, active military personnel. Therefore, the purpose of this study was to identify significant correlations between vitamin D and testosterone concentrations in male Special Operations Soldiers. This retrospective analysis examined unique cases of serum vitamin D assessments ordered at Womack Army Medical Center, Fort Bragg, NC, from January 2012 – September 2013. Inclusion criteria were male Special Operations soldiers who had a total testosterone assessment within 21 days of vitamin D assessment, yielding 312 service member records. Mean serum vitamin D concentrations were 29.9±9.6 ng/ml (range 10‐63), with 52% of subjects in the deficient range of <30 ng/ml according to the Army Medical Department guidelines. In vitamin D deficient subjects, serum vitamin D concentrations were a significant positive predictor of total testosterone when controlling for age and body mass index.

Independent Variable
Testosterone in Vitamin D Deficient Men (n=135)

β
Ρ

Vitamin D
8.429
0.008

Age
‐2.604
0.183

Body Mass Index
‐1.569
0.678

Model R2
0.062

These data indicate that deficient vitamin D concentrations may inhibit testosterone production and potentially limit human performance. Operational stress of military training has been shown to suppress testosterone concentrations in healthy men, causing muscle catabolism and fatigue, symptoms also characteristic of vitamin D deficiency. Given that more than half of male soldiers had insufficient vitamin D status in a southern latitude, measuring vitamin D concentrations in collaboration with testosterone assessments may be warranted.No funding was used to support this work.