bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fluorescence lifetimes as a monitor of conformational distributions in NOS
Authors: Umejiego Ezigbobiara N , Ghosh Dipak K , Hopper Benjamin L , Enweani Chioma M , Lester Army , Salerno John C 
Published: June 2021

The effects of calmodulin activation on the fluorescence of nitric oxide synthases are well established and parallel the activation of the enzyme by stimulation of electron transfer from NADPH to heme via the flavin containing domains. During this process, the FMN binding domain undergoes obligatory large scale conformational movements, resulting in the production of states in which the fluorescence lifetime of FMN, typically ~4.5 ns, is modified by interactions with FAD or heme, producing shorter lived states with lifetimes of ~90 ps or 1 ns.Changes in fluorescence have been observed by previous workers in response to calmodulin binding, but salt and chaotrope effects have also been observed. The enzyme has a pronounced dependence on ionic strength, characteristic of shuttle mechanisms, and some activities are greatly enhanced by chaotropes. Ionic strength has a relatively small effect on the distribution of lifetime states in the absence of calmodulin, consistent with the inability of salt to initiate NO synthesis or greatly enhance cytochrome c reduction in eNOS or nNOS. Chaotropes including guanidinium and urea, on the other hand, are effective at promoting the long lifetime states assigned to the free FMN domain (~4.3 ns) and the FMN‐oxygenase complex (1ns) at the expense of the short lifetime ‘input’ state in which FMN is in close association with FAD. The temperature dependence of the fluorescence decay indicates that increasing the temperature from 4C to 37C progressively favors the input state, in which the FMN binding domain has a large surface area of interaction with the FAD and NADPH binding domains. This suggests an entropy driven association in which reduction in size of a shell of ordered water provides the free energy.
Fatty acid elongase‐5 (Elovl5) regulates the mTORC2‐FoxO1 pathway in obese‐diabetic C57BL/6J mice
Authors: Tripathy Sasmita , Jump Donald B 
Published: June 2021

Elevated fatty acid elongase‐5 (Elovl5) activity in livers of high fat diet induced obese‐ diabetic C57BL/6J mice corrects fasting hyperglycemia by suppressing gluconeogenesis (GNG) through the regulation of Akt2 and FoxO1 phosphorylation status. This report examines Elovl5 regulated signaling mechanisms controlling Akt2 & FoxO1 phosphorylation. Elevated hepatic Elovl5 activity in obese‐diabetic mice and in HepG2 cells increased Akt2‐Serine473 (an mTORC2 site) phosphorylation. Increased Akt2‐Serine473 correlates with increased FoxO1 phosphorylation and suppressed Pck1 gene expression. In HepG2 cells, Akt inhibitor (Akt1/2i) blocked Elovl5 mediated FoxO1 phosphorylation while the PP2A inhibitor (mycrocystin‐LR) augmented FoxO1 phosphorylation. The mTORC1 and mTORC2 inhibitor (PP242), but not the mTORC1 inhibitor (rapamycin), blocked Elovl5 regulated Akt2‐Serine473 and FoxO1 phosphorylation. In vivo, high fat diets suppressed raptor (a mTORC1 component), but induced rictor (a mTORC2 component). Induction of hepatic Elovl5 activity augmented rictor protein abundance ~2‐fold and stimulated rictor‐mTor interaction. These studies establish rictor as a target for Elovl5 mediated suppression of GNG by regulating Akt2‐Serine473 and FoxO1 phosphorylation.Research support: NIH (DK43220) and NIFA/USDA (2009‐65200‐05846).
The Effects of the O‐xylosyltransferase (oxt) Mutation on Wg, Hh, and Dpp Signaling
Authors: Liu Alicia 
Published: June 2021

The o‐xylosyltransferase (oxt) encodes a glycosyltransferase needed for the first step of proteoglycan glycosaminoglycan (GAG) biosynthesis. Disruptions in heparan sulfate proteoglycan (HSPG) biosynthesis change the extracellular environment, which has been shown to block Wingless (Wg)/Wnt, Hedgehog (Hh), and Decapentaplegic (Dpp)/TGF‐β intercellular signaling. Therefore, mutations in oxt are predicted to disrupt normal Wg, Hh, and Dpp signaling due to the absence of heparan sulfate (HS) and chondroitan sulfate (CS) modified extracellular proteoglycans. Characterization of the oxt mutant in Drosophila wing discs shows this mutation has an unexpectedly mild phenotype as compared with other mutants acting further down in the HS biosynthetic pathway, such as sulfateless (sfl), which is responsible for specific downstream sulfation and activation of only HS chains. The double mutant oxt‐sfl was made to address why the oxt phenotype is less severe. Clonal analysis shows that the double mutant looks more like sfl, suggesting the oxt null mutation does not yield a fully penetrant phenotype, allowing some xylose addition to the core proteoglycan upon which sfl can act. Because the oxt mutation is a nonsense mutation that eliminates its active site, the lack of penetrance can be attributed to either perdurance of residual Oxt protein or mRNA in mutant cells.2P20RR01645 NIH/NCRR Administrative Supplement University of Delaware Summer Undergraduate Research Fellowship
Characterization of the EMT response in Crybb2Phil mutants
Authors: Roop Victoria Helen 
Published: June 2021

The most abundant protein in the adult lens fiber cells is βB2‐crystallin (Crybb2). Mutations in the Crybb2 gene cause cataracts in mice and humans. A twelve nucleotide deletion in mouse lens epithelial cells (LEC), known as the Crybb2Phil mutation, leads to cataracts associated with lens fiber cell defects as well as epithelial to mesenchymal transition (EMT) of the LEC in adulthood. However, the mechanism behind this phenotype is poorly understood. The aim of this project is to characterize the EMT response caused by the Crybb2Phil mutation.Tissues obtained from Crybb2Phil mutants were immunostained for alpha and beta integrin subunits, phospo‐SMAD3 (p‐SMAD3), transforming growth factor β‐induced (BIGH3), and EMT the markers E‐cadherin, vimentin, follistatin, and α‐smooth muscle actin (α‐SMA).Compared to the wildtype lens, β1, αV, and α5 integrins, vimentin, α‐SMA, and p‐SMAD3 were upregulated in the Crybb2Phil mice. No changes were seen in α2, α3, and α6 integrin subunits or follistatin. Consistent with EMT, E‐cadherin was downregulated in the Crybb2Phil mice.Upregulation of α‐SMA signifies that Crybb2Phil mice undergo EMT around two months of age. Furthermore, over expression of β1 and αV integrins along with p‐SMAD3 in the Crybb2Phil mutant suggests that the EMT seen in Crybb2Phil mice may be occurring via the TGFβ‐SMAD interacting pathway. This project is funded by National Eye Institute grant EY015279.
Silent scaffolds: inhibition of JNK3 activity in living cells by a dominant‐negative arrestin‐3 mutant
Authors: Gurevich Vsevolod V , Breitman Maya , Gimenez Luis E , Kook Seunghyi , Lizama Britney N , Palazzo Maria C , Gurevich Eugenia V 
Published: June 2021

We established a new in vivo arrestin‐3‐JNK3 interaction assay based on bioluminescence resonance energy transfer (BRET) between JNK3‐luciferase and Venus‐arrestins. We tested the ability of WT arrestin‐3 and its 3A mutant, both of which readily bind the β2‐adrenergic receptor, as well as two mutants impaired in receptor binding, Δ7 and KNC, to interact with JNK3 and to promote JNK3 phosphorylation in cells. Both receptor binding‐deficient mutants bind JNK3 significantly better than WT and 3A arrestin‐3. WT arrestin‐3 and the Δ7 mutant robustly promoted JNK3 activation, whereas the 3A and KNC mutants did not. Thus, receptor binding, JNK3 interaction, and JNK3 activation are three distinct functions of arrestins that can be independently manipulated by appropriate mutations. We found that the KNC mutant, which tightly binds JNK3 without facilitating its phosphorylation, functions as a dominant‐negative, competitively decreasing JNK3 activation by WT arrestin‐3. Thus, KNC is a silent scaffold, a novel type of molecular tool for selective suppression of MAPK signaling. These data provide the first demonstration that this mechanism of MAPK regulation can be used in living cells.NIH grants GM081756, GM077561, and EY011500 (VVG).
TCTP as a Regulator of Rheb: Impact on mTOR Signaling
Authors: Grabiak Mary , Cortez Jessica , Parmar Nitika 
Published: June 2021

TCTP (Translationally Controlled Tumor Protein) is a highly conserved protein that has been suggested as a tumor associated antigen as shown by its deregulation in many different types of cancers. TCTP has been shown to interact with Rheb, a key player of the PI3 Kinase/mTOR signal transduction cascade. This pathway regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. In this study the contribution of TCTP toward the mTOR pathway was investigated by over‐expression and gene silencing of TCTP in HEK293 and HeLa cells. The role of TCTP as a guanine nucleotide exchange factor (GEF) for Rheb has been controversial and this study highlights potential contribution of TCTP as a regulator of Rheb. Random mutant forms of TCTP were made and over‐expressed in cell lines. Additionally, cells were treated with multiple TCTP siRNAs in a dose and time dependent manner. The effects of TCTP over‐expression and silencing on mTOR signaling were monitored via analysis of S6 phosphorylation and cell viability assays. Results were compared to wild‐type TCTP expression and treatment with non‐specific siRNAs respectively. The impact of TCTP expression on cell viability under serum starved conditions was also explored.This work was supported by a Faculty Development Minigrant to Nitika Parmar.
Cellular localization of tenomodulin isoforms
Authors: Qi Jie , Dmochowski Jack , Banes Ashely , Tsuzaki Mari , Bynum Donald , Gomez Shawn , banes Albert J 
Published: June 2021

Tenomodulin, as a major tendon biomarker, TNMD is highly expressed in tendons. However, the functions of TNMD in tendons are not clear although it was suggested to play a role in anti‐angiogenesis in eye and its single nucleotide polymorphisms are associated with obesity. To investigate the function of TNMD in tendons, we performed studies on TNMD isoforms. Data mining showed that three different transcripts exist in human tissues, with deduced molecular weights of 37.1, 20.3 and 25.4 kDa. To clone TNMD isoforms, the cDNAs of human TNMD isoforms were amplified from human total RNA isolated from human tendon specimen and cloned into pEGFP‐C1 vector. GFP‐fused TNMDs were expressed in human and porcine tenocytes. Knockdown of TNMD expression was performed with a RNA interference technique. In the present study, we showed the existence of multiple Tnmd isoforms in tendons with Real time PCR and western blot. Knockdown of all TNMD isoforms with siRNA reduced cell proliferation. Each TNMD isoform showed different localization in tenocytes. Prediction of each isoform in I‐TASSER gave different functions, TNMD I was predicted to be a cytosine‐specific methyltransferase, TNMD II was classified as a SUMO protease, SENP1, and TNMD III was similar to á‐syntrophin. These results suggest that each Tnmd isoform may play distinct roles in tendons.This study was supported by Flexcell International Corp.
in vitro and in vivo investigation of modulators of hyperactivated ion channel induced necrosis in C. elegans
Authors: Kamat Shaunak , Bianchi Laura , Yeola Shrutika , Driscoll Monica 
Published: June 2021

Exposure of tissue to extreme temperature, corrosive chemicals, ischemia or other noxious stimuli can induce necrotic degeneration that is characterized by cellular swelling, loss of organellar integrity, accumulation of reactive oxygen species, activation of proteases and ultimately cell lysis. Necrosis can be genetically induced in C. elegans neurons by expression of mutant hyperactive ion channels. Mutant DEG/ENaC channels cause entry of excess calcium into the cell, triggering release of ER calcium stores and subsequent activation of calpain proteases. Due to the role of calcium in the induction and progression of this type of necrotic cell death we screened genes that encode calcium binding proteins and assayed for potential modulation of the cell death phenotype. RNAi and deletion mutant data indicates the involvement of EF hand containing proteins localized to ER and mitochondria. We will report on progress in deciphering their roles in neuronal necrosis.To extend mechanistic dissection, we have also been studying properties of hyperactivated ion channel‐induced necrosis in C. elegans embryonic cell culture and show that cultured neurons behave similarly to their counterparts in vivo. We have been pioneering the use of total internal reflection fluorescence microscopy to measure the MEC‐4/MEC‐10 channel at the neuron surface. We find changes in the surface expression and distribution of mechanotransduction complex proteins when necrosis is induced using a temperature‐sensitive mec‐4(d) allele. We are also studying the effects of mutant DEG/ENaC channels on mitochondrial morphology by imaging mitochondrially localized GFP in vitro and in vivo, as data suggest a relationship between maintenance of mitochondrial integrity and susceptibility to necrotic stimuli.
Endogenous and Psychostimulant Substrates but not Blockers Stimulate Dopamine Transporter Phosphorylation at a Proline‐Directed Site
Authors: ChallaSivaKanaka Sathyavathi , Foster James D , Vaughan Roxanne A 
Published: June 2021

The dopamine transporter (DAT) clears extraneuronal dopamine (DA) and thus controls the spatio‐temporal dynamics of dopaminergic neurotransmission. DAT is the major target for psychostimulants such as cocaine (COC) and amphetamine (AMPH). DAT has a serine cluster on the distal N‐terminus that is phosphorylated in a PKC‐dependent manner and a proline‐directed site, Thr (T) 53, that is phosphorylated in vitro by the MAP kinases. COC and AMPH impact DAT regulatory properties including uptake activity and surface expression. Although, the mechanism of drug action on DAT is unclear, DAT phosphorylation is associated with altered surface expression and DAT activity. In this study, we examined the effect of DA and several psychostimulant drugs on the phosphorylation of DAT using a newly developed phospho‐specific antibody that detects pT53. Immunoblotting revealed that DAT substrates led to increased DAT T53 phosphorylation. This increase was transport or binding dependent as COC blocked the AMPH effect. In rat striatal synaptosomes METH‐stimulated DAT T53 phosphorylation was rapid, occurring within 60 sec. In contrast, uptake blockers did not influence T53 phosphorylation. Because phosphorylation at proline‐directed sites causes cis‐isomerization of the protein backbone, our findings indicate that translocation or binding of substrates induces major structural rearrangements of the membrane‐proximal N‐terminus. We hypothesize that substrate‐mediated phosphorylation‐dependent structural rearrangement resulting from prolyl cis‐isomerization could thus act as a mechanism affecting DAT function.Supported by NIDA R01 DA 13147‐09
Development of biosensor for serine/threonine kinases
Authors: Lee Jhao-hong , Yuan Chiun-Jye 
Published: June 2021

Protein serine/threonine kinase has several important functions in cells, including regulation of glycogen, sugar and lipid metabolism. Many human diseases are found to be associated with the dysregulation of various protein serine/threonine kinases. There are several disadvantages of conventional methods for the mearurement of the activity of serine/threonine kinase, such as requirement of specific instruments, expensive, labor intensive and high risk of radioactive contamination. In this study, an impedance‐based biosensor was proposed for the detection of protein serine/threonine kinases was proposed and developed. Accordingly, the PKA (Protein kinase A) activity could be detected by seting the voltage at non‐redox potentials and record the impedance of electrode surface under different frequency. The peptide substrates of PKA (Protein kinase A) were coated on the electrode surface (working electrode with a diameter of 0.4 μm). After PKA phosphorylation at specific amino acid residue(s) of the substrate, the net impedance difference between non‐phosphorylated and phosphorylated peptide was determined. The results obtained in this study suggest that the activity of protein serine/threonine kinase can be detected by the developed electrochemical biosensor.Financial supported by NSC project NSC 100‐2311‐B‐009 ‐001
Functional validation of human pigmentation SNPs in zebrafish
Authors: Tsetskhladze Zurab , Canfield Victor A. , Copper Jean , Johnson Stephen L. , Kawakami Koichi , Cheng Keith C. 
Published: June 2021

Pigmentation is a quantitative and polygenic trait with high diversity within and between human populations. Two coding single nucleotide polymorphisms (SNPs), A111T in SLC24A5 and L374F in SLC45A2 have been linked to normal pigmentation variation in European populations, and one SNP ‐ E272K in SLC45A2 has been potentially linked to the East Asian skin color. To directly test the effect of each of these polymorphisms on pigmentation, we used a direct functional assay based on mutant rescue in the zebrafish. For this assay, we first identified slc45a2 as the mutated gene in zebrafish albino mutants. Zebrafish mRNA containing mutant zebrafish allele did not rescue the mutant phenotype, while wild‐type slc45a2 mRNA did. In this study we describe how we were able to ascertain that A111T and L374F mutations appear to be hypomorphic mutations and that the E272K mutation is not. The method may also be applicable to the analysis of other polymorphisms linked to human phenotypes by genome‐wide association studies or DNA sequencing.NIH funding: 5R01 AR052535
Effect of propionylcarnitine on mitochondrial energy metabolism in elderly rat heart
Authors: Kerner Janos , Virmani Ashraf , Koverech Aleardo , Hoppel Charles 
Published: June 2021

Background and objectivePropionyl‐L‐carnitine (PLC) has been shown to have an anaplerotic effect in rat heart. The goal of the present study was to test if PLC attenuates the age‐associated decreased myocardial carnitine content and improves mitochondrial oxidative phosphorylation.Approach and methodsTo achieve our objective we isolated subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) from hearts of 24 month old elderly Fisher 344 rats injected intraperitoneally with PLC or saline. We determined (a) the myocardial carnitine and acylcarnitine contents; (b) the oxidative capacity of isolated mitochondria with non‐lipid and lipid substrates; and (c) the activity of electron transport chain complexes.Results(a) Exogenous PLC is readily taken up and metabolized by the heart as indicated by the increased carnitine and acetylcarnitine content. (b) PLC has no effect on oxidative phosphorylation as assessed by unaltered oxygen consumption with isolated mitochondria. (c) PLC significantly increased the cytochrome oxidase (COX) activity in both mitochondrial populations as reflected by the increased first order rate constant.ConclusionThe data demonstrate that exogenous PLC corrects the age‐associated loss of myocardial carnitine and selectively and significantly increases COX activity.Supported in part by POI AG15885 and a gift from RostaQuo S.p.A..
Cholecalciferol increases 7‐dehydrocholesterol reductase activity in adult human epidermal keratinocytes
Authors: Zou Ling , Porter Todd D 
Published: June 2021

7‐Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis by reducing the 7–8 double bond of 7‐dehydrocholesterol. In skin cells 7‐dehydrocholesterol also can be converted to cholecalciferol (vitamin D3) by ultraviolet radiation, providing a second pathway for 7‐dehydrocholesterol metabolism in these cells. We hypothesized that keratinocytes might regulate cholecalciferol levels by modulating the activity of DHCR7, a hypothesis supported by evidence that DHCR7 is a phosphoprotein, and is activated by the addition of ATP to liver microsomes.To test this hypothesis, DHCR7 activity was measured in lysates of rat hepatoma cells (McA‐RH7777), immortalized human keratinocytes (HaCaT), and adult human epidermal keratinocytes (HEKa) after treatment of the cells with cholecalciferol or calcitriol for 3 h. Cholecalciferol had no effect on DHCR7 activity in hepatoma cells or HaCaT cells, but increased DHCR7 activity in HEKa cells by 3‐fold at 1 μM and 4‐fold at 10 μM. Calcitriol (100 nM), the active form of vitamin D, had no effect on DHCR7 activity in HaCaT cells at 3 or 24 h, but decreased DHCR7 activity in HEKa cells after 3 h by 40%.These findings suggest that cholecalciferol regulates its own levels in keratinocytes by increasing the activity of DHCR7, thereby decreasing the levels of the cholecalciferol precursor, 7‐dehydrocholesterol.
Apoptotic signaling of the Amblyomin‐X involves endoplasmic reticulum stress, cell cycle regulation and survival pathways
Authors: Chudzinski-Tavassi Ana Marisa , Morais Kátia Luciano Pereira , Souza Jean Gabriel , Simons Simone Michaela , Berra Carolina Maria , Lameu Claudiana 
Published: June 2021

The Amblyomin‐X, a Kunitz‐type inhibitor obtained in a recombinant protein form, promotes apoptosis in murine renal adenocarcinoma (RENCA), decreased proteasomal activity and increased pool of poly‐ubiquitinylated proteins in some tumor cell lines, suggesting an endoplasmic reticulum (ER)‐stress, in addition to D1 cyclin expression decrease. However, the mechanistic effects of this protein are still unclear.ObjectiveEvaluate the involvement of ER‐stress, cell cycle regulation and/or survival in tumor cells treated or not with Amblyomin‐X.MethodsThe genic expression was perfomed by ABI 7500 Real Time PCR System (Life Technologies) using forward and reverse specific genes primers. The intracellular calcium mobilization was measured by confocal microscope and microfluorometry.ResultsRENCA cells treated with Amblyomin‐X showed significant differences in genes related to ER‐stress and cell cycle regulation, as observed for caspase‐12, which showed increased expression and for mTOR, which, in turn, showed reduction in its expression. Moreover, Amblyomin‐X did not show instantly and transiently intracellular calcium mobilization, but showed increase in this intracellular ion concentration after 5h of protein treatment.ConclusionThe results suggest that ER‐stress, cell cycle regulation and survival pathway proteins are involved in this process pro‐apoptotic.Supported by FAPESP
Proteolytic Processing of Hendra Virus Fusion Protein in Cells of the Bat Reservoir Host
Authors: Najjar Farah El , Dutch Rebecca E. 
Published: June 2021

Hendra and Nipah are recently emerged, zoonotic paramyxoviruses with high mortality rates. While the majority of paramyxovirus fusion (F) proteins are proteolytically activated by the host protease furin, Henipavirus F proteins utilize the protease cathepsin L. The primary natural reservoir of Henipaviruses is fruit bats. Interestingly, fruit bats act as reservoirs for SARS‐like coronavirus and Ebola virus, both of which use cathepsins during their life cycles. We analyzed proteolytic processing of viral glycoproteins in recently generated cell lines from the fruit bats Pteropus alecto and Rousettus aegyptus. Both the cathepsin‐dependent Hendra virus F and the furin‐dependent parainfluenza virus 5 F proteins were properly cleaved, suggesting that furin and cathepsin‐like proteases function in these cells. Syncytia formation was observed with expression of either F protein, confirming that proteolytic activation resulted in fusogenic molecules. Addition of cathepsin L inhibitors inhibited Hendra F, but not PIV5 F cleavage in all cell types. There results demonstrate that proteolytic activation of Henipavirus F proteins by cathepsin is not due to lack of furin‐like proteases in Pteropus bat cells.This work was supported by NIAID/NIH grant R01AI051517 and NIH grant #U54 AI057157 from the Southeastern Regional Center of Excellence for Emerging Infections and Biodefense.