bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Effect of Aldosterone on the miRNA Content of a Murine Inner Medullary Collecting Duct Cell
Authors: Jacobs Mollie Elizabeth , Jeffers Lauren A. , Welch Amanda K. , Wingo Charles S. , Cain Brian D. 
Published: June 2021

MicroRNAs (miRNAs) are a family of small noncoding RNAs that have been shown to regulate gene expression by inhibiting translation or targeting specific mRNAs for degradation. miRNAs play important roles in the regulation of metabolism in the healthy cell and in the pathogenesis of many diseases including diabetic nephropathy and hypertension‐related renal disease.Aldosterone is the major known hormone responsible for sodium retention in the kidney collecting duct. Our working hypothesis is that aldosterone action alters the miRNA content in the inner medullary collecting duct resulting in increased translation and stabilization of mRNAs involved in sodium reabsorption.Here we report the miRNA landscape in mIMCD‐3 cells using Toray 3D‐GeneTM miRNA microarray analysis to evaluate 1080 miRNAs. Microarray analysis identified 35 highly expressed miRNAs, as well as several miRNAs whose levels appear to be affected by the presence of aldosterone. Two aldosterone responsive miRNAs affected levels of serum/glucocorticoid regulated kinase 1and sodium/potassium‐transporting ATPase subunit beta‐1 mRNA. Argonaute pull‐down experiments also demonstrated that endothelin‐1 mRNA is a target of the RNA‐induced silencing complex indicating miRNA action on this transcript.This work was supported by NIH NIDDK 1RO1DK082680 to B.D.C and C.S.W. and a 5T32DK076541–04 pre‐doctoral fellowship to M.E.J. Additional funding support and miRNA microarrays were provided by Toray Industries, Inc.
Mechanism for recruitment of the endosome‐associated deubiquitinating enzyme, AMSH
Authors: Davies Christopher W , Paul Lake N , Das Chittaranjan 
Published: June 2021

The endosomal‐sorting complexes required for transport (ESCRT) is a ubiquitin‐mediated process that is implicated in endosomal sorting, trafficking, and viral budding. The ESCRT machinery regulates signaling events by internalizing cell surface receptors and targets them for degradation by the lysosome. Two deubiquitinating enzymes (DUBs) regulate the activity of the ESCRT complex, STAM‐binding protein (AMSH), and ubiquitin specific protease Y (UBPY), by counteracting the ubiquitination of the cargo. Recently, we characterized AMSH's mode of substrate recognition through mutational analysis and found that AMSH's recognition of distal ubiquitin is significantly different from the homologous, but ESCRT‐independent protein, AMSH‐like protein (AMSH‐LP). In the current study, we are aiming to understand how AMSH's mode of substrate recognition in conjunction with AMSH's recruitment to the initial ESCRT complex affects the enzyme's activity using a combination of biochemical and biophysical analysis. We have recapitulated AMSH recruitment to the initial ESCRT complex using the minimal domains and have shown that the enzyme's activity is enhanced. This result supports AMSH's role as a major regulator of ubiquitin mediated degradation by the ESCRT machinery. Results from our studies could further provide insight into defining a clear role for AMSH within the ESCRT machinery.Support: NIH (1R01RR026273) and American Heart Association (12PRE12060249)
J‐protein requirements of multiple prion variants in two distinct yeast strains reveal functional specificity in yeast prion propagation
Authors: Harris Julia , Patel Milan , Hines Justin K 
Published: June 2021

The objective of this study was to determine the importance of prion variant and yeast strain variation on prion‐chaperone requirements. The J‐protein Sis1 and the prions [PSI+] and [RNQ+] were used as a model system to explore the impact of amyloid conformation (prion variants) and yeast strain polymorphisms (yeast genetic background) on chaperone requirements. The primary method used in this study was complementation by plasmid‐shuffling in the budding yeast Saccharomyces cerevisiae.Summary of ResultsYeast strain background had a small but noticeable effect on chaperone requirements; this effect is likely due primarily to altered chaperone protein levels between strains. While many prion variants shared similar chaperone requirements, some significant differences were found. Strikingly, despite these variations, our investigation revealed that individual prions have distinct, non‐overlapping Sis1 domain specificity, an observation which implies that Sis1 has prion‐specific functionality.
Thermal manipulation during chicken embryogenesis results in enhanced HSP70 gene expression and the acquisition of thermotolerance
Authors: Al‐Zghoul Mohammad Borhan 
Published: June 2021

SUMMARYThe aim of this study was to evaluate the effect of thermal manipulation (TM) at 38.5°C for 6 h or 18 h during embryonic days (ED) 10–18 on the muscle mRNA levels of heat shock protein 70 (HSP‐70) and the chick's ability to cope with the thermal challenge (TC; 41°C for 6h) at day 3, 7 and 42 of age. 750 fertile chicken eggs were obtained and divided randomly into 3 different treatment groups: control group was maintained at 37.8°C and 56% relative humidity (RH), TM1 was subjected to TM at 38.8°C for 6 h and 65% RH during ED 10–18 and TM2 was subjected to TM at 38.8°C for 18 h and 65% RH during ED 10–18. After hatching, the chicks from each treatment groups were randomly divided into 2 sub‐treatment groups (Naive and challenged). Chick's response to thermal challenged (41°C fro 6 hrs) was evaluated by determination of body temperature, plasma thyroid hormone levels (T3 and T4), plasma HSP70 and muscle mRNA levels of Hsp70. Our results indicated a long‐term improvement in the gene expression of HSP70 in the thermal manipulated chicks when compared with control, as manifested by a significant increase in the muscle mRNA levels of Hsp70 in thermally manipulated chicks during embryogenesis and during thermal challenge in post‐hatch chickens.
Dihydrodipicolinate Synthase from E. coli: Characterization of Arginine 138 by Site‐Directed Mutagenesis
Authors: Vann Tyler , Karsten William E. , Chooback Lilian 
Published: June 2021

Dihydrodipicolinate synthase catalyzes the formation of dihydropicolinate from pyruvate and L‐aspartate‐β‐semialdehyde (ASA). The enzyme catalyzes the first committed step for the biosynthesis of L‐lysine in bacteria and plants. The enzyme from Escherichia coli is feedback inhibited by lysine, the end product of the pathway. A study of the pH dependence of the kinetic parameters was done to elucidate the acid‐base chemical mechanism of the enzyme. The kinetic mechanism is ping pong with pyruvate binding to free enzyme. The ε‐amino group of lysine 161 attacks the carbonyl of pyruvate and forms a Schiff base intermediate. The loss of a proton from this intermediate leads to the formation of an enamine intermediate, with the loss of the proton accounting for the irreversible step and the ping pong kinetic mechanism. ASA binds to the enzyme:enamine covalent intermediate. Site‐directed mutagenesis was done to investigate the role of the active site arginine 138. The R138A and R138K mutants were created and the identity of the mutants was confirmed. Kinetic studies will be done to characterize the mutant enzymes.This work was supported by grant P20RR016478 from the National Center for Research Resources a component of the National Institutes of Health and a grant from the University of Central Oklahoma office of Research and Grants to L.C.
A click chemistry‐mediated approach to understanding survivin:caspase‐9 protein‐protein interactions
Authors: Bishop Stephanie , Lampe Jed N 
Published: June 2021

Survivin, a 16.5 kDa mammalian cytosolic protein, is upregulated in multiple cancers and is a promising therapeutic target. As its name implies, survivin facilitates cell survival by inhibiting apoptosis. Survivin is thought to inhibit the apoptotic initiator caspase‐9, however, the specific mechanisms of and amino acids involved in caspase‐9 inhibition have yet to be fully elucidated. Identifying the amino acid residues involved in the interaction between survivin and caspase‐9 will facilitate the rational design of small molecule protein‐protein interaction inhibitors. Click chemistry provides a facile approach to covalently link disparate azide‐ and terminal alkyne‐ containing molecules, thereby allowing for site‐specific incorporation of a fluorescent tag into a polypeptide sequence. Using site‐directed mutagenesis with the unnatural amino acid para‐azidophenylalanine (paF), we synthesized an azide‐containing mutant survivin protein. We then used copper‐free click chemistry to covalently modify the mutant survivin azide with an alkyne‐containing fluorophore dibenzylcyclooctyne‐488 (DBCO488) to generate a triazole‐containing cycloadducted product. Using western blotting, UV illumination, and Coomassie blue staining, we demonstrated the ability to site‐specifically cycloadduct the para‐azidophenylalanine in the survivin protein to the fluorescent cyclooctyne, DBCO488. This technology will now enable us to perform fluorescence analyses of the survivin:caspase‐9 complex in order to identify the specific amino acid residues involved in this interaction. This novel approach to monitor protein‐protein interactions holds untapped potential to improve rational drug design for survivin:caspase‐9‐targeted anticancer therapeutic agents.Funding: Environmental Toxicology Training Program 5T32ES007079–32 (SCB), COBRE P20 RR021940‐06 (JNL), R01 GM099959‐01 (PI, Collaborator John Karanicolas)
A neutron can make the difference: The 15N stable isotope effect on metabolic pathways
Authors: Filiou Michaela , Webhofer Christian , Nussbaumer Markus , Landgraf Rainer , Maccarrone Giuseppina , Turck Christoph W 
Published: June 2021

We have investigated the effects of metabolic labeling with the 15N stable isotope in different organisms.Mice and bacteria were metabolically labeled using a 15N‐labeled diet or 15N‐labeled media, respectively. The 15N‐labeled organisms were then compared with their unlabeled counterparts at the phenotypic and molecular levels with the help of behavioral tests, quantitative proteomics and targeted enzymatic, metabolic and immunochemical assays.At the phenotypic level, differences in behavioral traits between 15N‐labeled and unlabeled mice were observed. At the molecular level, changes in protein expression, growth rates, pathway activities, metabolite levels and chromatographic peak patterns were found in the 15N‐labeled specimens.Our results indicate for the first time a global 15N isotope effect on labeled organisms. This information can be used to correlate phenotypic and molecular alterations and track relevant metabolic pathways.This work was supported by a QuantPro grant and the Max Planck Society
Designing a unique reporter based microarray sensor platform for monitoring cell signalling.
Authors: Gibriel Abdullah 
Published: June 2021

PurposeBiological processes in mammalian cells are driven by gene expression that is in turn regulated by transcription factors (TFs). The latter are controlled by a series of complex network of biochemical pathways that represent the core for cell signalling. There has been little success in monitoring cell signalling using traditional methods such as proteomics. In this report we demonstrate the possibility of deconvoluting such complexity using a multiplexed library of plasmids carrying a TF binding site upstream of a unique reporter sequence (UR). A microarray sensor platform was then used to monitor dynamic changes in cells by measuring UR expression levels.MethodsHEK293 cells, treated with various drugs and inducers, were transfected with the multiplexed plasmid library. mRNA was then purified and reverse transcribed to produce first strand cDNA. The latter was then amplified by PCR using a universal fluorescently labelled primer. Purified PCR amplicons were then hybridized with antisense oligonucleotide captures at 53°C. Epoxy slides were then washed and quantified using the ScanArray Express Scanner (Perkin Elmer).ResultsThe microarray platform was optimized to detect down to 1 attomol with a maximal degree of specificity and a minimal probe volume of 2μl. This platform was able to detect changes in the TF activity following treatment of cells with dexamethazone, cadmium, forskolin, TPA, cAMP analogues, cGMP analogues and PDEIs. The data was consistent with qPCR and western blot analysis.ConclusionThe microarray platform was shown to be robust, sensitive and reproducible in monitoring dynamic changes in mammalian cells. The system can be easily tailored to include transcription factors of interest to fit with the required purposes.
SELDI‐TOF MS Combined With Magnetic Beads for Detecting Serum Protein Biomarkers for Diagnosis of Active Tuberculosis
Authors: Liu Jiyan , Wei Liliang , Jiang Tingting , Zhang Xing , Xu Dandan , Wang Chong , Li Zhongjie , Zhong Jianping , Li Ji‐Cheng 
Published: June 2021

New technologies for the early detection of tuberculosis (TB) are urgently needed. The aim of the present study was to screen for the potential protein biomarkers in serum for the diagnosis of TB using proteomic fingerprint technology. The surface‐enhanced laser desorption ionization time of flight mass spectrometry (SELDI‐TOF‐ MS) combined with WCX magnetic beads was used to profile the serum proteins from 180 patients with TB, 91 cases of healthy volunteers, 40 cases of lung cancer patients, 40 cases of pneumonia patients, and 40 cases of chronic obstructive pulmonary disease. The protein fingerprint expression of all the serum samples and the resulting profiles between TB and control groups were analyzed with the Biomarker Wizard system. A total of 35 discriminating m/z peaks were detected that were related to TB (p < 0.01). The model of biomarkers based on the four biomarkers (2554.6, 4824.4, 5325.7, and 8606.8 Da) generated excellent separation between the TB and control groups. The sensitivity was 83.3% and the specificity was 84.2%. Blind test data indicated a sensitivity of 75.0% and a specificity of 83.5%. The data suggested a potential application of SELDI‐TOF MS as an effective technology to profile serum proteome, and with pattern analysis, a diagnostic model comprising three potential biomarkers was indicated to differentiate people with TB and healthy controls rapidly and precisely.This work was supported by grants from National Special Sci‐Tech Projects (No. 2012ZX10005001–006), National Natural Science Foundation of China (No. 81072724, No. 81273882), and Zhejiang Province Special Sci‐Tech Projects (No. 2009C03011–3).
Moonlighting Proteins
Authors: Jeffery Constance , Mani Matt , Amblee Vaishak , Chen Chang , Zhang Zhou 
Published: June 2021

The ability of a protein to have multiple functions and be involved in different multi‐protein complexes or pathways can complicate the prediction of the protein function from sequence or structure and the annotation of sequence and structure databases. We are studying moonlighting proteins, a class of multifunctional proteins in which a single polypeptide chain has multiple biochemical functions that are not due to gene fusions, multiple splice variants or multiple proteolytic fragments. Many of the known moonlighting proteins are cytosolic enzymes that are also transcription factors, crystallins, chaperones, extracellular growth factors, or cell surface adhesins. The variety of known moonlighting proteins, the multiple ways in which one protein can have multiple functions, the potential benefits to the organism of combining two functions in one protein, and the methods proposed for a protein to evolve a second function suggest that moonlighting proteins might be common.To date, most moonlighting functions have been found by serendipity. There is currently no straightforward method to identify which proteins have moonlighting functions, or for determining if a protein of interest is a moonlighting protein. In addition, sequence homologues of moonlighting proteins often do not perform both functions. We are currently organizing information about the sequences, structures, functions and functional sites of the over 200 known moonlighting proteins with the goal of preparing an online, searchable database of moonlighting proteins
Quantum analysis/improvement of antipsychotic's docking results
Authors: Nunes Gustavo , Zanatta Geancarlo , Bezerra Eveline M. , da Costa Roner F. , Freire Valder N. , Gottfried Carmem C. 
Published: June 2021

Research support: CNPq‐BrazilHuman dopamine receptors are important targets in treatment of Parkinson's disease and Schizophrenia. Thus, the understanding of the binding mechanisms involving selective ligand agents and dopamine receptors D2 and D3 may contribute to pharmacologic strategies. Recently, we have applied quantum mechanics methods to calculate the contribution of each amino acid residue in the binding pocket of D3 receptor in complex with eticlopride (Zanatta, G et al, 2012). In this study, to understand the mechanism of haloperidol interaction, we employed the Molecular Fractionation with Conjugated Caps (MFCC) followed by quantum calculations to analyze the structure of haloperidol docked in D3 receptor using Autodock 4.0 (Fig 1).It was not possible to obtain the stabilization in total binding energy for a radius of 16 Å (Fig. 2), suggesting that this pose is not yet in the best binding conformation. In order to improve the results, we are performing a series of energy minimization steps using quantum mechanics/molecular mechanics (QM/MM). The establishment of a protocol to improve docking results is important to obtain representative structures of selective ligands to D2 and D3 receptors.
Signalling and cholesterol metabolism
Authors: Luu Winnie , Sharpe Laura J. , Stevenson Julian , Brown Andrew J. 
Published: June 2021

Cholesterol is a vital lipid and performs diverse functions on a whole body and cellular level. However, excess cellular cholesterol is toxic, and thus, elegant mechanisms have evolved to tightly regulate this important lipid. Cellular homeostasis is governed by two major transcriptional factors, the sterol regulatory element binding proteins (SREBPs) and liver X receptor (LXR). SREBP‐2 is up‐regulated when cholesterol levels are low, promoting genes that are involved in synthesis and uptake, whilst LXR up‐regulates efflux genes such as ATP‐binding cassette transporter A1 (ABCA1). While there is a large body of work elucidating these cholesterol‐related pathways, less is known about the role of signalling in these processes. We found that Akt activates SREBP‐2 and its target genes, whilst tyrosine phosphatase inhibition down‐regulates gene expression of ABCA1. These findings offer new insights into the regulation of cholesterol metabolism.The Brown lab is funded by grants from the National Health and Medical Research Council and National Heart Foundation of Australia.
The role of O‐fucosylated glycans in Notch signaling
Authors: Shim Jeongsup , Petryniak Bronislawa , Lowe John B. 
Published: June 2021

Signaling from the Notch receptor family is important in development, and its role in disease has assigned the Notch signaling pathway as a therapeutic target. Mammalian Notch receptors are single‐pass transmembrane glycoproteins containing 29–36 EGF‐like repeats. Notch receptors on “receiving” cells signal when they interact with Notch ligands expressed on “sending” cells. Glycosylation of some Notch EGF‐like repeats modulates Notch‐dependent signaling. EGF repeat glycosylation includes constitutive fucosylation of some EGF repeat‐localized serine/threonine residues, and elongation of fucose‐initiated glycans regulated by the Fringe family of glycosyltransferases.To understand how glycosylation controls Notch signaling, we sought to determine how O‐fucosylated glycans modulate Notch receptor‐ligand interactions, using (1) surface plasmon resonance, (2) cell‐based binding assays, and (3) cell‐based Notch signaling assays. We find that lunatic Fringe modification of Notch1 receptors enhances Dll1 and Dll4 binding affinities and signaling, while it reduces Jag1‐mediated binding affinity and signaling. Fringe modification of Notch1 does not significantly alter Jag2‐mediated binding affinity and signaling. We also find that Dll1 binding and signaling is enhanced by lunatic Fringe primarily when Notch1 expression is low, and this enhancement is blunted when Notch1 expression is high.
Effects Of Dextromethorphan Mediated Bitter Taste Receptor Activation In Pulmonary Artery Smooth Muscle Cells
Authors: Upadhyaya Jasbir Deol , Singh Nisha , Sikarwar Anurag Singh , Bhullar Rajinder Pal , Dakshinamurti Shyamala , Chelikani Prashen 
Published: June 2021

Activation of bitter taste receptors (T2Rs) in human airway smooth muscle cells lead to muscle relaxation and bronchodilation. However, the presence of T2Rs in human pulmonary artery smooth muscle cells (hPASMCs) has not been established. The previous finding in airway smooth muscle led to our hypothesis that T2Rs, if present in hPASMCs, might be involved in regulating the vascular tone. RT‐PCR analysis revealed the expression of multiple T2R transcripts in hPASMCs. Functional analysis showed that these cells responded to many bitter‐tasting compounds, by increasing intracellular calcium concentration, indicating that T2Rs in hPASMCs are functional. Since the response of T2R1 to dextromethorphan (DXM) has been characterized in our previous studies, T2R1 was selected for further analysis in this study. Knockdown with T2R1‐specific shRNA decreased mRNA levels and DXM‐induced calcium responses by up to 40%. ELISA data obtained from in vitro studies using hPASMC showed that DXM‐treatment led to increased levels of endothelin‐1, biomarker of systemic hypertension. To analyze if T2R1 is involved in regulating the pulmonary vascular tone, ex vivo studies using pulmonary arterial rings are being pursued. Furthermore, it remains to be analyzed if this effect of DXM in hPASMCs is T2R1 specific or due to the blockade of NMDA receptors.Supported by MHRC, NSERC and a New Investigator Award from HSFC.
The involvement of prohibition in Akt and Raf co‐regulated apoptosis of HaCaT keratinocytes upon UVB irradiation
Authors: Wu Qiong , Wu Shiyong 
Published: June 2021

Prohibitin (PHB) is involved in regulation of many cell functions, such as cell‐cycle control, cell proliferation, senescence, apoptosis, and tumor suppression. However, the roles of PHB in regulation of UVB‐induced apoptosis of keratinocytes are largely unknown. In this study, we evaluated the role of PHB in regulation of Akt and Raf activity in UVB‐irradiated HaCaT cells. Our data showed that PHB was localized to the membrane from the cytosol upon UVB irradiation. A co‐immunoprecipitation analysis indicated that UVB altered the interaction among PHB, Raf, and Akt. Using siRNA‐mediated knockdown as well as stable shRNA‐mediated knockdown, we demonstrated that upon UVB irradiation, the cells with reduced expression of PHB showed an increase in apoptosis, as well as an increased phosphorylation of Akt and a decreased phosphorylation of ERK. In addition, our data showed that the effect of L‐NAC on UVB‐induced apoptosis is independent of PHB knockdown, which suggests that the PHB‐knockdown‐mediated cell apoptosis is independent of UVB‐induced oxidative stress. Based on these results, we propose that PHB regulates UVB‐induced apoptosis of keratinocytes via Akt and Raf, not the oxidative‐stress signaling pathway.This work is funded by NIH RO1 CA086928 (to S. W.)