bluuebunny/crossref_metadata_2025_split huggingface dataset:
Motif Q and I of SWI2/SNF2 family proteins are not involved in ATP binding but are required for hydrolysis
Authors: Gupta Meghna , Nongkhlaw Macmillan , Komath Sneha Sudha , Muthuswami Rohini 
Published: June 2021

ObjectiveDeciphering the role of motif Q and I in the catalysis by Active DNA‐dependent ATPase A Domain (ADAAD).MethodsSite‐directed Mutagenesis, Deletion Mutagenesis, Fluorescence Spectroscopy, Circular Dichroism Spectroscopy, and ATPase assays.ResultsADAAD a representative member of SWI2/SNF2 family of proteins possesses the conserved helicase motifs including motif Q that are a characteristic feature of this family. It hydrolyzes ATP in the presence of stem‐loop DNA which mimics the double‐strand to single‐strand transition regions of DNA in a cell. In this study we have investigated the role of motif Q and motif I in ATP hydrolysis in ADAAD. The motif Q directs the catalytic rate while motif I is absolutely necessary for mediating catalysis. But neither of these motifs is required for ATP and DNA binding. The interaction of the protein with ATP appears to be mediated via adenine ring alone both in the absence and presence of DNA. The γphosphate of ATP is required only for inducing ATPase‐competent conformation.ConclusionThe function of both motif Q and I appear to be at variance from other helicases.Source of research support: Defense Research and Development Organization, Council of Scientific and Industrial Research, Department of Biotechnology.
Differential histone modifications induced by chronic methamphetamine exposure in the rat striatum
Authors: Jayanthi Subramaniam , McCoy Michael T , Ladenheim Bruce , Martin Tracey , Beauvais Genevieve , Krasnova Irina N. , Hodges Amber B. 
Published: June 2021

Methamphetamine (METH) is a very addictive drug. Repeated METH injections cause increases in spine density in striatal medium spiny neurons that receive inputs from corticostriatal glutamatergic projections. We thus undertook the present study to test the possibility that repeated injections of METH might influence the expression of AMPA glutamatergic receptors and of scaffolding proteins. We also measured the effects of METH on the enrichment of acetylated histone 4 on the promoters of AMPA receptors and Homer1. Repeated injections of increasing doses of METH caused significant decreases in the expression of AMPA glutamate receptors (GluA1 and GluA2). There were also METH‐induced decreases in the mRNAs that code for Homer1 and PSD95, proteins that participate in the regulation of the functional effects of glutamate receptors. ChIP‐PCR experiments using antibodies against histone H4 acetylated at lysine 5 (H4K5ac) or lysine 16 (H4K16ac) documented chronic METH‐mediated decreases in the enrichment of H4K5ac and H4K16ac proteins on the promoters of AMPA GluA1 and GluA2 receptors, and on the Homer1 promoter. These results suggest that dynamic regulation of histone H4 acetylation might be involved in the regulation of GluA1 and GluA2. These findings suggest that changes in AMPA receptors might underlie METH‐induced changes in striatal spine density.This work is supported by DHHS/NIH/NIDA/IRP.
Alternative Splicing of chrng gene generates Novel Isoforms with different N‐terminals
Authors: Azim Shafquat , Banday Abdul Rouf , Tabish Mohammad 
Published: June 2021

The γ‐subunit of nicotinic acetylcholine receptor is encoded by chrng gene of mouse genome. It plays an important role in maturation of the receptor. The gene contains 12 exons and 11 introns. Two transcripts of chrng have already been reported that arise due to the presence or absence of 5th exon. The aim of this study is to analyze the gene structure and identify any other transcript that may produce different isoforms.A combination of bioinformatics tools like gene finders, blast searches, alignment tools, etc. were used to study chrng gene. Using these methods three new coding exons namely N1, N2, and N3 were identified from 5′ UTR and 1st intron of chrng. Each one of these is capable of alternatively replacing the 1st exon of the reported transcript making three new transcripts differing at the 5′ end. Est database searches also revealed the presence of an Est corresponding to N3 exon. To confirm the presence of these transcripts RT‐PCR followed by semi‐nested PCR of RNA isolated from mouse brain was performed. Sequencing of the products further confirmed the results.Thus, using bioinformatics and molecular biology techniques we have identified three new transcripts of chrng encoding γ‐subunit and having different N‐terminal sequences. The sequence encoded by N3 exon is predicted to have a signal peptide, phosphorylation and glycosylation site which lack in all other isoforms. Funding from CSIR, India is acknowledged.
Calnexin levels are regulated by the ER localized ubiquitin ligase Nixin
Authors: Neutzner Albert , Neutzner Melanie , Benischke Anne‐Sophie , Ryu Seung‐Wook , Youle Richard J , Karbowski Mariusz 
Published: June 2021

To identify novel regulators of ER linked protein degradation and ER function, we determined the entire inventory of membrane spanning RING finger proteins localized to the ER. We identified twenty four ER membrane anchored ubiquitin ligases and found Nixin to be central for the regulation of calnexin turnover. Using fluorescence protease protection and hydrophobicity plotting, we established that Nixin spans the ER membrane once, thus exposing its RING domain to the cytosol.Ectopic expression of wild type Nixin induced a strong downregulation of the ER localized chaperone calnexin that was prevented by inactivation of the Nixin RING domain as well as by proteasomal inhibition. Furthermore, Nixin physically interacts with calnexin in a glycosylation independent manner and enhances calnexin ubiquitination.Taken together, the ER membrane‐anchored ubiquitin ligase Nixin regulates calnexin levels through proteasomal degradation and thus might play a role during ER stress recovery when calnexin levels are adjusted to pre‐stress levels.
Overexpression of myristoylated methionine sulfoxide reductase A in the mouse protects the heart against ischemia‐reperfusion injury
Authors: Zhao Hang , Sun Junhui , Deschamps Anne M. , Kim Geumsoo , Murphy Elizabeth , Levine Rodney L. 
Published: June 2021

Methionine sulfoxide reductase A (MsrA) has been reported to function as an antioxidant protein, either by scavenging reactive oxygen species via cyclic interconversion of the reduced and oxidized forms of methionine residues or by protecting critical protein residues from oxidation. However, the mechanisms and requirements for providing an effective oxidative defense in vivo are not known. In mammalian cells, ~75% of MsrA is located in the cytosol and is myristoylated while ~25% is in the mitochondria and not myristoylated. We created several strains of transgenic mice overexpressing MsrA directed to the cytosol or mitochondria or both. Quantitative Western blotting and activity measurements demonstrated a high level of functional MsrA in most tissues tested, with the highest in the heart. We tested whether the increased MsrA in heart protected against ischemia‐reperfusion injury in a Langendorff model. We found that MsrA overexpression in the cytosol but not in the mitochondria protected. We then examined whether myristoylation affected this protection. We created another transgenic mouse overexpressing MsrA only in the cytosol, but in this strain glycine at position 2 was mutated to alanine to prevent myristoylation. Analysis of heart tissue confirmed that the overexpressed MsrA was active but not myristoylated. However, this non‐myristoylated MsrA heart experienced no protection. These experimental results indicate that myristoylation of cytosolic MsrA is key to its ability to provide cardioprotection.This research was supported by the Intramural Research Program of the NIH, NHLBI.
Protein kinase‐D1 overexpression in mice prevents lipid‐induced insulin resistance and cardiomyopathy by upregulation of glucose uptake
Authors: Dirkx Ellen , Schwenk Robert W. , Steinbusch Laura KM , Hoebers Nicole , Janssen Ben J , Brans Boudewijn , Basel‐Duby Rhonda , Olson Eric N , Bonen Arend , van Eys Guillaume JJM , Glatz Jan FC , Luiken Joost JFP 
Published: June 2021

Disturbances in metabolic substrate uptake promote the development of cardiomyopathy and eventually lead to heart failure. Understanding the pathways that control substrate uptake could enable therapeutic approaches to prevent maladaptive cardiac remodeling.We identified protein kinase‐D1 (PKD1) as a contraction‐activated kinase, which promotes GLUT4‐mediated glucose uptake, without affecting long chain fatty acids (LCFA) uptake. This corroborated with in‐vitro experimentation using PKD1 siRNA in HL‐1 cells and in‐vivo by microPET scan results showing a higher glucose uptake in the hearts of mice overexpressing constitutively active PKD1 (caPKD1). The increased glucose uptake in the heart of caPKD1 mice results in dilated cardiac hypertrophy and heart failure. In contrast, mice on a high fat diet with lipid‐overloaded hearts displayed concentric cardiac hypertrophy, accompanied by insulin resistance. However, hearts of caPKD1 mice on a high fat diet displayed neither dilated nor concentric hypertrophy. Further, these hearts did not show increased glucose uptake nor increased LCFA uptake, demonstrating the role of PKD1 to balance cardiac substrate utilization, which is crucial in preventing pathological cardiomyopathy. In conclusion, in the lipid‐overloaded heart, upregulation of PKD1 offers protection against the development of insulin resistance and hypertrophic remodeling.Grant Funding Source: NWO (Dutch Organisation for scientific research)
Differential Expression of Cholecystokinin Receptors and Their Ligands in Normal and Neoplastic Pancreas
Authors: Ortega Emily E , Smith Jill P. 
Published: June 2021

BackgroundThe gastrointestinal peptide gastrin has been identified in fetal pancreas where it is thought to play a role in growth and differentiation. Gastrin has also been identified as a growth factor that stimulates pancreatic cancer through the CCK‐B and CCK‐C receptors by an autocrine mechanism.HypothesisIt is hypothesized that gastrin re‐expression is important to pancreatic carcinogenesis.MethodsEndpoint RT‐PCR was performed on normal human pancreas mRNA to detect RNA levels of gastrin and CCK peptide. PCR products were visualized on an ethidium bromide stained gel after electrophoresis. Normal human pancreatic tissue samples were evaluated for gastrin and CCK by immunohistochemistry.ResultsThe end‐point RT‐PCR showed significant levels of CCK‐B receptor and CCK peptide in normal pancreas samples. The gastrin end‐point RT‐PCR showed low levels in all of the normal pancreas samples. Gastrin immunoreactivity was not detected in normal pancreas, however, staining of the positive control was reactive; the negative control showed no staining.ConclusionsNormal pancreas possesses the receptor CCK‐B and the peptide CCK. Gastrin is not present in normal pancreatic tissues, but is detected in neoplastic pancreatic samples. Therefore, the re‐expression of gastrin plays a pivotal role in the development of pancreatic cancer and could be a potential trigger to pancreatic carcinogenesis.
Dynamics of human hepatic cells metabolome during dengue virus infection
Authors: El‐Bacha Tatiana , Galina Antonio , Almeida Fabio C , Da Poian Andrea T 
Published: June 2021

The metabolic resources crucial for viral replication are provided by host cells. The mechanisms by which viruses interact with cell metabolism, driving biosynthetic molecules for their own replication, however, are unknown. We employed nuclear magnetic resonance to study metabolic fluxes and identify metabolites in dengue virus (DenV) infection of human hepatic cells since liver dysfunction plays a central role in DenV pathogenesis. Metabolomics were done at 24h of infection. 13C spectra were acquired after cell incubation with D‐[U‐13C]glucose. Results showed that glycolytic (fructose‐6‐phosphate, fructose‐1,6‐bisphosphate, dihydroxyacetone phosphate and glyceraldehyde‐3‐phosphate) and pentose phosphate pathway (PPP; 6‐phosphogluconate) intracellular intermediates were decreased in DenV infection and so was lactate accumulation in culture medium. Intracellular L‐glutamate and L‐alanine were increased upon DenV infection. The decrease in glycolytic and PPP intermediates indicates an enhancement of glucose utilization for biosynthetic purposes; Increased contents of L‐aminoacids indicates decreased oxidative metabolism suggesting impaired mitochondrial function. The use of metabolomics to evaluate carbon fluxes represents a key step towards the elucidation of the dynamics of DenV‐human hepatic cells metabolic interactions.Supported by: CAPES, CNPq and FAPERJ.
Structural and sequence analyses of an infrared fluorescent tissue marker
Authors: Baker Anna W. , Salgado Daniel , Kennis John T. M. , Stojkovic Emina A. 
Published: June 2021

Within the mammalian body infrared (IR) radiation is not scattered by tissues and fluids as much as visible light, making an IR fluorescent marker ideal for imaging applications in mammals. Recently, bacteriophytochromes (Bphs), red‐light sensing proteins from different bacterial strains, have been shown to contain significant levels of IR fluorescence. A Bph from Deinococcus radiodurans, after extensive mutagenesis, has been successfully expressed as IR fluorescent tissue marker in mice. Bph response to light depends on an organic cofactor, biliverdin (BV), which is abundant in the mammalian body. BV, upon the absorption of a photon, induces conformational changes in the surrounding Bph allowing it to reversibly photoconvert between spectrally distinct Pr (red light absorbing) and Pfr (far red light absorbing) states. Bph from Rhodopseudomonas palustris, RpBphP3 (P3), is unique among the Bphs in that its ground state is Pr and its excited state is Pnr (near red light absorbing). P3 and its mutant variants have been shown to emit significant IR fluorescence due to P3's unusual photoconversion properties. A single mutation D216A or Y272F in the BV binding region or C‐terminal truncation of the photosensory module of P3 increases IR fluorescence over the wild‐type protein. Using site‐directed mutagenesis we created mutant variants of P3 that contain combination of D216A or Y272F mutation with C‐terminal truncation of the photosensory module. We report that truncated P3 with D216A mutation displays higher levels of near IR fluorescence relative to wild type P3. Expression and visualization of IR fluorescent P3 mutants in mammalian cell lines followed by in vivo expression in mice are the subsequent goals of this research.Supported by NIH EARDA pilot grant 5G11HD049644 – 04 to E.A.S..
Adhesion of lysozyme, albumin and transferrin to two types of FDA Group II contact lenses
Authors: Solanki Darshan , Chandrasekaran N. , Chodhry R. , Cuprillnilson S. , Desai S. , Liberman B. , Lee V.S. , Lin Y.J. , Nguyen K. , Roughi L. , Shah S. , Sharma V. , Thomas P. , Janoff A. , Keith E.O. 
Published: June 2021

Tears contain ~60 different proteins that adhere to contact lenses, causing lens deterioration and ocular pathology. We examined the adhesion of three tear proteins to two different types of FDA Group II contact lenses (hilafilcon and omafilcon). Lenses were incubated in 2.0 mg/ml solutions of human lysozyme, albumin and transferrin for 1–4 days. Protein adhesion was determined by bicinchoninic acid assay. Lysozyme adhered to hilafilcon lenses in an up‐down pattern, with a maximum on day 3. Lysozyme adhesion to omafilcon lenses was high after 1 day and remained high on day 4. Albumin adesion to both types of lenses was high after 1 day, declined, and increased on day 4. Transferrin adhesion to both lenses was initally low, increasing to a maximum on day 3 and declining on day 4. These results are due to differences in lens material and tear protein structure. Hilafilcon, more negatively charged than Omafilcon, absorbed more lysozyme (+ charged at physiological pH). Omafilcon lenses are coated with phosphorylcholine, reducing adhesion by the hydrophobic domains of albumin. Human apo‐transferrin has few positive charges and thus bound more reluctantly to both materials.Supported by the Farquhar College of Arts and Sciences and the Health Professions Division, NSU.
Lipid droplets promote survival of nutrient‐deprived cells
Authors: Cabodevilla Ainara G , Nintou Eleni , Gubern Albert , Claro Enrique 
Published: June 2021

Lipid droplets (LD) are intracellular organelles formed in most eukaryotic cells. They are a core of triacylglycerols (TAG) and choresteryl esters surrounded by a phospholipid monolayer with which a variety of proteins interact. Most of the present knowledge on LD biology is derived from experimental models where cells are loaded with external TAG and/or fatty acids. Little is known, however, about the physiological significance of LD formed under stress and in the absence of external lipids, conditions that lead eventually to cell death. Here we show that cells kept in isotonic buffer devoid of nutrients form LD whose TAG derive from membrane phospholipids in a process that depends on iPLA2 and cPLA2 activities. In this experimental model, inhibition of LD biogenesis accelerates cell death and increases the expression of markers of autophagy. And conversely, inhibition of autophagy increases the occurrence of LD. Taken together, our results show that LD biogenesis under stress has a cell protective role.Supported by BFU2009‐0783 grant from the Spanish Ministry of Science.
Role of phospholipase D in adrenal glomerulosa cell function
Authors: Seremwe Mutsa Patience , Bollag Wendy B 
Published: June 2021

ObjectiveTo study the role of agonist‐induced phospholipase D (PLD) activation in modulating aldosterone secretion in human adrenocortical carcinoma cells (HAC15).MethodsHAC15 cells were treated with elevated extracellular potassium (K+) and angiotensin II (AngII) in the presence and absence of 5‐fluoro‐2‐indolyl des‐chlorohalopemide (FIPI), a selective PLD inhibitor. To verify that FIPI was not cytotoxic, we used 22(R)‐hydroxycholesterol [22(R)OH‐cholesterol], a cholesterol analog that can bypass signal transduction pathways and be directly used for aldosterone production. Supernatants were collected and assayed for aldosterone. [3H]Oleate‐prelabeled HAC15 cells were stimulated with AngII in the presence of ethanol, and lipids extracted and assayed for the PLD product, phosphatidylethanol.ResultsFIPI inhibited agonist‐induced aldosterone secretion in a manner unrelated to cytotoxicity, as shown with 22(R)OH‐cholesterol. Furthermore, AngII stimulated PLD activity in HAC15 cells.ConclusionAldosterone secretion in HAC15 cells in response to secretagogues was inhibited by FIPI, suggesting that PLD plays a role not only in AngII‐ but also in K+‐induced aldosterone secretion. Experiments are in progress to determine the time course and dose dependence of the PLD response to AngII and lastly, to define its mechanism of action. Funded by NIH award#HL70046 and a VA Merit award.
Extracellular matrix (ECM) protein lumican regulates innate immune signals at the host cell surface and promotes bacterial phagocytosis
Authors: Chakravarti Shukti , Lee Seakwoo 
Published: June 2021

Here we show that lumican aids phagocytosis of gram− bacteria through cell surface interactions with integrins and CD14, an adaptor protein for LPS signaling. Lum−/− peritoneal macrophages (MF) were tested for response to LPS (induction of TNF‐α), and phagocytosis of gfp‐E. coli in the presence or absence of wild type (wt) and mutant recombinant lumican (rLum). Lum−/− MF showed poor induction of TNF‐α, and impaired phagocytosis. Pretreatment of Lum−/− MF with wt rLum rescued LPS response and phagocytosis completely, while mutated rLumY20A rescued only 30% of wild type functions. Phagocytic functions in Lum−/−Cd14−/− MF could not be rescued with wt rLum, indicating complete CD14 dependence. CD14‐antibody blockade reduced phagocytosis in Lum+/+ MF to Lum−/− levels, with no further decrease in Lum−/− MF. CD18 blockade reduced phagocytosis in Lum+/+ and Lum−/− MF, indicating some CD18 phagocytic functions to be lumican‐independent. Lumican, β2 integrin (CD18) and CD14 co‐localize on the surface and phagosomes of Lum+/+ MF. Surface Plasmon resonance indicated specific binding between CD14 and rLum (KA=2.15 ×106 M−1) but weaker affinity for rLumY20A (KA= 3.50 × 104 M−1). These studies identify a novel role for lumican in innate immunity and bacterial phagocytosis, in a CD14 and CD18 dependent manner. An N‐terminal tyrosine in lumican is critical for binding CD14, LPS response and phagocytosis.Supported by NEI11654 to SC
RNAi screening reveals new players in the defense against RNA viruses
Authors: Xu Jie , Sabin Leah , Zhou Rui , Hannon Greg , Cherry Sara 
Published: June 2021

Virus‐host interactions are a delicate interplay of opposing forces: the virus subverts cellular machinery to promote its replication while the host mounts an immune response to eliminate the infection. Innate immunity is the first and most ancient line of defense, yet little is known about the cellular factors that restrict viral infection. Our lab uses RNAi screening in cells of Drosophila melanogaster, a model organism that only has an innate immune system, to identify novel antiviral factors. Of particular interest to us are pan‐antiviral factors that restrict arthropod‐borne viruses, for which there is significant morbidity and mortality worldwide due to the lack of vaccines or therapeutics. Using this approach, we previously identified Ars2, which restricts human arthropod‐borne viruses via the antiviral RNA silencing pathway. To find additional antiviral factors, we screened a panel of genes involved in various aspects of RNA metabolism against 4 RNA viruses, including 2 arthropod‐borne pathogens. We focused on genes whose depletion by RNAi leads to increased infection by at least 3 of the 4 viruses tested. We found that factors involved in various steps of RNA biogenesis and metabolism had antiviral effects. We are now characterizing the antiviral activity of these factors and their role in small RNA silencing.Funded by RO1AI074951 to SC.
Valproic acid suppresses interleukin‐1β‐induced microsomal prostaglandin E2 Synthase‐1 expression in chondrocytes
Authors: Zayed Nadia , Chabane Nadir , Elmansouri Fatima Ezzahra , Martel‐Pelletier Johanne , Pelletier Jean‐Pierre , Fahmi Hassan 
Published: June 2021

INTRODUCTIONMicrosomal prostaglandin E2 Synthase (mPGES)‐1 catalyzes the terminal step in the biosynthesis of PGE2. Early growth response factor‐1 (Egr‐1) is a key transcription factor in the regulation of mPGES‐1. In the present study we examined the effects of valproic acid (VA), a histone deacetylase (HDAC) inhibitor, on interleukin (IL)‐1β‐induced mPGES‐1‐expression in human chondrocytes.METHODSChondrocytes were stimulated with IL‐1 in the absence or presence of VA, and the level of mPGES‐1 protein and mRNA expression were evaluated using Western blotting and real‐time reverse‐transcription polymerase chain reaction, respectively. The mPGES‐1 promoter activity was analyzed in transient transfection experiments. Egr‐1 recruitment to the mPGES‐1 promoter were evaluated using chromatin immunoprecipitation (ChIP) assays.RESULTSVA dose‐dependently suppressed IL‐1β‐induced mPGES‐1 protein and mRNA expression as well as its promoter activation. Treatment with VA did not alter IL‐1‐induced Egr‐1 expression, nor its recruitment to the mPGES‐1 promoter, but prevented its transcriptional activity.CONCLUSIONOur study demonstrates that VA inhibits IL‐1‐induced mPGES‐1 expression in chondrocytes. The suppressive effect of VA was not due to reduced expression or recruitment of Egr‐1 to the mPGES‐1 promoter.