bluuebunny/crossref_metadata_2025 huggingface dataset:
AMINO ACIDS REGULATE DE NOVO PURINE SYNTHESIS VIA AKT REGULATION OF PENTOSE PHOSPHATE PATHWAY ENZYMES
Authors: Saha Arindam , Amador Deron , Fridman Alla , Pilz Renate , Boss Gerry
Published: June 2021

The focus of this study is to investigate how starvation for any single essential amino acid reduces de novo purine synthesis in cultured human cells. As little as 3 h of starvation for lysine reduces de novo purine synthesis by 70% in HeLa cells. Decreased purine synthesis results largely from a 45% decrease in phosphoribosylpyrophosphate (PRPP) production via the non‐oxidative pentose phosphate pathway (PPP); flux through the oxidative PPP is unchanged. The activity of two key non‐oxidative PPP enzymes transketolase (TK) and transaldolase (TA)‐decreased by 50–60% during amino acid starvation. Restoring lysine in the medium for 30 min completely returns purine synthesis, PRPP production, and TK and TA activities to control values. Expressing TK and TA individually has only a small effect on de novo purine synthesis, but expressing both enzymes simultaneously doubles the rate of purine synthesis in starved cells. Amino acid starvation causes a 50% reduction in Akt activity, a serine‐threonine kinase and a master regulator of intermediary metabolism. Constitutively active, myristoylated Akt increases TK and TA activities and completely restores de novo purine synthesis in starved cells, while kinase‐dead Akt prevents reversal by amino acid restoration. We conclude that amino acid starvation decreases de novo purine synthesis through Akt‐mediated regulation of TK and TA.
Arginine metabolism and sensing by mTOR in T cells
Authors: Senkevitch Emilee , Frauwirth Kenneth , Tuchman Mendel , Bequette Brian
Published: June 2021

Arginine has been shown to play an essential role in T cells, as arginine depletion in the immune microenvironment has a negative effect on T cell function. The goal of this study was to characterize how T cells sense and metabolize arginine.

Experiments were carried out using murine T cells stimulated by CD3 and CD28 as well as the lymphoma cell line EL4. Proliferation and IL‐2 ELISA was used to assess T cell function. Western Blots were performed to determine amino acid sensing by the mTOR pathway. To establish pathways of arginine metabolism, a stable arginine isotope was used and metabolic products were analyzed using GC‐MS. Metabolomic analysis by GC‐MS was used to identify any unsuspected signaling and metabolic pathways regulated by arginine. RT‐PCR was used to analyze expression of enzymes known to be involved in arginine metabolism.

T cells stimulated in the absence of arginine fail to proliferate or increase production of IL‐2, and activated T cells increase their uptake of arginine. T cells differentially sense arginine, citrulline, and ornithine through mTOR. Urea cycle enzymes are not expressed in T cells. GC‐MS analysis showed that arginine is not a substrate for the Krebs cycle and potentially plays a role in fatty acid synthesis. This research was supported by a Howard Temin Award from NCI/NIH and a New Investigator Grant from the Leukemia Research Foundation.
An inhibitory role of quercetin on adipocyte differentiation by mechanisms involving p27 protein stability and suppression of G1/S phase transition during preadipocyte proliferation
Authors: Nam Heesun , Ferguson Bradley S , Rainey Claire F , Morrison Ron F
Published: June 2021

With recent interest in bioactive food components as therapeutic tools in the prevention and treatment of obesity and related morbidities, the flavonol, quercetin has garnished considerable attention for its potent anti‐oxidant and anti‐inflammatory effects. Using 3T3‐L1 differentiation as a model of adipocyte hyperplasia, we observed that quercetin potently inhibited adipogenesis, but only when treated during the first 48 hrs of differentiation when preadipocytes (PA) transition through an obligatory phase of synchronous cell cycle progression. We also demonstrated that quercetin arrested PA proliferation during late G1 phase in a dose‐dependent manner with no measurable indices of toxicity. To determine a mechanistic role for quercetin in suppressing PA proliferation, we examined its effects on p27, a cyclin dependent kinase inhibitor known to regulate G1‐to‐S phase transition. We showed that quercetin inhibited the decrease in p27 protein abundance that is required for S phase progression, independent of changes in mRNA expression. We further demonstrated that quercetin increased p27 protein half‐life by mechanisms involving regulated proteolysis. Collectively, these data demonstrate that quercetin inhibits adipocyte differentiation by mechanisms involving p27 protein degradation and suppression of G1/S phase transition during PA proliferation. Supported by NIH‐NIDDK (R15DK082799)
Sequence Independent Phylogenetic Footprinting Analysis Identifies Novel Transcription Regulation for Orexin Receptors
Authors: Perez‐Leighton Claudio Esteban , Buterick Tammy Angeline , Billington Charles , Kotz Catherine
Published: June 2021

The orexin receptors (OX1/2R) are G‐protein coupled receptors involved in the regulation of energy metabolism (Tsujino et al. 2009). Current evidence suggests that changes in expression of the OX1/2R are implicated in the development of obesity (Teske et al. 2006). However, there is limited information about the transcriptional regulation of these receptors. A sequence‐independent phylogenetic footprinting bioinformatic analysis of the OXR promoters from six species (human, rat, mouse, chimpanzee, dog and cow) identified several transcription factor binding sites (TFBS). Our preliminary results indicate the transcription factors (TFs) SP1, NFATc2, EGR1 and PPARγ are involved in the transcriptional regulation of OX1/2R. These TFs have been implicated in several rodent models of obesity, yet their specific role in the brain is undefined (Plagemann et al. 2009, van Beekum et al. 2009, T. C. Yang et al. 2006). This study is ongoing and we are currently analyzing TFBS co‐occurrences and OXR promoter's enhancer structures. Simultaneously, we have started working towards validation of the TFBS predictions in set of new immortalized rat hypothalamic cell lines, which either contain or do not contain both OXR. These findings will provide novel insight into the transcriptional regulation of the OX1/2R.

Funding: The Department of Veterans Affairs and the National Institute of Health, NIDDK, DK078985‐02S2.
Mechanism of interaction between YY1, YAF2 and Polycomb recruitment
Authors: Vargas Jose M. , Basu Arindam , Atchison Michael
Published: June 2021

Polycomb group (PcG) proteins are essential epigenetic regulators of gene expression. The mammalian transcription factor, YY1 has been shown to recruit Polycomb proteins to specific DNA sequences. A domain of YY1 compound of 25 amino acids (REPO domain) is necessary and sufficient for the recruitment of PcG proteins to DNA. Recently it has been shown that YY1 interacts with YAF2. Studies in our lab have shown that YY1‐REPO domain interacts with YAF2 to recruit PcG proteins in flies. The current research aims at delineating whether REPO domain of YY1 physically interacts with YAF2 by co‐immunoprecipitation and GST pull down assays. Transient transfections are being carried out in HEK293‐T cells with GAL‐YY1, GAL‐YY1DREPO and GAL‐REPO with FLAG‐YAF2 expression plasmids followed by co‐immunoprecipitation with nuclear extracts. Western blot analyses were performed to detect for the interaction between YY1 and YAF2. Our preliminary studies indicate that the deletion of the REPO domain reduced interaction with YAF2 suggesting that the REPO domain of YY1 is involved in interaction with YAF2.
Identification of host cell factors common to both mosquito and human cells in response to Dengue virus infection
Authors: Rout Subrat Narayan , Ramkissoon Kevin , Anderson David , Perrone Joseph , Vaidyanathan Rajeev , Perrone Joseph , Vaidyanathan Rajeev
Published: June 2021

Dengue virus (DENV) is an enveloped, positive‐stranded RNA virus with four antigenically distinct serotypes. The virus causes dengue fever which is endemic in tropical and subtropical regions of the world. Over 2.5 billion people are at risk for dengue fever with 50 to 100 million annual cases of DENV infection occur worldwide, of which 500,000 cases are life‐threatening dengue hemorrhagic fever and dengue shock syndrome. At present, there are no effective vaccines, specific therapeutics, or natural animal models for DENV. Functional genomics and proteomic analyses have been used to identify the host responses in virus infected cells or hosts providing a better understanding of the molecular basis of pathogenesis of viral infections. In this study, two mosquito cell lines C6/36, CCL‐125 and one human cell line (HUVEC) were used to infect with DENV and identified several factors up‐regulated or down‐regulated in response to virus infection. We used a 2‐DE/MS proteomics approach followed by Western blot analysis to identify differential expression of protein profiles that are common to mosquito and human cell lines following DENV challenge. Further analysis of these data will allow for a greater understanding of DENV pathogenesis and vector‐pathogen interactions.
In silico characterization and homology modeling of cyanobacterial phosphoenolpyruvate carboxylase enzymes with computational tools and bioinformatics servers
Authors: Smith Aubrey Allen , Plazas Manuela
Published: June 2021

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) catalyzes the irreversible β‐carboxylation of phosphoenolpyruvate (PEP) to yield oxaloacetate (OAA) and inorganic phosphate (Pi). PEPC is found in higher plants and most types of bacteria including cyanobacteria. Several PEPCs have been purified from plants and bacteria, including thermophilic bacteria. PEPC contributes to photosynthetic and anaplerotic CO2 fixation. In this study we computed the physicochemical properties of cyanobacterial PEPCs and compared sequences from fresh water and marine sources. The alignment of cyanobactyerial enzymes and secondary structure analysis revealed that there are conserved amino acid substitutions and polymorphisms between PEPCs from marine and fresh water organisms. Furthermore, some marine subgroups seem to possess unique amino acid stretches that may modulate various aspects of catalysis and regulation. Phosphoenolpyruvate carboxylase from Synechococcus PCC 7002, a marine organism, most closely resembles PEPC from fresh water organisms; for this reason, the enzyme was chosen for homology modeling alongside PEPCs from the fresh water strain Anabaena variabilis and the marine cyanobacterium Synechococcus RS 9917. The physicochemical characteristics and the 3D models provide a framework for the purification and characterization of cyanobacterial PEPCs.
Expression, Purification, and Characterization of a Putative Acetylcholine Binding Protein from Nostoc punctiforme
Authors: Johnson Kasey , Pisciella Victoria , Selinsky Barry S.
Published: June 2021

Nostoc punctiforme contains in its genome a series of three open reading frames encoding for putative ligand‐gated ion channels (LGICs). Between the first and second ORF lies a 121 amino acid sequence with over 40% homology to the first 120 amino acids of the three LGIC homologs. The objective of this work is to clone and express this peptide, and possibly perform a structural analysis. A 91‐residue peptide (minus a predicted signal sequence) was cloned into an expression plasmid with an N‐terminal small ubiquitin related modifier (SUMO) fusion partner, and overexpressed in BL21(DE3) cells. The largely soluble fusion protein was purified by Ni2+‐affinity chromatography, cleaved with SUMO‐hydrolase, and further purified by removal of the SUMO fusion partner. Characterization of the purified protein is ongoing, including an attempt to generate protein crystals for structural analysis.
Expression, Purification, and Characterization of a Putative Cyclooxygenase from Nostoc punctiforme
Authors: Tang Huasong , Backus Kevin , Selinsky Barry S
Published: June 2021

The use of sequence‐dependent profile searches of bacterial and fungal genomes have uncovered the existence of open reading frames encoding proteins predicted to be similar to mammalian cyclooxygenase (COX) proteins. In this study, a putative COX from Nostoc punctiforme was identified, expressed, purified, and characterized. The gene (Npun_R5469) was cloned into an expression plasmid with an N‐terminal small ubiquitin related modifier (SUMO) fusion partner, and overexpressed in BL21(DE3) cells. The largely soluble fusion protein was purified by Ni2+‐affinity chromatography, cleaved with SUMO‐hydrolase, and further purified by removal of the SUMO fusion partner. Kinetic characterization of the Nostoc protein product is in progress, and should provide insight into the evolutionary origin of mammalian COX enzymes.
Discovery of the Nitro‐Proteome in Human Myometrium
Authors: Ulrich Craig , Nordmeier Akira , Schegg Kathleen , Quilici David , Buxton Iain L. O.
Published: June 2021

NO nitrosylates one or more regulatory proteins in myometrial smooth muscle to produce relaxation. In order to examine nitrosylation of candidate proteins, we developed a method for showing S‐nitrosylation in whole tissue lysates. This method will allow us to elucidate the human myometrial smooth muscle nitroproteome and to test the effect of these post‐secondary modifications on relaxation. Our method, which we are calling Nitro‐DIGE selectively labels S‐nitrosylated proteins in a cell extract using spectrally resolvable Alexa Fluor maleimide dyes. Labeled extracts are then analyzed using 2‐dimensional in‐gel electrophoresis (2D DIGE). With this powerful technique we can identify differences in levels of S‐nitrosylation between term and preterm myometrium. These identified proteins are regulatory in nature and preliminary results have identified candidates such as heat shock protein 27 and alpha actinin which have been shown to regulate contraction in smooth muscle tissue. Further work is directed at both hypothesis‐directed examination of a select relaxation pathway, as well as identifying prominent proteins that are selectively nitrosylated by pregnancy progression and/or in response to experimental NO‐stimulation of tissues in functional assays where relaxation/contraction can be correlated with protein nitrosylation. Supported by March of Dimes Prematurity Initiative Grant 21‐FY10‐176 and NIH HD053028 to ILOB
Improved purification scheme for Riboflavin Binding Protein
Authors: Milligan Michael , Turner Geraldine , Smith Sheila Rose , Benore Marilee Ann
Published: June 2021

Riboflavin Binding Protein (RBP) is a glycophosphoprotein found in the eggs of oviparous species. Responsible for transporting riboflavin for the developing embryo,

RBP also binds copper, suggesting other potential functions for this protein. Because of its stability and functional assets, RBP has been employed for use in structure‐activity studies and other uses. RBP is abundant in chicken egg albumen and yolk and can be isolated from eggs as well as from recombinant sources; several purification methods have been reported in the literature. Most procedures require one or more steps requiring high salt, low pH, or organic solvent extraction. Improved purification procedures that minimize stringent steps would allow for faster isolation as well as retention of all structural features. We have made improvements to the procedure for isolation of RBP, including modifications to the fractionation and chromatographic steps in the protocol.
Bcl‐w promotes cell invasion by inducing mitochondrial ROS production
Authors: Um Hong‐Duck , Kim Eun Mi
Published: June 2021

Bcl‐w, a member of the Bcl‐2 family, acts in the mitochondria to block apoptosis. We have previously shown that the upregulation of Bcl‐w in cancer cells also enhances their migratory and invasive potentials by stimulating signaling components, such as phosphoinositide 3‐kinase and Akt. To better understand the mechanism underlying this new function of Bcl‐w, we deleted its C‐terminal hydrophobic tail. This treatment abolished the ability of Bcl‐w to localize in the mitochondria and promote cell invasion, suggesting that Bcl‐w requires its mitochondrial localization for enhancing cellular invasiveness. It was also found that Bcl‐w stimulates mitochondrial ROS production, which then mediates the ability of Bcl‐w to induce the invasion pathway. All these results were obtained using either human cancer cells or mouse embryonic fibroblasts. Therefore, it appears that ROS are involved in Bcl‐w‐induced invasion not only in cancer but also normal cells as well as in both human and mouse cells.
KrsB: A Novel Regulator of D. discoideum Development, Adhesion and Chemotaxis
Authors: Artemenko Yulia , Borleis Jane S. , Lee Josephine , Sanséau Doriane , Willard Stacey , Devreotes Peter N.
Published: June 2021

Studies in the social amoeba D. discoideum have contributed extensively to the current understanding of chemotaxis. To identify novel regulators of directed migration, we conducted a screen of restriction enzyme mediated integration mutants using the chemotaxis‐dependent process of multicellular development as a readout. A mutant with an insertion between krsB and aph1 had a pronounced “streamer” phenotype when plated on a bacterial lawn, and reduced chemotaxis compared to wild‐type cells. Expression of krsB, but not aph1, rescued these defects, suggesting that krsB was the gene affected by the insertion. Disruption of krsB in wild‐type cells also led to a “streamer” phenotype when developed on a bacterial lawn or non‐nutrient agar, and reduced chemotaxis to cAMP. KrsB‐null cells had reduced motility and chemotactic speed, although chemotactic index and persistence were not affected. Evidence suggests that this behavior is due to increased adhesion of KrsB‐null cells to the substrate. KrsB is a Ste20 family protein kinase and is predicted to consist of an N‐terminal kinase domain, followed by four calpain III domains. Expression of KrsB with mutations and/or truncations in these regions revealed that the catalytic domain is essential, whereas calpain III domains are dispensable for KrsB function in regulating cell development and adhesion. This work was supported by NIH grants GM34933 and GM28007 to PND.
PKCδ localization at the membrane causes increased cytoskeleton reorganization associated with cell spreading and protrusions
Authors: Jamison Joshua A , Wells Alan
Published: June 2021

In wound repair, fibroblasts coordinate asymmetric signaling for cell motility and contractions of the extracellular matrix. Systems analyses show a central role in motility for transcellular contractility as a process involving PKCδ as a mediator. In addition, PIP2 hydrolysis by PLCγ1 activates novel protein kinase c delta (PKCδ), which leads to phosphorylation of MLC‐2. As PLCγ1 is immediately activated by chemo‐kinetic signals from EGFR at the membrane, the partial activation of PKCδ would be closely localized to its activity. To directly investigate PKCδ activation and its association to MLC‐2, PKCδ constructs were created with farnesylation moieties targeting PKCδ to the membrane, denoted as (PKCδ‐CaaX) and a non‐targeting control (PKCδ‐SaaX). NR6‐WT fibroblast with PKCδ‐CaaX displayed a protrusive and expansive cell phenotype, whereas cells with PKCδ‐SaaX were similar in morphology to cells expressing GFP (transfection control). These results were further supported by PKCδ‐CaaX increased redistrubition to the leading edge with activation of phospho‐MLC and actin redistribution. From these results, it appears that membrane tethering of PKCδ leads to direct activation of MLC‐2 to affect cell morphology; interestingly, this does not require global MLC2 phosphorylation. As PKCδ localizes to the membrane, the mechanism driving cytoskeleton reorganization is dependent on membrane activated PKCδ.
Distinct roles of two lipid kinases in lysosomal transport of the Gaucher disease enzyme glucocerebrosidase
Authors: Jovic Marko , Szentpetery Zsofia , Kean Michelle , Gingras Anne‐Claude , Brill Julie , Balla Tamas
Published: June 2021

Lysosomal function in lipid metabolism relies on the efficient trafficking of glucocerebrosidase (GBA), a key enzyme in the breakdown of lysosomal glucosylceramide, through binding to its receptor LIMP2. Mutations in GBA result in mislocalization and onset of Gaucher disease. Surprisingly, the mechanism regulating GBA/LIMP2 transport to lysosomes remains elusive. In this study we have assessed the role of phosphatidylinositol 4‐phosphate (PtdIns4P), a key regulator of the Golgi sorting machinery, in LIMP2 transport. Compartment‐specific depletion of PtdIns4P results in Golgi accumulation of LIMP2. However, PtdIns4P synthesis at the Golgi has been attributed to more then one PtdIns4P kinase, including PI4KIIIβ and PI4KIIα. Inhibition of the PI4KIIIβ kinase activity resulted in the decreased lysosomal delivery of LIMP2. Depletion of PI4KIIα, however, affected trafficking at a post‐Golgi compartment, resulting in LIMP2 accumulation on enlarged endosomal compartments. This effect was decreased following the PI4KIIIβ inhibition, as LIMP2 exit out of Golgi towards lysosomes became impaired. Collectively, LIMP2 exit was found to depend on the PI4KIIIβ activity at the Golgi, while transport at the late endosomal compartment requires PI4KIIα, demonstrating sequential roles of two kinases in lysosomal transport.
Identification Of A Potential Receptor That Couples Ion Transport To Protein Kinase Activity
Authors: Ye Qiqi , Li Zhichuan , Tian Jiang , Xie Jeffrey X , Liu Lijun , Xie Zijian
Published: June 2021

In our previous studies, we have demonstrated that the Src‐coupled α1 Na/K‐ATPase works as a receptor for cardiotonic steroids, such as ouabain, to regulate cellular protein kinase cascades. Here, we further explore the structural determinants of the interaction between the α1 Na/K‐ATPase and Src, and demonstrate that the Src‐coupled α1 Na/K‐ATPase allows the cell to decode the transmembrane transport activity of the Na/K‐ATPase to turn on/off protein kinases. The α1 Na/K‐ATPase undergoes E1/E2 conformational transition during an ion pumping cycle. The amount of E1 and E2 Na/K‐ATPase is regulated by extracellular K+ and intracellular Na+. Using purified enzyme preparations we find that the E1 Na/K‐ATPase can bind both the Src SH2 and kinase domains simultaneously, and keep Src in an inactive state. Conversely, the E1 to E2 transition releases the kinase domain and activates the associated Src. Moreover, we demonstrate that changes in E1/E2 Na/K‐ATPase by either Na+ or K+are capable of regulating Src and Src effectors in live cells. Together, the data suggest that the Src‐coupled α1 Na/K‐ATPase act as a Na+/K+ receptor, allowing salt to regulate cellular function through protein kinases.
The role of the alpha‐tocopherol transfer protein during zebrafish embryogenesis
Authors: Miller Galen William , Labut Edwin M , Lebold Katie M , Barton Carrie , Tanguay Robert L , Traber Maret G
Published: June 2021

The alpha‐tocopherol transfer protein (TTP) is required for vitamin E bioavailability and transfer to the blood stream. The finding that TTP is expressed in mammalian uterine and placental cells emphasizes the significance of TTP during embryogenesis. To study TTP's role in fetal development, a non‐placental model is essential. The zebrafish meets these requirements. Expression of TTP mRNA in zebrafish embryos increases 10‐fold within the first 10 h of zebrafish development and increases again between 24 and 36 h post fertilization (hpf). TTP was knocked down using morpholinos (MO) designed to inhibit TTP mRNA processing by interfering with proper splicing or translational initiation. Attenuating TTP by blocking proper mRNA splicing in the zebrafish embryos caused a distinct “pinched” phenotype in >50% of the embryos, observed at 8 hpf, resulting from an irregular yolk sac engulfment, suggesting membrane instability. Corresponding with the second increase in TTP mRNA, both MOs at 24 hpf caused severe cell death in the developing brain (>50% and >95%, splice‐blocking and translation inhibition respectively) while concentration matched controls caused less (<5%). To control for non‐specific effects we injected a MO against p53, which resulted in less mortality by 8 hpf while preserving the TTP knockdown effects. Thus, TTP is vital for embryonic survival.

Funding: NICHD 5R01HD062109‐02
Detection of phosphoinositide‐binding proteins in Tetrahymena vorax using liposomes as an affinity binding matrix
Authors: Yarbrough Thomas Michael , Ryals Phillip E
Published: June 2021

Tetrahymena vorax, a unicellular eukaryotic organism, was surveyed for the presence of proteins that bind to inositol‐containing phospholipids. Cultures of T. vorax were homogenized at logarithmic growth phase, and microsomal fractions isolated by ultracentrifugation. All fractions were assayed for the microsomal marker cytochrome c reductase. Fractions enriched in microsomes were carbonate extracted and dialyzed. Microsomal protein samples were incubated with control liposomes containing biotin‐tagged phosphatidylethanolamine and lacking any phosphoinositides, and experimental liposomes contained the same lipids and an inositol phospholipid. After the liposomes were allowed to bind to streptavidin‐agarose beads, protein was electrophoresed and stained with SYPRO‐Ruby. Preliminary data show at least two bands (44 and 49.5 kDa) in the experimental samples that have greater staining intensity than the corresponding control bands, suggesting the presence of phosphoinositide‐binding proteins in the microsomal samples. Further investigation of phosphoinositide‐binding proteins is underway using protein from macrostomal and microstomal cell types of Tetrahymena patula.
Fatty acyl‐CoA interaction with LXRα
Authors: Kaliappan Alagammai , Kelzer Matthew S. , Rider S. Dean , Hostetler Heather A.
Published: June 2021

Liver X Receptor‐α (LXRα), is a ligand activated nuclear receptor predominantly expressed in liver, kidney, intestine, adipose tissue and adrenal glands. Binding of oxysterols to LXR activates transcription of genes which are involved in cholesterol, lipid and carbohydrate metabolism. Previous studies suggested that LXR can also bind unsaturated fatty acids (FA) and inhibit transcription of sterol regulatory element binding protein (SREBP‐1C). However, the significance of this finding is unclear, since another study showed that unsaturated FA inhibition of SREBP‐1c activity is independent of LXRα. Since increased levels of FA and fatty acid derivatives (fatty acyl‐CoAs, FA‐CoA) are associated with several metabolic disorders, the purpose of this study was to examine the interaction of FA and FA‐CoA with recombinantly expressed full‐length, human LXRα. LXRα displayed high affinity for fluorescently labeled saturated C12 and C16 FA‐CoAs (Kd = 15–32nM). Circular dichroic spectra confirmed structural changes of LXRα upon binding of unlabelled saturated fatty acyl‐CoA, suggesting that FA‐CoA serve as endogenous ligands for LXRα. Effects of these ligands (FA‐CoA) could alter the regulation of genes involved in metabolism and energy homeostasis. Since FA are readily metabolized in cells, these data may help explain the inconsistency of previous studies. This work was supported by USPHS NIH grant DK77573.
Phosphorylation of peroxiredoxin 6 changes its conformation and increases its binding to phospholipids
Authors: Rahaman Hamid , Zhou Suiping , Shuvaeva Tea , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

Peroxiredoxin 6 (Prdx6), a unique non‐seleno mammalian peroxidase, is a bifunctional protein with GSH peroxidase and acidic‐calcium independent phospholipase A2 (aiPLA2) activities. It has been shown previously that phosphorylation of Prdx6 by mitogen activated protein kinase regulates its aiPLA2 activity. This study focused on the equilibrium and kinetic aspects of the interaction of recombinant native and phosphorylated human Prdx6 with liposomes at pH 7.4 using circular dichroism (CD) and fluorescence techniques. Phosphorylation of Prdx6 decreased its negative far‐UV CD peak and tryptophan fluorescence quenching indicating a conformational change of the protein. Using native tryptophan fluorescence emission or fluorescence from DNS‐PE labeled liposomes, Prdx6 bound only to oxidized liposomes (apparent binding constant, K, 30.3 ± 11.1 μM) while phosphorylated Prdx6 bound to both reduced and oxidized liposomes (K= 18.5 ± 5.8 and 32.9 ± 11.8 μM, respectively). These specific interactions were confirmed by analysis of the far‐UV CD spectrum of Prdx6. Our results indicate that change in the conformation of Prdx6 upon its phosphorylation allows binding to its lipid substrate as the basis for enhanced PLA2 activity at pH 7.4.
Identification of a common motif for the recognition of moieties containing polyunsaturated fatty acids
Authors: Shulga Yulia V , Topham Matthew K , Epand Richard M
Published: June 2021

The pattern of residues L/I‐X (2–4) ‐R‐X (2) ‐L‐X (3–4) ‐G, in which ‐X(n) ‐ represents n residues of any amino acids, is found in several enzymes acting on polyunsaturated fatty acids. For enzymes that do exhibit preference for polyunsaturated fatty acid‐containing lipids, generally only one isoform has this specificity and it is also the only isoform with this motif. One example we study is diacylglycerol kinase epsilon (DGKε). It is the only one of the 10 mammalian isoforms of DGK that exhibits arachidonoyl specificity and is the only isoform with the above motif. Mutations of the essential residues in this motif result in loss of arachidonoyl specificity. Furthermore, DGKα can be converted to an enzyme having this motif by substituting only one residue. When DGKα was mutated to gain the motif, the enzyme also gained some arachidonoyl specificity. This motif is also present in an isoform of phosphatidylinositol‐4‐phosphate‐5‐kinase (PIP5K) that we showed had arachidonoyl specificity for its substrate. Single residue mutations within the motif of this isoform result in loss of activity against an arachidonoyl substrate. We also demonstrate the importance of acyl chain specificity for the phosphatidic acid activation of PIP5K, and that this activation is dependent on the substrate. This is the first demonstration of a motif that endows specificity for an acyl chain.
Effects of Fasting on Food Choices
Authors: Tal Aner , Wansink Brian C. , Shimizu Mitsuru
Published: June 2021

How does food‐deprivation effect food choices? The study reported here suggests that people who have suffered mild food deprivation lean more towards higher calorie foods and consequently consume more of them. Participants (N = 81) were instructed to avoid eating for 18 hours prior to participating in the study during lunch. Importantly, only participants who reported not normally skipping breakfast were chosen for the study, as it was hypothesized that the manipulation would be more efficacious for them. Since not all participants obeyed instructions we only counted those who skipped breakfast as our fasting condition. We examined the order in which they chose to eat 6 different foods belonging to 3 different food groups: carbohydrates (dinner rolls and fries), protein (chicken fingers and cheese), and vegetables (green beans and carrots). Analyses revealed that fasting participants started with carbohydrates first (41.03%) to considerably greater extent than did non‐fasting participants (20%): χ2= 4.37, p = .04. Carbs replaced vegetables as the first chosen food. Choice of starting with vegetables went down from 52.5% for control participants to 23.08% for fasting participants. Importantly, eating a food first led to consuming more of a food, r = .24 (p < .0001). Effects were particularly marked for females. No funding

Grant Funding Source: None
The origins and consequences of heterogeneous neurons in a molecular oscillator model of the mammalian circadian clock
Authors: Webb Alexis Brooke , Taylor Stephanie , Petzold Linda , Doyle Frank , Herzog Erik
Published: June 2021

The mammalian suprachiasmatic nuclei (SCN) contain 20,000 neurons capable of generating near 24‐h rhythms. Recent data from functionally isolated SCN neurons show that single cells can exhibit a range of oscillatory phenotypes when removed from their network. The implications of variability in the quality of oscillations between circadian cells remain unknown. We exploited known bifurcations in a set of parameters from an existing deterministic model to reveal their contributions to different behaviors in SCN neurons. By simulating single cells with a range of parameter values, the authors (1) produced a range of oscillatory phenotypes similar to biological cells, (2) found that phenotype was more sensitive to changes in rates of transcription and degradation of Period than to changes in rates of translation and phosphorylation, and (3) determined phenotype‐dependent properties using velocity response curves. The authors next looked for potential biological consequences of building networks from these heterogeneous oscillators and (4) observed that the rate of resynchronization in the network depends on the number and location of damped cells, such that when coupling is reinstated, network topologies in which damped oscillators are highly connected restore rhythmicity and synchrony faster. These results indicate that heterogeneity in oscillatory ability amongst neurons is beneficial and their position within the network can influence the behavior of the system overall. Thus, a range of oscillation types is crucial for a resynchronizing network and damped cells in particular aid the speed of synchronization.
Studies of the molecular mechanism of Salmonella mediated the caspase‐independent apoptotic pathway
Authors: Wu Hung‐Yi , Lin Chia‐Ying , Chang Ching‐Dong , Shen Perng‐Chih , Chang Tsung‐Chou , Liao Ming‐Hui
Published: June 2021

Apoptosis plays an important role in infection, cancer, embryo development and tissue growth balance. In intracellular mechanism of infection avoiding, apoptosis could be induced to prevent intracellular proliferation of virus or bacteria. Apoptosis included caspase‐dependent and caspase‐independent pathways. Due to the previous studies revealed that the caspase‐dependent pathway is not the only one apoptotic pathway in Salmonella‐infected cells, the other apoptotic pathway in Salmonella‐induced apoptosis needs to be manifested. The TUNEL and Annexin V assay on mice tissues to evaluate apoptosis, and immunohistochemistry and immunofluorescence on mice tissues to evaluate the effect of AIF, Endo G and caspases. After the cells were treated with Salmonella, the Salmonella‐infected cells were treated with caspase inhibitor for the studies of TEM and immunofluorescence to analyze the intracellular distribution of Salmonella. We realized the intracellular effects of AIF and Endo G and the relationships between them by Western Blot analysis. Thus, we clarify Salmonella induced apoptosis via caspase‐independent pathway. The changes and interaction of apoptotic proteins, AIF and Endo G. in Salmonella‐ induced apoptosis.
Defining a mechanism of circadian output in Neurospora crassa: temporal regulation of a MAP kinase pathway
Authors: Goldsmith Charles S , Lamb Teresa , Bennett Lindsay , Finch Katelyn , Bell‐Pedersen Deborah
Published: June 2021

In the filamentous fungus Neuropora crassa, the Osmotically Sensitive (OS) signal transduction pathway is rhythmically activated and functions as an output pathway from the circadian clock. This conserved fungal pathway is composed of a sensory phosphorelay coupled to a mitogen activated protein kinase (MAPK) cascade. Our goal is to define the mechanism utilized by the FREQUENCY/WHITE COLLAR (FRQ/WCC) circadian oscillator to mediate rhythmicity of OS pathway activity that subsequently affects transcription of clock controlled genes. We show that expression of two components in the OS pathway, the histidine phosphotransferase encoded by hpt‐1 and the downstream MAPK kinase kinase encoded by os‐4, is regulated by the FRQ/WCC oscillator. We also demonstrate that the promoter of os‐4 is rhythmically bound by the White Collar Complex (WCC), a transcription factor and core component of the FRQ/WCC oscillator, with a period and phase that correlate with the rhythmic accumulation of os‐4 transcript. Furthermore, deletion of the promoter region bound by the WCC results in arrhythmic expression of os‐4 indicating that direct clock interaction is required for rhythmicity of os‐4. Interestingly, hpt‐1 expression rhythms peak in an opposite phase relative to os‐4 expression, and the hpt‐1 promoter is not bound directly by the WCC. Our research tests how clock regulation of OS pathway components affects the activation state of the pathway and expression of downstream genes. Our working model predicts that the activity of HPT‐1 inhibits pathway activation, while the activity of OS‐4 promotes pathway activation; therefore, by expressing these two components in opposite phases, the clock coordinates their activity to set the activation state of the pathway. By variably expressing the levels of these two components, we hypothesize that the clock can tune the basal level of pathway activation resulting in rhythmicity. This work is funded by a grant from the NIH.
Cellular reprogramming by bacterial lipoprotein rescues ST2‐deficient mice from lethal sepsis via acceleration of phagosome maturation
Authors: Wang Jiang Huai , Buckley Julliette M. , Liu Jinghua , Blankson Siobhan , Di Wu Qiong , Redmond H. Paul
Published: June 2021

Background

ST2, a member of the Toll‐IL‐1 receptor (TIR) superfamily, negatively regulates both TLR2 and TLR4 signaling. However, it remains undefined whether or not ST2 influences phagocyte‐mediated antimicrobial responses.

Methods

Polymicrobial sepsis was induced by cecal ligation and puncture in wild‐type and ST2‐deficient mice. Phagosome maturation was assessed by detecting phagosome acidification and phagosome/lysosome fusion.

Results

ST2‐deficient mice were more susceptible to polymicrobial sepsis than their wild‐type littermates, with an increased production of proinflammatory cytokines. Bacterial clearance from the circulation and visceral organs following polymicrobial infection was significantly delayed in ST2‐deficient mice. This was associated with a reduced antimicrobial response and impaired phagosome maturation characterized in ST2‐deficient phagocytes. Cellular reprogramming by bacterial lipoprotein (BLP) tolerisation protected ST2‐deficient mice against polymicrobial sepsis‐related lethality and abrogated the proinflammatory effect of ST2‐deficiency. Moreover, bacterial clearance was accelerated in BLP‐tolerised, ST2‐deficient mice, with substantially enhanced bacterial uptake, phagocytosis and intracellular killing of either gram‐positive S. aureus or gram‐negative S. typhimurium, and accelerated phagosome maturation.

Conclusion

ST2‐deficient mice exhibit an increased susceptibility to polymicrobial infection, and cellular reprogramming by BLP rescues ST2‐deficient mice from lethal sepsis by attenuating the overwhelming proinflammatory response, and simultaneously accelerating bacterial clearance via enhanced phagosome maturation and bactericidal activity.
An in vivo hypoxic environment induces atypical responses in HUVEC derived from sickle cell anaemia pregnancy
Authors: Decastel Monique Lucie
Published: June 2021

While it is recognized that hypoxic stress could induce responses locally and/or remote to the initial sites, less attention has been devoted to the later, probably due to unavailability of adequate models. Accordingly, endothelial cells from umbilical veins in sickle cell disease pregnancy (HUVECSCDP), previously used to explore remote hypoxia in vivo, were isolated and characterized. Using fluorescence microscopy and Western blot analyses, we demonstrated that isolated HUVECSCDP retained, for at least 5 passages in culture, their main in vivo characteristics that are high level expression of von Willebrand factor, P‐selectin, ICAM‐1 and VEGF proteins. Furthermore, additional Western blot experiments indicated that, unlike normal HUVEC cultured under similar conditions, HUVECSCDP expressed higher NFkB (p65) level and solely expressed the phosphorylated/activated form of the extra cellular regulated kinase42/44(ERK42/44). Cobalt chloride, a mimetic of hypoxia, markedly increased protein tyrosine‐phosphorylation in HUVEC, whereas it did discretely in HUVECSCDP. After stimulation by tumour necrosis factor‐alpha, adhesion molecule levels peaked more rapidly in HUVECSCDP but at lower rate (36‐fold; p=0.03) than in HUVEC (128‐fold) as corroborated by flow cytometry. Taken together, HUVECSCDP constituted a natural human model to study hypoxic and inflammatory responses in macrovascular endothelial cells.
Interleukin‐1‐induced cyclooxygenase‐2 and inducible nitric oxide synthase expression in human OA chondrocytes is associated with histone H3K4 methylation
Authors: El mansouri Fatima Ezzahra , Chabane Nadir , Zayed Nadia , Martel‐Pelletier Johanne , Pelletier Jean‐Pierre , Fahmi Hassan
Published: June 2021

Objective

In the present study, we investigated the role of H3K4 methylation in interleukin‐1β (IL‐1)‐induced COX‐2 and iNOS expression in human OA chondrocytes.

Methods

Chondrocytes were stimulated with IL‐1 for various time periods and the expression of iNOS and COX‐2 mRNAs and proteins were evaluated using RT‐PCR and Western blotting, respectively. H3K4 methylation at the iNOS and COX‐2 promoters was evaluated using ChIP assays. The role of histone methylation was further evaluated using the methyltransferase inhibitor, 5′‐deoxy‐5′(methylthio) adenosine (MTA).

Results

IL‐1 induced iNOS and COX‐2 mRNA and protein in a dose‐ and time‐dependent manner. The induction of iNOS and COX‐2 expression by IL‐1 was associated with H3K4 di‐ and trimethylation at the iNOS and COX‐2 promoters, whereas the levels of H3K4 monomethylation remained unchanged. Treatment with MTA inhibited IL‐1‐induced H3K4 methylation as well as IL‐1‐induced iNOS and COX‐2 expression.

Conclusion

These results indicate that H3K4 methylation contributes to IL‐1‐induced iNOS and COX‐2 expression and suggest that this pathway could be a potential target for pharmacological intervention in the treatment of OA disease.
Mechanisms of the Anti‐inflammatory Action of Apocynin
Authors: Houser Kenneth Roy , Johnson David K , Ishmael Faoud T
Published: June 2021

Apocynin is a natural compound that has been used for centuries to treat a number of diseases, including asthma. We have shown that apocynin inhibits production of numerous cytokines (CCL2, CCL7, GM‐CSF, IL‐8, and IL‐6) in airway epithelial cells stimulated with TNF‐α. The mechanism of apocynin action is unclear; it can act as an anti‐oxidant, but has also been proposed to prevent generation of reactive oxygen species by inhibition of the p47 subunit of the NADPH oxidase. Using 15 amino acid synthetic peptides corresponding to cysteine‐containing regions of p47, we have shown by mass spectrometry that apocynin induces intermolecular disulfide bond formation. Similarly, apocynin induced the disulfide‐linked oligomerization of purified p47 protein. Furthermore, apocynin treatment altered the gel mobility of another NADPH oxidase component, Noxo1 in human airway cells. These effects do not seem to be specific, as apocynin also oxidizes cysteines in albumin and tubulin. We propose that apocynin can act as both an oxidant and anti‐oxidant, and can exert effects on cellular proteins in the inflammatory response. This work was supported by internal funding at the Penn State College of Medicine.
Pathogen induced upregulation of the lysosomal maturation protein, Melanoregulin (MREG) in macrophages
Authors: KAZI ALTAF S , Boesze‐Battaglia Kathleen
Published: June 2021

In professional phagocytic cells such as the retinal Pigment epithelial (RPE) cells, the product of the Mregdsu gene, melanoregulin (Mreg) plays a critical role in lysosome‐dependent phagosome maturation and degradation of phagocytosed photoreceptor debris. MREG null mice demonstrate defects in lysosomal maturation, alkalanization of lysosomal pH, incorrect processing of key degradative enzymes such as cathepsin‐D resulting in the accumulation of the engulfed cargo. We hypothesize that in macrophages Mreg plays a similar in the phagolysosmal dependent clearance of engulfed microbial pathogens. Treatment of macrophage cells, J774 with purified lipopolysaccharide (LPS) from E. coli and the periodontal pathogen, P. gingivalis resulted in the transcriptional up regulation of Mreg mRNA and protein. Treatment of J774 cells with the PI‐3‐kinase inhibitors, LY29004 and Wortmanin inhibited LPS induced expression of Mreg. Analysis of Mreg promoter indicated conservation in sequence and transcription factor binding sites such as SP1 and AP1. Mreg induction in macrophages challenged with bacterial endotoxins and its proposed role in lysosomal maturation suggests that Mreg may have a pivotal role to play in the clearance of microbial pathogens.
Cranberry Phytochemicals Inhibit Glycation of Human Hemoglobin and Serum Albumin by Scavenging Reactive Carbonyls
Authors: Liu Haiyan , Liu Haiwei , Wang Wei , Khoo Christina , Taylor Jim , Gu Liwei
Published: June 2021

The objective of this study was to investigate the inhibitory effects of cranberry phytochemicals on protein glycation. Cranberries were purified to yield a sugar‐free powder, which was fractionated into ethyl acetate fraction and water fraction. Water fraction was separated into water fraction‐I, II, and III. Antiglycation activities were tested using glycated hemoglobin (HbA1c), human serum albumin (HSA) ‐glucose, and HSA‐methylglyoxal assays. Cranberry powder and its fractions significantly inhibited the formation of HbA1c by 41.7–81.9 %. The concentrations of cranberry phytochemicals required to inhibit 50% of albumin glycation (EC50) in HSA‐glucose assay was lower than aminoguanidine except for water fraction‐I. The cranberry powder and fractions inhibited glycation of HSA by methylglyoxal. Cranberry phytochemicals scavenged 21.6–89.3% methylglyoxal within 6 hours. Fractions enriched with procyanidins showed higher antiglycation activities. Epicatechin, the constituent unit of procyanidins, were incubated with methylglyoxal and glyoxal at pH 7.4. Five adducts were detected and their structures were tentatively identified using HPLC‐ESI‐MSn. In conclusion, cranberry phytochemicals inhibited glycation of human hemoglobin and serum albumin by scavenging reactive carbonyls. This research is supported in part by Ocean Spray Cranberries, Inc.
Regulation of the dopamine transporter by dopamine D2 receptors and the AKT‐GSK3 signaling cascade
Authors: Miller Leah , Blakely Randy D.
Published: June 2021

Dopamine (DA) D2 receptors (D2R) modulate DA neurotransmission via actions on DA neurons and their targets. The DA transporter (DAT, SL6A3), is regulated by and forms a complex with D2 short autoreceptors. D2R signaling has been shown to involve the protein kinase B (Akt)‐glycogen synthase kinase‐3 (GSK3) cascade. As the interplay between D2R and Akt signaling to DAT is poorly understood, we sought to establish an in vitro model for their functional interactions using real‐time assessment of DAT activity with a novel non‐DA substrate of DAT that provides for measurements of DAT without activation of the D2R. In parallel, [3H]DA uptake studies are pursued, along with western blot analyses. Results indicate that Akt inhibition decreases basal DA uptake whereas GSK3 inhibition lead to increased basal DA uptake in HEK‐293T cells transiently transfected with hDAT and FLAG‐tagged D2R. This work has been supported by NIH grant #T32 NS061201.