bluuebunny/crossref_metadata_2025 huggingface dataset:
Progress towards development of an ELISA for quantifying avian CBG
Authors: Antunes Ivy , Johnson Elizabeth , Malisch Jessica , Mertz Pamela S.
Published: June 2021

Corticosterone (CORT) is the avian equivalent to mammalian cortisol, one of the stress hormones expressed as part of the acute stress response. The biochemical response to environmental stressors is highly conserved amongst vertebrates, allowing for use of avian models in mammalian stress research. Corticosterone binding globulin (CBG) is a carrier protein for CORT which binds ~80% of CORT in the bloodstream. Current research indicates that only unbound CORT is active, suggesting that CBG acts as a regulator as well as a carrier of CORT. CBG declines under chronic stress which would correlate with higher levels of free CORT during the acute stress response. There are also studies which indicate that the CBG‐CORT complex has a biological role separate from CORT delivery or inactivation, and studies which indicate that unbound CBG can play a role in cell signaling pathways independent of CORT binding. There is evidence in humans that the CBG‐CORT complex can increase expression of cyclic adenosine monophosphate, but it is unknown if it has the same effect in birds. Routine quantification of CBG could lead to a more clear definition surrounding its role in the stress response and clarify some of its other biological functions. This project reports on the progress of refining an avian CBG enzyme linked immunosorbent assay (ELISA) using a novel CBG antibody against zebra finch (Taeniopygia guttata), and developing a standard for use in the ELISA for quantifying CBG. The current accepted technique for quantifying CBG is a radioactive assay which is not accessible to all researchers, highlighting the necessity of development of an ELISA to be an easier, safer, and more accessible method for quantification

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondrial Oxidative Stress And Adipocyte Protein Carbonylation
Authors: Huang Yimao , Hauck Amy , Chen Yue , Bernlohr David A
Published: June 2021

At the molecular level, obesity‐linked type 2 diabetes is characterized by chronic low‐grade inflammation driven by resident macrophages resulting in potentiated adipocyte oxidative stress, mitochondrial dysfunction, lipid oxidation and altered gene expression. Among the many mechanisms linking inflammation to metabolic disease, protein carbonylation has been considered a primary event linking oxidative stress to insulin resistance. Protein carbonylation refers to oxidative posttranslational modification of proteins with reactive lipid aldehydes such as 4‐hydroxy trans 2,3 nonenal (4‐HNE), 4‐hydroxy trans 2,3 hexenal (4‐HHE), or 4‐oxo trans 2,3 nonenal (4‐ONE). Lipid aldehydes are formed at high levels under conditions of increased oxidative stress in visceral, but not subcutaneous, fat tissue and has long been considered a biomarker of oxidative stress in adipocytes, neurons, and muscle cells. Since 4‐HNE and 4‐ONE are derived from lipid peroxidation, C11‐BODIPY581/591, a peroxidation‐sensitive sensor, was used to profile which organelle in 3T3‐L1 exhibited the greatest oxidation. Using C11‐BODIPY581/591, the mitochondrion exhibited the greatest probe peroxidation and is therefore the likely source of reactive lipid aldehydes in fat cells. Moreover, carbonylation is increased coincident with lipogenesis suggesting that ROS sources in the mitochondrion drive reactive lipid aldehyde formation. Despite the localization of lipid hydroperoxides to the mitochondrion, carbonylated proteins accumulate in the nucleus of visceral, but not subcutaneous, white adipose tissue. Proteomic analysis of carbonylated nuclear proteins reveals that zinc‐finger proteins (ZFP) including the estrogen related receptors a and g are primary targets and result in loss of DNA binding activity. Mapping of carbonylation sites reveals that the DNA/RNA binding domain of ZFPs are primary targets. These results suggest that oxidative stress and lipid peroxidation in the mitochondrion may lead to retrograde signaling of aldehydes to the nucleus culminating in the carbonylation‐dependent inactivation of nuclear zinc‐finger proteins.

Support or Funding Information

Supported by NIH DK053189 to DAB

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sterols promote atlastin‐mediated endoplasmic reticulum membrane fusion in Saccharomyces cerevisiae
Authors: Jun Youngsoo , Lee Miriam , Moon Yeojin
Published: June 2021

Sterols play critical roles in various membrane fusion events, including SNARE‐mediated membrane fusion, mainly by modulating the physical properties of biological membranes; however, it remains unclear whether they also function in atlastin‐mediated endoplasmic reticulum (ER) membrane fusion. Although ergosterol, the major sterol in yeast, is essential for fusion of Sey1p (yeast atlastin)‐containing liposomes with an ER‐mimicking lipid composition, fusion of phosphatidylcholine/phosphatidylserine liposomes does not require sterols. Here, we examined whether sterols are important for Sey1p‐mediated ER fusion in Saccharomyces cerevisiae using an in vitro ER fusion assay with isolated yeast ER microsomes. Ergosterol‐specific ligands inhibited microsome fusion, indicating that ergosterol is critical for ER fusion. However, microsomes isolated from yeast strains lacking genes that encode enzymes involved in synthesis of ergosterol from lanosterol still fused, suggesting that other sterols can replace ergosterol and support Sey1p‐mediated ER fusion. Importantly, disruption of sterol‐binding motifs in the transmembrane regions of Sey1p markedly reduced ER fusion. Furthermore, Sey1p physically interacted with Erg11p, which converts lanosterol into an ergosterol precursor, suggesting that Sey1p recruits Erg11p to fusion sites and thereby enriches ergosterol, which, in turn, may recruit more Sey1p. This positive feedback loop may facilitate ER membrane fusion by concentrating fusion factors at fusion sites.

Support or Funding Information

This work was supported by a grant from the National Research Foundation (NRF) of Korea (2016R1A5A1007318) awarded to the Cell Logistics Research Center and by a grant from the GIST Research Institute at the Gwangju Institute of Science and Technology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epac1 is required for cAMP‐mediated endothelial barrier stabilization
Authors: Radeva Mariya Yosifova , Schuster Katharina , Ponce Alexander Garcia , Døskeland Stein‐Ove , Curry FitzRoy E , Reed Rolf K. , Waschke Jens
Published: June 2021

Regulation of intercellular junctional complexes is critical for the control of endothelial barrier function and dependent on cAMP‐mediated Rac1 activation. Recently, the exchange protein activated by cAMP (Epac1) has been shown to serve as a tonic stabilizer of the endothelial barrier. Here, we further elucidate the role of Epac1 in cAMP and Rac1‐mediated endothelial barrier regulation.

Transendothelial‐electrical‐resistance (TER) measurements in newly generated immortalized myocardial endothelial cells derived from Epac1‐knockout (KO) mice revealed significant reduction in the baseline TER, when compared to wild‐type (WT) cells, indicating that Epac1 is required for maintenance of endothelial barrier function. This effect was associated with fragmented VE‐cadherin staining and reduced localization of tight junctional proteins ZO‐1 and occludin along cell borders. In WT‐cells, application of forskolin (F) and rolipram (R) resulted in increased TER associated with linearization and augmentation of VE‐cadherin immunostaining at adherens junctions. In contrast, Epac1‐KO‐cells did not respond to F/R treatment indicating that Epac1 is crucial for cAMP‐mediated endothelial barrier stabilization. In comparison to WT‐cells, Epac1‐KO‐cells revealed significantly increased protein levels of Rac1, whereas Rac1 basal activity was not enhanced, indicating a defect in Rac1 activation. However, in Epac1‐KOcells, cAMP‐mediated Rac1 activation was not abolished and the basal cAMP‐concentration was significantly increased. Additionally, in Epac1‐deficient cells F/R‐mediated increase in cAMP levels was less compared to WT cells. Taken together, our data show that Epac1 is crucial for cAMP‐mediated barrier stabilization by mechanisms which at least in part are independent of Rac1 regulation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Specificity Phosphatase and Pro Isomerase Domain Containing 1 (Dupd1) is Upregulated During Neurogenic Skeletal Muscle Atrophy and is Differentially Expressed in MuRF1‐null Mice
Authors: Cooper Lisa , Waddell David
Published: June 2021

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Dual Specificity Phosphatase and Pro Isomerase Domain Containing 1 (Dupd1) has been identified as a novel atrophy‐induced gene in skeletal muscle and has been found to be differentially regulated in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice in response to denervation. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has previously been shown to be upregulated under almost all conditions of skeletal muscle wasting. Preliminary data described in this study provides evidence that MuRF1 may play a more complex role in the muscle atrophy cascade than previously believed and appears to be necessary for full activation of a subset of neurogenic atrophy‐induced genes, including Dupd1. A recent study that utilized microarray analysis of skeletal muscle isolated from wild‐type and MuRF1‐null mice revealed that Dupd1 expression is significantly induced at 3 days and 14 days post‐denervation in wild‐type mice, while Dupd1 expression is dramatically blunted in the MuRF1‐null mice at both 3 days and 14 days post‐denervation. qPCR analysis revealed that Dupd1 is significantly upregulated during differentiation of cultured muscle cells. To determine how Dupd1 might be transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dupd1 were cloned, fused to a reporter gene and found to be highly inducible in response to ectopic expression of MyoD and myogenin. Furthermore, site‐directed mutagenesis of conserved E‐box elements in the proximal promoter of Dupd1 rendered the Dupd1 reporter genes incapable of being induced by overexpression of myogenic regulatory factors. The Dupd1 gene codes for a dual specificity phosphatase (DUSP) that can recognize and dephosphorylate tyrosine, serine and threonine amino acids of substrate proteins. Finally, we have found that Dupd1, an atypical DUSP, can modulate the ERK branch of the MAP Kinase signaling in muscle cells and inhibits muscle cell differentiation when ectopically expressed in proliferating muscle cells. Elucidating the role of Dupd1 in muscle will help contribute new insight into the molecular mechanisms for skeletal muscle atrophy.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stem Cell‐Based Modelling of Brown Adipogenesis Using Hypothermal Conditions
Authors: Leija Hilda Anaid Lugo , Velickovic Ksenija , Bloor Ian , Law James , Sacks Harold , Symonds Michael , Sottile Virginie
Published: June 2021

In vivo cold exposure is the main stimulus to activate the unique mitochondrial uncoupling protein (UCP)1, known for mediating heat production in brown and beige adipocytes. In vitro, UCP1 adipogenic response under hypothermic conditions requires an extended evaluation in order to be comparable to an in vivo cellular function. We have developed a new in vitro culture system to induce adipogenic browning in bone marrow (BM) derived mesenchymal stem cells (MSC), using exposure to reduced temperature during differentiation. Incubation at 32°C instead of 37°C over the course of the culture treatment was seen to promote brown adipogenesis with increased adipocyte response and upregulation of adipogenic and thermogenic factors including UCP1. Adipocytes differentiated under hypothermic conditions displayed enhanced uncoupled respiration and metabolic adaptation, as well as significant durable changes in cellular and molecular features. These results suggest that MSC available from accessible tissue sources can be differentiated in vitro using cold temperature conditions to favor the appearance of functional beige‐like adipocytes. This cell‐based model represents a new valuable tool to screen for pro‐browning factors in vitro and elucidate the mechanism of metabolic adaptation relevant to the browning process.

Support or Funding Information

This work was funded by the EU‐CASCADE fellowship scheme funded by the EU's 7th FP PCOFUND‐GA‐2012‐600181 (K.V.), CONACyT, Mexico (H.A.L.L.), and received support from The Cardiometabolic Disease Research Foundation (Los Angeles, USA) and the Biotechnology and Biological Sciences Research Council (BBSRC, Multidisciplinary Super Resolution Microscopy Facility grant BB/L013827/1).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysophosphatidic Acid Regulates Erythropoiesis at Different Hematopoietic Hierarchy
Authors: Chiang Jui‐Chung , Lin Kuan‐Hung , Chen Wei‐Min , Lee Hsinyu
Published: June 2021

Hematopoiesis, a complex developmental process to form blood components and replenish the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor has opposing regulatory effects on erythrocyte differentiation through activating LPA receptor 2 and 3, yet the underlie mechanisms in vivo remained unclear. Examining the LPA receptors expression profile in murine myeloid cells indicates that LPA2 is highly expressed on common myeloid progenitor (CMP), whereas expression of LPA3 increases with later erythrocyte differentiation. Therefore, we hypothesize that the expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian hematopoietic progenitors. In vitro colony forming unit (CFU) assays of murine sorted progenitor cells reveal that LPA2 agonist, GRI977143, reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist,1‐oleoyl‐2‐O‐methyl‐rac‐glycerophosphothionate (OMPT), increases the production of erythrocyte from megakaryocyte erythrocyte progenitor (MEP). In addition, LPA2 agonist treatment reduces erythroid, CMP and MEP populations in mice. These results indicate that activation of LPA2 predominantly inhibits myeloid differentiation at the early stage of myelopoiesis. By contrast, activation of LPA3 increases production of terminally differentiated erythroid cells. We also demonstrate that LPA3 agonist successfully restored phenylhydrazine (PHZ)‐induced acute hemolytic anemia in mice. Our results reveal distinct roles of LPA2 and LPA3 at different hematopoietic hierarchy in vivo, suggesting potential therapeutic strategies for the treatment of anemia.

Support or Funding Information

This research was supported by a grant from Ministry of Science and Technology, Taiwan (MOST 107‐2311‐B‐002‐009‐)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Galectin‐3 Levels in Hepatic Tissue from Lean and Obese Zucker Rats
Authors: Schuster Jane E , Motawe Zeinab Y. , Trujillo Andrea N. , Breslin Jerome W.
Published: June 2021

Obesity and metabolic syndrome are of major concern for physicians worldwide. Galectin‐3 is a β‐galactoside‐binding protein that has been shown to have regulatory functions in inflammatory responses as well as fibrosis of the liver, lung, heart and blood vessels. Galectin‐3 is widely expressed in multiple types of immune cells as well as epithelial and endothelial cells, and its properties have been explored as a therapeutic target for nonalcoholic fatty liver disease liver fibrosis, heart disease and other obesity‐associated diseases. We previously observed elevation of galectin‐3 in mesenteric fat from male and female 12‐week‐old obese Zucker rats compared to their lean counterparts. We hypothesized that galectin‐3 expression levels would be elevated in the liver in obese animals. Livers were collected from 20 Zucker rats (5 male, 5 female lean rats and 5 male, 5 female obese Zucker rats) for histology and protein analysis. After protein lysates were collected and total protein levels were normalized, galectin‐3 levels were quantified with the Wes Simple Western capillary electrophoresis assay. The results showed that livers of obese rats of both sexes display typical fatty liver pathology and have a significantly higher level of Galectin‐3 expression. The results show that increased galectin‐3 expression accompanies fatty liver in obese rats. Galectin‐3 may represent a future target for preventing tissue dysfunction in obesity and metabolic syndrome

Support or Funding Information

APS Frontiers in Physiology Fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystallographic Structural Elucidation of E. coli HU and a Four‐Way Junction
Authors: Khun Charya , Mukerji Ishita
Published: June 2021

E. coli histone‐like HU protein binds with high affinity to DNA recombination intermediates, such as the Holliday junction, to facilitate prokaryotic DNA transcription, compaction, recombination, and replication. While previous Förster resonance energy transfer (FRET) studies have determined possible structural models of HU‐junction interaction, X‐ray crystallographic data is needed to confirm those models. Studies have shown that crystals can be obtained with a dodecamer junction complex. However, we believe that HU can also be successfully crystallized with J20, that is, a junction with 20 base pairs per strand. We believe that 20 base pairs per arm is sufficient in limiting the flexibility of our junction. Our investigation aims to determine the most favorable conditions for HU‐J20 crystallization by using various solvents, pH, and viscosities. In our study, we show that our HU samples are pure, nuclease‐free, and bind with high affinity to J20. In addition, we show that our J20 samples are at least 90% properly annealed via a non‐denaturing polyacrylamide gel. Moreover, we have observed the formation of small crystals in a few of our wells, primarily around pH 8.5 in 20% polyethene glycol with 0.2 M ammonium chloride, and 0.005 M calcium dichloride. Going forward, we will screen around our successful conditions with higher protein concentrations. We will also use magnesium dichloride solutions in addition to calcium dichloride solutions, as previous studies have shown that magnesium improves the stabilization of DNA. Upon the development of crystals, we hope to solve the structure of HU‐J20 via diffraction.

Support or Funding Information

American Society of Biochemistry and Molecular Biology Undergraduate Research Award, Wesleyan Math and Science Scholars Program, Wesleyan University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increasing Methionine in the Lectin Erythroagglutanin (PHA‐E) of Phaseolus vulgaris (Black Bean)
Authors: Madhwani Ravi , Morales Celina , So Sokuntheavy , Zavala MariaElena
Published: June 2021

Many developing countries around the world do not have access to regular sources of protein (e.g. meats, fish, dairy products, etc). Beans have become a staple food in many of these communities' diets for their daily protein and calorie intake due to their availability. This crop is a great agronomic choice because it requires minimal inputs. It can be grown at small and large‐scale farms. Though it is a reliable source of protein, the most abundant proteins are low in an essential amino acid, methionine. Nutritional deficiencies can be attributed to the two major stores of protein in the bean, phaseolin and lectin; Lectin is the second most abundant seed protein. Its biochemical characteristics make it an effective anti‐herbivory agent, and if the beans are not prepared properly, lectin can affect the nutritional value. We selected several sites containing isoleucine to convert to methionine in the common black bean to improve the methionine content. Plasmids were prepared and transformed into wild type plants using Agrobacterium. The gene cassette was created and successfully introduced into T0 plants. The putative transformants were tested to confirm they carry the expected modified gene using PCR. The positive plants were allowed to grow, and their seeds were collected. The T1 plants are being tested for the expression and accumulation of PHA‐E. We will begin to investigate how the conversion of isoleucine to methionine affects the structure and function of the PHA‐E protein lectin. This research could elucidate whether we can improve the nutritional value of essential foods in third world countries.

Support or Funding Information

This research was made possible by funding through the NIH grant #GM063787.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acetylcholinesterase inhibitors enhance its folding and assembly in the endoplasmic reticulum
Authors: Medina Jessica , Kot Taylor Corbin , Rossi Susana , Rotundo Richard Lee
Published: June 2021

Acetylcholinesterase (AChE) is responsible for terminating synaptic transmission by the hydrolysis of acetylcholine (ACh) in the synaptic cleft. AChE is synthesized at a constant rate in the rough endoplasmic reticulum with only an estimated 20% of all synthesized enzyme assembling into catalytically active oligomeric forms. The overwhelming majority of the enzyme undergoes degradation through the ERAD. It is this large inactive pool we seek to understand through in‐vitro analysis using human embryonic kidney cells expressing mouse acetylcholinesterase (HEKmAChE). In particular we wished to determine whether the inhibitors could rescue inactive AChE protein into catalytically active enzyme. Galantamine and Donepezil, two commonly used therapeutics for Alzheimer's disease (AD), act as active site AChE inhibitors with the purpose of allowing ACh to persist for prolonged synaptic transmission. To understand the effects of Galantamine on inactive AChE, HEKmAChE cultures were first treated with diisopropylfluorophosphate (DFP), an irreversible and membrane permeable AChE inhibitor, and then allowed to synthesize new AChE in the presence of Galantamine during a 60 minute recovery period. Preliminary results indicated a significant increase in active AChE when exposed to Galantamine in comparison to control (defined medium alone). To focus on the mechanism by which Galantamine enhances AChE folding and assembly, HEKmAChE cultures were incubated with DFP and subsequently treated with Puromycin alone or in combination with either Galantamine or Donepezil. Using Ellman's assay for AChE we observed a significant increase in catalytically active AChE in the presence of Donepezil or Galantamine. These results suggest current therapeutic treatments may be acting as molecular chaperones during the protein folding process, enabling otherwise inactive AChE bound for degradation to become catalytically active. This research was supported by the Florida Department of Health, Biomedical research program, grant 7AZ04.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetics of Mutations in the Active and Allosteric Sites of Fumarate Hydratase
Authors: Heng Rithy Sakk , Nguyen Nhan , Weaver Todd , Taylor Ann TS
Published: June 2021

Fumarase, also known as fumarate hydratase (FH), is an enzyme that catalyzes the reversible interconversion of fumarate to malate. Fumarase has two sites: active A‐site and allosteric B‐site. Mutations in these sites especially the active site may disrupt the enzyme's efficiency and activity. The main focus of this experiment was characterizing different fumarase mutations in both the active and allosteric sites. The kinetic parameters were determined in both forward and reverse reactions. Some mutants showed a decrease in both KM and Vmax while others showed an increase in either or both KM and Vmax. The experimental results suggest that different mutations can have different effects on fumarase efficiency and its overall activity. Further studies are required to address the specific interaction of these mutations involving in those changes in fumarase activity.

Support or Funding Information

Thanks to the Haines Fund for the Study of Biochemistry at Wabash College.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Homologous Trans‐editing Factors with Broad Substrate Specificity Prevent Global Mistranslation
Authors: Musier‐Forsyth Karin , Bacusmo Jo Marie , Bakhtina Marina , Cantara William A. , Goto Yuki , Kuzmishin Alexandra B. , Suga Hiroaki
Published: June 2021

Accurate translation of genetic information is determined, in part, by aminoacyl‐tRNA synthetases (ARSs), which are responsible for the correct pairing of amino acids with their cognate tRNA adaptors. However, these reactions are susceptible to errors due to structural similarities of amino acids that challenge the specificity of some synthetases and promote the incorporation of amino acids at wrong codons during protein synthesis (mistranslation). To maintain translational fidelity, many synthetases have expanded their aminoacylation capabilities to include editing mechanisms that involve deacylation of the mischarged tRNA (“post‐transfer editing”) in a second catalytic site. In addition, single‐domain homologs of some ARS editing domains catalyze post‐transfer editing in trans. A global bioinformatics analysis has uncovered distinct trans‐editing domains related to the bacterial prolyl‐tRNA synthetase (ProRS) post‐transfer editing domain, which is known to clear Ala‐tRNAPro. While a subset of these trans‐editing proteins correct ProRS‐dependent errors Ala‐ and Cys‐tRNAPro recent studies have revealed surprising substrate specificities that include non‐proteinogenic amino acids. In addition, whereas some editing domains have borrowed some aspects of tRNA recognition from the parent aminoacyl‐tRNA synthetase, semi‐promiscuous editing may offer advantages to cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular analysis of putative virulence genes in Russian wheat aphid biotypes 1 and 2
Authors: Cui Feng , Dittmer Neal , Pierson Matthew , Smith C. Michael , Reese John , Edwards Owain , Reeck Gerald
Published: June 2021

The Russian wheat aphid (Diuraphis noxia) (RWA) is a particularly noxious pest of wheat. Our objective is to find virulent genes in RWA. Our work is guided by studies in the pea aphid, Acyrthosiphon pisum, a model aphid for which the genome project is nearing completion. Seventeen transcripts were selected for investigation from the salivary gland of Biotypes 1 and 2 of the RWA. Using RT‐PCR primers based either directly on pea aphid cDNA sequences or on a range of orthologous sequences from insect species, we have amplified and cloned regions of these 17 transcripts. We will present a summary of the results, which vary widely from transcript to transcript. Examples are transcripts for C002, trehalase and sucrase. For that encoding protein C002, which has been shown in the pea aphid to be required for feeding on a host plant, we have found 9 different haplotypes, 8 of which are apparently unique to either Biotype 1 or 2. For transcripts putatively encoding the enzyme trehalase, we have found 8 different haplotypes, 7 of which are unique to one biotype or the other. For transcripts putatively encoding the enzyme sucrase, we have found 15 different haplotypes, 8 unique to Biotype 1 and 6 unique to Biotype 2. In conclusion high level of genetic diversity in salivary gland transcripts are observed between biotypes 1 and 2 of RWA, which correlates molecular variation in salivary gland proteins with the virulence phenotype.

Supported by CRC, Australia.
Development of a non‐isotopic saturation binding assay for analysis of dynamic interactions of flagellar export apparatus proteins
Authors: Kraft Lewis , Nguyen Viet Q. , McMurry Jonathan L.
Published: June 2021

FliI is the flagellar export ATPase in Salmonella enterica. It is negatively regulated by FliH, which also likely targets FliI to the membrane‐bound export apparatus. To understand the dynamics of flagellar export, a Type III secretion process, a saturation binding study has been undertaken to ascertain affinity constants for FliI interaction with FliH and other export protein interactions using an apparatus of our own design and construction. The apparatus utilizes an affinity resin, positive pressure and a dot‐blot style immunoassay to analyze specific binding between a His‐tagged protein and an impure binding partner, eliminating the need for radiolabeling. 96 samples may be processed simultaneously, allowing for replicates and nonspecific binding to be performed under identical conditions. The apparatus has been used to determine KD for FliH (and truncations thereof) binding to FliI and is being utilized to analyze other flagellar protein interactions. This work was supported by grants from the NIH (R15GM080701), the Research Corporation (Cottrell College Science Award) and the KSU Mentor‐Protégé Program.
Structural and functional characterization of the dehydratase domains of the animal cytosolic fatty acid synthase
Authors: Pasta Saloni , Witkowski Andrzej , Joshi Anil , Smith Stuart
Published: June 2021

The objective of the study was to determine the region of the type I animal fatty acid synthase (FAS) multifunctional polypeptide that comprises the dehydratase (DH) domain and to identify residues essential for catalysis. The 4.5 Å electron density map of porcine FAS can accommodate two subunits of type II DH of E. coli (FabA) suggested that this domain may be twice as long as originally anticipated. Expression of FAS constructs of various length revealed that residues 1–809 are required for ketoacyl synthase and malonyl/acetyl transferase activity and 824–1154 for DH activity. Sequence alignment, modeling and mutational studies reveal that animal DHs consist of two pseudosubunits formed by contiguous regions of the same polypeptide, with one active site per pseudodimer, formed by cooperation of histidine 878 on one subunit and aspartate 1032 on the other; glutamine‐1036 is important in positioning the catalytic aspartate. Thus the type I DH resembles its type II counterpart, except that the former has one active site per pseudodimer as opposed to two per dimer in the latter. These studies also assign a role to part of the previously uncharacterized ‘central core’ region of type I FAS and some modular polyketide synthases as contributing to the catalytic region of the DH domain. (Supported by NIH DK16073).
Laforin and Malin: Novel Regulators of Glycogen Metabolism
Authors: Worby Carolyn A , Gentry Matthew S , Dixon Jack E
Published: June 2021

Lafora disease (LD) is an autosomal recessive neurodegenerative disease that results in progressive myoclonus epilepsy and death. LD is caused by mutations in either the E3 ubiquitin ligase, malin, or the dual‐specificity phosphatase, laforin. A hallmark of LD is the accumulation of insoluble complex carbohydrates similar to amylopectin in the cytoplasm of cells from most tissues. Classically, glycogen metabolism was shown to be regulated by phosphorylation of key metabolic enzymes. One regulator of phosphorylation status is protein targeting to glycogen (PTG/R5), a scaffold protein that binds glycogen and many of enzymes involved in glycogen metabolism, including protein phosphatase 1 (PP1), glycogen synthase, phosphorylase, and laforin. Overexpression of PTG markedly increases glycogen accumulation, and decreased PTG expression decreases glycogen stores. To investigate if malin and laforin play a role in glycogen metabolism, we overexpressed PTG, malin, and laforin in tissue culture cells. We found that expression of malin or laforin decreased PTG‐stimulated glycogen accumulation by 25%, and co‐expression of malin and laforin abolished PTG‐stimulated glycogen accumulation. Consistent with this result, we found that malin ubiquitinates PTG in a laforin‐dependent manner, both in vivo and in vitro, and targets PTG for proteasome‐dependent degradation. These results suggest a novel mechanism, involving laforin and malin, in regulating glycogen metabolism.
The Metabolic Role of PEPCK‐C in the Epithelial Cells of the Mammary Gland
Authors: Hsieh Chang‐Wen , Millward Carrie , Huang Charlie , Reineke Erin , Seachrist Darcie , Croniger Colleen
Published: June 2021

PEPCK‐C has been proven that plays an important role in both carbohydrate and lipid metabolism, and involves in type II diabetes. PEPCK‐C gene is expressed primarily in the liver, kidney, adipose tissue, and the mammary gland. Because the mechanism of PEPCK‐C in mammary gland is still unclear, we proposed the role of PEPCK‐C gene in the mammary gland is glyceroneogenesis. By the deletion analysis of PEPCK‐C promoter, we found the region from −490 to −206 is required for the expression of PEPCK‐C gene in NMuMG cells and T47D cells. According to the previous studies, we suggested the PEPCK‐C gene might be regulated by peroxisome proliferator activator gamma. Therefore, we investigated the regulation of the PEPCK‐C gene by utilizing site‐directed mutagenesis. We found that the PPAR‐gamma binding site overlaps the retinoic acid response element and is required for the basal expression of PEPCK in T47D cells. In addition, RARE1 and RARE2 are required for the promegestone induction of PEPCK expression in T47D cells. Moreover, we investigate the role of RARE1, RARE2, and RARE3 in PEPCK expression by EMSA and ChIP assays. To determine the metabolic function of PECPK‐C in mammary gland epithelial cells, we measured the synthesis of triglyceride from pyruvate using 2H2O. These data suggests that PEPCK‐C is regulated by progesterone and plays a key role in the synthesis of triglyceride in the mammary gland during lactation.
Development of Algorithms for High Throughput “Omics” Data Mining and Classification
Authors: Hammamieh Rasha , Hellman Matthew , Jett Marti
Published: June 2021

An essential part of data analysis involves the ability to identify patterns present throughout the entire omic dataset. Excel2SVM was developed as a stand alone Python mathematical analysis tool to grant researchers access to this complicated, high level algorithm. The software provides a simple graphical user interface with the capability to create a classification kernel model through minimal steps. Excel2SVM offers the ability to convert data to the proper sparse format while performing a variety of kernel functions along with cost factors/modes, grids, cross‐validation and several other functions, and the results from this software have proven to be more accurate than other currently available software in several instances. This program functions with any type of quantitative data making Excel2SVM the ideal tool for analyzing a wide variety of input.

Genecite, a stand alone open source tool for high throughput literature and pathway mining, was developed to provide a unified automated high throughput search platform to yield a summary providing literature citation counts for each given search key that can be sorted as well as hyperlinks for each query connecting the appropriate result page of the data source.
In utero exposure to HAART induces oxidative stress in adult mice offsprings
Authors: Lee Yun Kyung , Nerurkar Vivek , Frank Jennifer E , Nerurkar Pratibha
Published: June 2021

Use of nucleoside analogue reverse transcriptase inhibitors (NRTI) during pregnancy reduces HIV infection among newborns and prevents vertical transmission of the virus. The fact that alterations in fetal environment can have long‐term consequences on metabolic and endocrine pathophysiology in adult life raises concern about long‐term safety of uterine exposure to NRTI. Since NRTI can induce mitochondrial toxicity and oxidative stress, we hypothesize that infants exposed to NRTI in utero may be at increased risk of oxidative stress‐associated diseases in adult life. To test our hypothesis, pregnant mice were treated with the NRTI, combivir, two weeks before mating, during mating, pregnancy, delivery and lactation. After weaning the pups also received combivir for two additional weeks. The untreated control mothers received the vehicle, apple juice. Our data indicates that 3 months after birth, the adult offsprings of NRTI‐treated mice had significantly higher levels of blood glucose, total cholesterol and calculated LDL‐cholesterol and reduced levels of hepatic and plasma glutathione, as compared to age‐matched offsprings born to control mothers (p<0.05). Since oxidative stress is implicated in pathophysiology of insulin resistance and diabetes, studies are being conducted to address the effects of oxidative stress on glucose metabolism and mitochondrial toxicity in adult offspring. [Supported by grants in part by the Clinical Research Center of Hawaii (P20 RR011091) and the U.S. Public Health Service grants from the RCMI Program (G12 RR003061)].
Hepatic Stearoyl‐CoA desaturase 1 Deficiency Promotes Liver Inflammation Under High Carbohydrate Low Fat Diet Feeding
Authors: Liu Xueqing , Flowers Matthew , Miyazaki Makoto , Sampath Harini , Chu Kiki , Ntambi James
Published: June 2021

High carbohydrate diet causes liver steatosis and inflammation. To understand the role of stearoyl‐CoA desaturase 1 (SCD1) in liver steatosis, we generated liver‐specific SCD1 deficient mouse models (SCD1‐LKO). Previously we showed that SCD1‐LKO mice were protected from liver‐steatosis with long term high carbohydrate feeding. In this study we further analyzed the effects of a short term (10 days) feeding of high‐sucrose‐very‐low‐fat (HSVLF) diet on SCD1‐LKO mice. Surprisingly, the HSVLF feeding led to a dramatic increase in liver inflammation in SCD1‐LKO but not in Lox control mice. Histological analysis on liver sections revealed no sign of steatosis in LKO and Lox mice fed with HSVLF. The expression of macrophage specific genes Emr1 (F4/80) and CD68 in liver was significantly higher in HSVLF‐fed LKO than Lox mice. However, macrophage infiltration was not detected. We subsequently observed an increased liver ER stress response in HSVLF‐fed LKO mice, which was consistent with a dramatic elevation of the plasma ALT level. Furthermore, supplementation of the SCD1 product oleate rescued liver inflammation, ER stress response and liver injury in SCD1‐LKO mice, whereas supplementation of the SCD1 substrate–stearate to HSVLF did not reverse these effects. These data indicate that hepatic SCD1 is both necessary and sufficient to prevent the high carbohydrate low fat diet induced liver inflammation.
RGS14 is a multifunctional scaffold that binds C‐Raf‐1 and either Rap2 or H‐Ras to modulate MAPkinase signaling
Authors: Shu Fengjue
Published: June 2021

RGS14 is a multifunctional protein that contains an RGS domain, which binds Gi/oa‐GTP, a GoLoco/GPR domain, which binds Gi1/3‐GDP, and a tandem (R1, R2) Ras binding domain (RBD) which binds Rap2, a Ras‐like GTPase. However, little is known about functions of the RBD domain. Recent reports indicate that the RBD of RGS12, a close relative of RGS14, serves a scaffolding role in MAPK signaling. To determine signaling roles for the RGS14 RBD domains, we investigated the relationship between RGS14 and Rap2A. RGS14 is recruited from the cytosol to the plasma membrane to form a stable complex with wild type Rap2A, whereas constitutively activate Rap2A(GV) forms a stable complex with RGS14 in the cytosol. RGS14 also binds C‐Raf‐1 and is recruited to the plasma membrane to form a heterotrimeric complex with wild type Rap2A and C‐Raf‐1. H‐Ras and activated H‐Ras(GV) also form a stable complex with RGS14 at the plasma membrane, which is blocked by mutation (R333L) of the R1‐RBD in RGS14. These findings suggest that RGS14 switches between Rap2A or H‐Ras to modulate C‐Raf‐1and linked MAPkinase signaling.
Molecular Mechanisms for Reciprocal Regulation of the PDGF Receptor and G Protein‐coupled Receptor Kinase‐5
Authors: Cai Xinjiang , Wu Jiao‐Hui , Freedman Neil J.
Published: June 2021

Seryl (Ser) phosphorylation of the platelet‐derived growth factor receptor‐β (PDGFRβ) by G protein‐coupled receptor kinase‐5 (GRK5) diminishes the PDGFRβ signaling in vascular smooth muscle cell (SMCs). This work tests the hypothesis that the PDGFRβ tyrosine (Tyr)‐phosphorylates and activates GRK5

Using immunoprecipitated PDGFRβs, purified bovine GRK5 and [ã‐32P]ATP, we found that the PDGFRβ Tyr‐phosphorylated GRK5 with a stoichiometry of 0.8±0.1, a value 4‐fold higher than that obtained with a Y857F PDGFRβ mutant, which autophosphorylates normally but fails to phosphorylate exogenous substrates. GRK5 was activated only by the PDGF‐stimulated WT, but not the Y857F PDGFRβ. We found 26±2% more substrate peptide phosphorylation with PDGFRβs IP’d from grk5−/− than WT SMCs (p<0.05). In vitro peptide phosphorylation assays showed that PDGFRβ‐mediated Tyr phosphorylation of GRK5 increased GRK5 activity by 1.6±0.2‐fold, by increasing GRK5 Vmax (3.4±0.6‐fold, p<0.05) without altering GRK5's KM for substrate. Conversely, GRK5‐mediated Ser phosphorylation of the PDGFRâ decreased PDGFRβ activity by 40±10%.

In conclusion, the PDGFRβ and GRK5 are functionally coupled in a novel feedback regulatory mechanism.
Telling a molecular story: Tangible models of Cdc42 interacting with Intersectin
Authors:   Lincoln SMART Team, Jeske Sabine , Chau Angela H. , Giles Kurt , Colton Shannondoah A. , Reis Julie
Published: June 2021

Information transfer within the cell relies upon signaling pathways made up of interacting proteins. Rho‐family GTPases are GTP‐binding proteins, which function as molecular switches initially in the “off” state. Interactions with activators turn these GTPases “on” and they then interact with other proteins, leading to a variety of cellular behaviors such as directed movement and changes in cell shape.

Traditional methods for visualizing protein structures and interactions are often limited in the amount of information that can be conveyed in two dimensions. Using the rapid prototyping technology at the Milwaukee School of Engineering's Center for BioMolecular Modeling, we have built tangible 3D models of the Rho GTPase Cdc42 in complex with one of its activators. Our model shows the interaction of Cdc42 with the catalytic domains of intersectin, based upon the published crystal structure solved by Snyder, et al (PDB , Nature Structural Biology 2002; 9(6): 468–475). Along with computer visualization tools, tangible 3D models allow students and scientists alike to more fully explore the intricacies of protein interactions.

The SMART (Students Modeling A Research Topic) program was established by the Center of BioMolecular Modeling at the Milwaukee School of Engineering and involves partnerships between university researchers and local high schools. This work is funded by NIH‐NCRR‐SEPA and HHMI.
The phosphatase PHLPP1 is a key regulator of insulin signaling in vivo
Authors: O’Neill Audrey Kathleen , Gao Tianyan , Reyes Gloria , Newton Alexandra
Published: June 2021

The recently discovered PHLPP phosphatases, PHLPP1 and PHLPP2, control the amplitude and duration of signaling by Akt by directly dephosphorylating and thus inactivating all three Akt isoforms, Akt1, Akt2, and Akt3. The three Akt isozymes are selectively regulated by the two PHLPP isoforms: Akt1 is complexed with, and dephosphorylated by, PHLPP2; Akt2 is complexed with, and dephosphoryalted by, PHLPP1, and Akt3 is regulated by both PHLPP isoforms. To explore the in vivo role of PHLPP in Akt signaling, we have disrupted the PHLPP1 locus in mice. Mouse embryonic fibroblasts derived from PHLPP1 −/− mice display increased proliferation rates and enhanced insulin sensitivity. Specifically, insulin‐evoked phosphorylation of Akt is increased three‐fold in PHLPP1 −/− MEFs compared to +/+ MEFs. Histological analysis reveals that the pancreatic islets of PHLPP1 −/− mice are markedly smaller and considerably more apoptotic than those of their wild‐type littermates. Thus, lack of PHLPP1 results in enhanced insulin sensitivity and causes defects in β‐cell survival. These data are consistent with PHLPP1's regulation of Akt2, the predominant Akt isoform in insulin‐sensitive tissues. This work was supported by NIH 067946.
Biochemical characterization of the PP2C domain of PHLPP
Authors: Sierecki Emma , Newton Alexandra C
Published: June 2021

PHLPP (PH domain Leucine rich repeat Protein Phosphatase) dephosphorylates the hydrophobic motif of Akt/protein kinase B (Ser 473) thus terminating the Akt pathway, a major system regulating the balance between cell death and cell survival. Its phosphatase domain belongs to the PP2C family, characterized by a metal‐dependent activity and insensitivity to known phosphoprotein phosphatase inhibitors such as okadaic acid. The PP2C domain of one of the isozymes of PHLPP, PHLPP2, was expressed and purified from insect cells. This domain effectively dephosphorylates p‐nitrophenylphosphate (pNPP), peptides substrates, and recombinant Akt. Using pNPP as substrate, we determine the kinetic parameters (kcat and KM). The influence on the catalytic activity of factors such as the nature/concentrations of divalent metals, of various lipophilic compounds, nature of the substrate were determined and the results compared to those obtained for another member of the PP2C family: PP2Cα. A screen for potential inhibitors identified molecules with some selectivity for PHLPP2. This first biochemical characterization of the phosphatase domain of a PHLPP family member reveals that it is a thermally stable domain with phosphatase activity towards diverse substrates; its activity is modulated by metals, lipophilic compounds and heteroaromatic compounds.

This work was supported by GM NIH 067946.