bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationThanks 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 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.
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 GRK5Using 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.
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.
Microwave‐assisted synthesis of N‐vanillylformamide
Authors: Keller Brent , Bobylev Mikhail 
Published: June 2021

The Leuckart reaction is a fast and convenient method of synthesis of various formamides and amines and has been successfully used for the synthesis of many biologically active compounds, including agrochemicals and pharmaceuticals. Surprisingly, it has never been used for the synthesis of vanillylamine (I) from vanillin (II) via the intermediate N‐vanillylformamide (III). In the literature, the intermediate III is described only once, when it was obtained via the formylation of I, not by the reductive amination of II. In this work, we report a successful synthesis of III from II via a microwave assisted Leuckart reaction. This method provides a new synthetic pathway to I, which is a valuable intermediate in the synthesis of a number of biologically‐active compounds. This method can also be used for the synthesis of other hydroxy‐substituted benzylamines.The project is supported by NIH grant P20 RR016741 from the NCRR.
Palmitoylation‐dependent plasma membrane transport, but lipid raft‐independent signaling function, of linker for activation of T cells (LAT)
Authors: Hundt Matthias , Harada Yoshuke , Hayashi Keitaro , De Giorgio Lauren , Tanimura Natsuko , Zhang Weiguo , Altman Amnon 
Published: June 2021

LAT is a dually palmitoylated transmembrane (TM) adaptor protein that is essential for T cell development and peripheral T cell activation. LAT‐deficient Jurkat T cells (J.CaM2) have been used to explore the role of LAT palmitoylation in its function. Consistent with previous reports, a non‐palmitoylated LAT mutant (LAT C26/29A) did not reconstitute TCR signaling in J.CaM2 cells and did not localize in lipid rafts. Surprisingly, this mutant was not found in the plasma membrane (PM) but, rather, was restricted to the Golgi apparatus. To further examine the importance of LAT palmitoylation, we analyzed a chimeric LAT construct (Src‐LAT) consisting of the membrane‐targeting N‐terminal region of Src kinase and the cytoplasmic tail of LAT. Src‐LAT localized in the PM (but not in lipid raft). Unexpectedly, and similar to another, TM LAT chimera (LAX‐LAT), Src‐LAT was capable of restoring TCR signaling in J.CaM2 cells, and T cell development in Src‐LAT‐reconstituted LAT−/− bone marrow chimeras, despite being expressed as a peripheral membrane protein. Thus, functionality of LAT correlates with its PM localization (even as a peripheral protein), but is independent of its lipid raft localization. Furthermore, LAT palmitoylation functions primarily as a sorting signal required for its transport to the PM.Supported by the Leukemia and Lymphoma Society.
The analysis of interrelationship of parameters of immune homeostasis with the system of LPO‐AOS
Authors: Dushanova Gavhar A 
Published: June 2021

Taking into account very important role of mebraine patology in processus of immunogenesis, investigation of existence of parallels between immune system status and indexes of intensity of AOS and enzymes activity of antiradical defence systems are subject of our research.Investigation of correlation analysis of indexes of immune system connected with parameters of LPO‐AOS of totally healthy pacients in general group, showed existence of negative correlation between serum Lg classes M with indexes of MDA.Further investigations on biological significance of system as a central genetic apparatus of immune system functioning, showed great interest of existence of some relationship of antigen HLA with the level of activity of metabolic processus, related to LPO‐AOS.Thus, the level of MDA differ in groups of HLA‐DR opposit pacients, representing more lower indexes among antigen positive individuals. Pacients with had HLA antigen B in their fenotypes had higher activity of SOD enzyme, as compared to pacients without this antigen in fenotype.
Mitogenic CD28 induces granzyme B and a cytokine storm in human TH1 cells
Authors: Medina Miguel A. , Martin Ashley , Couturier Jacob , Camell Christina , Rodgers John , Lewis Dorothy 
Published: June 2021

Phase I clinical trials with a humanized super‐agonistic CD28 monoclonal antibody resulted in multi‐organ failure with loss of lymphocytes in the periphery.We investigated the mechanism of mitogenic CD28 induced activation and cell death in T cells. Mitogenic but not conventional CD28 induced activation and apoptosis of the Jurkat but not H9 T cell line. In vitro polarized human T‐helper (Th1) cells were activated by mitogenic CD28, but unlike their TH2 counterparts, produced significant amounts of granzyme B (10 ug/ml) from 5 x 105 cells. Negatively selected human Th1 cells exhibit a Th1 like cytokine profile following PMA and ionomycin stimulation and express extracellular markers, CXCR3 and CCR5, a profile consistent with Th1 cells. Upon mitogenic CD28 stimulation more than half of the cells are activated and about 30% apoptotic similar to activation induced cell death seen after anti‐CD3 stimulation. Following mitogenic CD28 stimulation negatively selected TH1 cells produced a cytokine profile consistent with a cytokine storm. Therefore, activated inflammatory Th‐1 cells are potential producers of large quantities of granzyme B and inflammatory cytokines, which may be associated with multi‐organ failure in the phase I clinical trial.This study was supported by NIH grants R21‐AI054251, R37‐AI36682 and Center for AIDS Research grant P30 AI36211.
In vitro and in vivo activation of CD4+CD25−Foxp3+ Tregs in NZB/W mice
Authors: Scalapino Kenneth John , Daikh David I 
Published: June 2021

PURPOSE: CD4+Foxp3+ Tregs increase with onset of lupus in (NZBxNZW)F1 (B/W) mice, but many Tregs lack CD25 expression. Absence of CD25 is correlated with impaired Treg function. The ability to restore CD25 expression and enhance Treg function was assessed.METHODS: CD4+CD25−CD62LLO T cells were stimulated with anti‐CD3/anti‐CD28 and IL‐2. Cells up‐regulating CD25 were sorted and tested for Foxp3 expression, suppression capacity, and survival. In vivo up‐regulation of CD25, Foxp3, and suppression capacity following a brief course of IL‐2 to mice was then assessed.RESULTS: 44% of Tregs in sick B/W mice were CD4+CD25−CD62LLO. Stimulation of these cells in vitro resulted in up‐regulation of CD25 on 85% of Tregs. Activated Tregs expressed high levels of CD25, Foxp3, and had enhanced in vitro suppression and in vivo survival following adoptive transfer. Activation of CD4+CD25− Tregs in vivo by IL‐2 increased per cell Foxp3 expression and the fraction of Tregs that were CD25+ (75%). Purified CD4+CD25+ T cells from treated mice were highly suppressive when tested in vitro.CONCLUSION: Inadequate CD25 expression on B/W Tregs may correlate with impaired Treg function and disease development. This problem is reversible, producing Tregs with prolonged survival and suppression capacity. These results suggest the potential for lupus therapies directed towards Treg activation.Supported by Department of Veterans Affairs.
Targeted deletion of fgl2 leads to impaired regulatory T cell activity and development of autoimmune glomerulonephritis
Authors: Shalev Itay , Liu Hao , Koscik Cheryl , Bartczak Agata , Javadi Mojib , Wong Kit Man , Maknojia Asif , He Wei , Liu Ming Feng , Diao Jun , Winter Erin , Manuel Justin , McCarthy Doug , Gommerman Jennifer , Cattral Mark , Clark David A , Phillips James , Gorczynski Reginald R , Zhang Li , Downey Greg , Grant David , Cybulsky Myron I , Levy Gary 
Published: June 2021

Mice with targeted deletion of fibrinogen‐like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild‐type recipients reconstituted with fgl2−/− bone marrow. These data implicate FGL2 as an important immunoregulatory molecule and led us to identify the underlying mechanisms. Deficiency of FGL2, produced by CD4+CD25+ regulatory T cells (Treg), resulted in increased T cell proliferation to lectins and alloantigens, T helper 1 (Th1) polarization, and increased numbers of antibody‐producing B cells following immunization with T‐independent antigens. Dendritic cells (DC) were more abundant in fgl2−/− mice and had increased expression of CD80 and MHCII following LPS stimulation. Treg cells were also more abundant in fgl2−/− mice, but their suppressive activity was significantly impaired. Antibody to FGL2 completely inhibited Treg cell activity in vitro. FGL2 inhibited DC maturation and induced apoptosis of B cells through binding to the low affinity FcγRIIB receptor. Collectively, these data suggest that FGL2 contributes to Treg cell activity and inhibits the development of autoimmune disease.This work was supported in part by grants from the Heart and Stroke Foundation of Canada and the Canadian Institutes for Health Research.