bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparing the Solution Conformation and Activin‐binding of Follistatin Isoforms
Authors: Bhenderu Lokeshwar Sai Santosh , Murray Kyle , Stepurko Natalija , Wang Xuelu , Hyvönen Marko , D'Arcy Sheena 
Published: June 2021

Activins are members of the Transforming Growth Factor‐β superfamily. This superfamily of secreted growth factors activates transmembrane serine‐threonine kinase receptors, thereby stimulating Smad‐mediated intracellular signaling cascades. These cascades alter the gene expression profile of the target cells. Follistatin (FST) is a protein antagonist that directly binds to activins and other TGF‐β‐like proteins, preventing their interaction with cell‐surface receptors. Alternative splicing and proteolysis result in three isoforms of FST which differ in their C‐terminal regions. The three FST isoforms have different affinities for activins, as well as the extracellular matrix component heparan sulfate. We are focused on two FST isoforms, FST315 and FST288. FST315 contains an acidic C‐terminal region that is absent in FST288. Crystal structures of FST315 and FST288 with activin A are unable to explain the basis for isoform‐specific effects. The structures are similar and the acidic C‐terminal region of FST315 is not observed in the electron density. To identify differences between FST315 and FST288, we have used hydrogen/deuterium exchange coupled to mass spectrometry. We compare exchange of FST315 and FST288, both alone and in complex with activin A. The results will improve our molecular understanding of how FST isoforms regulate signaling by activin A.Support or Funding InformationStartup funds to S. D'Arcy from The University of Texas at Dallas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Beta‐cell‐specific loss of the inhibitory G protein, Gαz, prevents development of Type 1 Diabetes in NOD mice.
Authors: Fenske Rachel , Wienkes Haley , Peter Darby , Kimple Michelle 
Published: June 2021

The mechanisms that underlie the beta‐cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our lab has discovered a role for the unique inhibitory G‐protein, Gαz, in beta‐cell function, survival, and replication in both normal conditions and those of pathological stress. Gαz, when acting as a canonical inhibitory G‐protein, reduces production of cyclic adenosine monophosphate (cAMP) through inhibition of adenylate cyclase. cAMP acts as essential player in the amplification of glucose‐stimulated insulin secretion, while also mediating pathways that regulate beta‐cell death and replication. We've shown previously that removal of this inhibitory protein is sufficient to promote insulin secretion, beta‐cell growth and survival using chemically‐induced beta‐cell death and diabetes in C57BL/6J mice. Previous work in our lab has also shown that non‐obese diabetic (NOD) mice with global loss of Gαz are protected from developing T1D‐like hyperglycemia through a multi‐faceted maintenance of beta‐cell health. Whole body Gαznull animals exhibited a significant increase in beta‐cell function and replication and decreased beta‐cell death. Most interestingly, islets from Gαz‐null NOD animals exhibited a marked decrease in immune infiltration. In order to determine if Gαz in the beta‐cell was responsible for the protected phenotype, we generated a beta‐cell‐specific Gαz knockout by breeding Gz Flox/Flox mice with Rat Insulin Promoter (RIP) driven CreHerr mice, on the NOD background. After 10 generations of backcrossing, we began by determining if loss of beta cell Gαz was sufficient to protect NOD animals from developing T1D‐like hyperglycemia. Weekly random‐fed blood glucose measurements were taken beginning at weaning. In the initial cohort, we saw a 12% penetrance of hyperglycemia in male Cre‐positive NOD mice and a 23% penetrance of hyperglycemia in female Cre‐positive NOD mice by 30 weeks of age. In contrast, Gαzβ‐cell‐KO males and females were entirely protected from developing hyperglycemia during this period. Despite this stark contrast, fasting blood glucose was no different between genotypes, grouped by sex, at either 12 or 28 weeks of age. Glucose tolerance was also not different between Cre‐positive NOD controls and Gαzβ‐cell‐KO animals. Experiments are ongoing to further investigate other facets of beta‐cell health, including function, replication and survival. Additionally, it will be crucial to evaluate the presence and extent of insulitis in our Gαzβ‐cell‐KO animals. Thus far, our results show a critical role for beta‐cell Gαz in promoting T1D pathophysiology, although the precise mechanism remains to be determined.Support or Funding InformationNIH F31 DK109698 to RFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The PKC inhibitor BimI functions as a bi‐topic ligand that toggles a conserved allosteric switch to drive potent kinase inhibition
Authors: ma ning , Lee Sangbae , Devamani Titu , Sandhu Manbir , Sommese Ruth , Sivaramakrishnan Sivaraj , Vaidehi Nagarajan 
Published: June 2021

ATP‐competitive small‐molecule kinase inhibitors (SMKIs) are viewed primarily from the lens of tight and specific binding to the conserved catalytic domain. The allosteric effects of ATP‐competitive SMKIs are generally ignored, despite evidence that they can influence the potency and selectivity of kinase inhibition. We have recently showed that the ATP‐competitive PKC inhibitor BimI can displace substrate binding leading to potent inhibition of the kinase (Sommese and Sivaramakrishnan, JBC, 2016). In this study, we examine the substrate‐competitive behavior of a range of SMKIs derived from staurosporine and nucleotide analogs. We find that BimI functions like a bi‐topic ligand that acts on both the orthosteric ATP binding pocket and a remote allosteric site that determines overall kinase conformation. Combining novel FRET‐based sensors and multi‐scale MD simulations we identify a novel, conserved structural mechanism that regulates substrate binding in protein kinases. Bi‐topic ligands such as BimI disrupt a conserved ionic‐lock that drives global reorganization of the kinase domain through long‐range allosteric pathways that connect the N‐ and C‐terminal lobes. Together our studies identify a novel allosteric switch that regulates substrate binding in protein kinases that can be leveraged to develop highly selective kinase inhibitors.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rare Alzheimer's Disease‐Associated Protein Kinase C Variant Displays Altered Pharmacological Profile in a Cellular Environment
Authors: Yang Yimin , Callender Julia , Newton Alexandra 
Published: June 2021

Ever since protein kinase C (PKC) was discovered as receptors for tumor‐promoting phorbol esters in the early 1980s, efforts have been made to target PKC in the treatment of disease. Here, we show that PKCα‐M489V, a rare Alzeheimer's Disease (AD)‐associated PKC variant with enhanced agonist‐evoked signaling output, displays an altered pharmacological profile compared to wild type PKCα in a cellular environment. Using a genetically encoded PKC activity reporter and a phospho‐serine PKC substrate antibody as readouts for PKC activity, we find that overexpressed PKCα‐M489V in COS7 cells is more sensitive to inhibition by the ATPcompetitive PKC inhibitor Gö6976, but not by the uncompetitive PKC inhibitor bisindolylmaleimide IV (BisIV). However, PKCα‐M489V is equally sensitive to inhibition by Gö6976 in vitro, suggesting that the difference may result from protein‐protein interactions within the cell. The altered pharmacology of the M489V variant in vivo may provide a novel method of targeting PKCs in the treatment of AD.Support or Funding InformationThis work was supported by N!H R35 GM122523 and NIH GM43154 (AN) and the Cure Alzheimer's Fund (AN). JC was supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology (T32 GM007752).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deep Reading of the Literature and the Biosynthesis of Nucleotides
Authors: Anderson Ellen 
Published: June 2021

Biosynthesis of Nucleotides is a Master's level elective course that was developed to advance students' comprehension of peer‐reviewed articles using a detailed study of the biosynthesis of purine and pyrimidine ribonucleotides and their conversion to deoxyribonucleotides. Nucleotides are ubiquitous molecules essential for nearly every biochemical pathway serving as substrates, regulatory molecules or monomers for RNA and DNA synthesis. Many medical conditions are the result of abnormalities in these biosynthetic pathways and their enzymes are targets for pharmaceutical intervention. Unfortunately, one‐semester biochemistry courses rarely have time to cover nucleotide metabolism. Coupling this fundamental topic in biochemistry with in‐depth reviews of the literature enabled graduate students to learn how to effectively read and analyze the literature while learning nucleotide metabolism. A scaffoldedset of assignments, adapted with permission from B.D. Spielgelberg (2014), was created to deepen students' understanding of experimental details, interpretation of the results and development of conclusions from these results. These assignments consist of a mix of individual assessments, group‐generated analyses of figures and tables of data, and online discussions based on a peer‐reviewed paper for each section of the course. A final project, completed individually, combines these elements and is used to evaluate student progress in literature evaluation and comprehension. Examples of the assignments will be presented, as well as preliminary data demonstrating the improved performance over the semester, especially on a comprehensive analysis of a set of data or a figure from the assigned paper. Likewise, data from student evaluations will be presented to demonstrate the value of these assignments.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell‐Free Protein Synthesis: A Platform Technology for Education
Authors: Kao Wesley Y. , Gregorio Nicole E. , Oza Javin P. 
Published: June 2021

Hands‐on learning of transcription and translation remains limited due to a lack of classroom modules for early‐career students. Currently, classrooms implement animations, simulations, games, and bacterial transformation/protein expression to teach the genetic code. The cell wall poses the primary barrier for students to directly observe and manipulate the intracellular environment, limiting inquiry‐based learning of the genetic code. We have overcome this limit by levering transcription and translation in vitro. The open nature of cell‐free protein synthesis (CFPS) is achieved by removing the cell wall and genomic DNA, while isolating the cellular machinery involved in transcription and translation. This allows experimenter to harness the genetic code in a test tube and directly manipulate the reaction conditions for hands‐on learning. While reconstitution of cellular machinery in vitro for CFPS has been transformative for biotechnology, CFPS has not been accessible to early‐career students due to the complexity in reagent preparation and reaction set‐up. We have successfully adapted the CFPS biotechnology to develop a learning module accessible to early‐career STEM students focusing on learning objectives involving the genetic code. Here we report the reformulation of the CFPS reaction setup for simplicity and improved reagent shelf life in order to adapt CFPS for the classroom. First, our simplified platform requires the pipetting of only 3 pre‐mixed reagents, an 80% reduction in setup steps, making it more accessible to an early‐career student with little‐to‐no laboratory experience. Second, our simplified platform is shelf‐stable at −20°C rather than −80°C, making it accessible for storage and implementation in undergraduate and high school classrooms. Longer‐term experiments are in progress to validate shelf‐life at 4°C and room temperature. Our data show that these improvements are achieved without sacrificing CFPS performance in reaction kinetics or total protein yield. Third, our analysis of green fluorescent protein synthesis kinetics shows that 90% of maximum protein yields can be achieved within 3 hours at 37°C, making this platform compatible with most college laboratory courses. We believe that this educational technology will be transformative to the way the genetic code is taught in classrooms today. Toward this end, we have developed a CFPS classroom ‘kit’ for broad dissemination.Support or Funding InformationOur research is funded by the Center for Applications in Biotechnology @ Cal Poly SLO, Bill and Linda Frost Fund, and NSF‐1708919.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
14‐3‐3 Protein‐Dependent GATA Transcription Factor Control in Saccharomyces cerevisiae
Authors: Tate Jennifer J. , Rai Rajendra , Buford David , Cooper Terrance G. 
Published: June 2021

14‐3‐3 proteins, Bmh1/2, are major participants in the control of most nutrient‐ and stress‐sensitive regulons in Saccharomyces cerevisiae. However, the Nitrogen Catabolite Repression (NCR)‐sensitive regulon and its Gln3 and Gat1 GATA transcription activators have not been reported to be Bmh1/2‐regulated. Observations from proteomic studies identify Gln3 as a candidate Bmh1/2 interacting protein (Biochem 46:7781; Genomics 94:287), and NCR‐sensitive GAP1 expression is about 1.5‐fold higher in bmh2 than bmh1 mutants treated with TorC1 inhibitor, rapamycin (Biosci. Rep. 34;art:e0009). Therefore, we determined whether Bmh1/2 participates directly in Gln3 and Gat1 control or more indirectly via Bmh1/2‐dependent TorC1 regulation. The question of Bmh1/2‐dependent GATA factor regulation can only be effectively investigated in a ∑1278b background because in most other strains the double mutant is lethal. In poor nitrogen conditions, Gln3 and Gat1 are nuclear and support high level NCR‐sensitive transcription in wild type cells. In contrast, when abundant nitrogen is available, Gln3 and Gat1 relocate to the cytoplasm and GATA factor‐dependent transcription is repressed. This wild type regulation is achieved via post‐translational Gln3 regulation by the TorC1 and Gcn2 kinases, Sit4 and PP2A phosphatases and downstream negative regulator Ure2. In a bmh1Δbmh2Δ double mutant, we find that: (i) NCR‐sensitive transcription of three reporters, GDH2, DAL80 and DAL5, is almost completely abolished. (ii) Gln3 and Gat1‐mediated transcriptional responses to rapamycin‐treatment are detectable but severely diminished. (iii) Loss of Bmh1/2 results in Gln3 dephosphorylation similar to the levels observed following rapamycin treatment. (iv) The response of Gln3 and Gat1 nuclear localization parallels that observed for GATA‐factor mediated transcription. (v) Cells form pseudohyphae‐like chains when provided with nitrogen sources whose utilization requires NCR‐sensitive transcription. Prior to the point when bmh1Δbmh2Δ mother and daughter cells separate, only one contains detectable GFP‐Gln3. Either Bmh1 or Bmh2 will fulfill the 14‐3‐3 requirement as nuclear Gln3 localization is normal in single bmh1 mutants. Using bmh1Δbmh2Δ and bmh1Δbmh2Δure2Δ mutants, we demonstrate that a bmh1Δbmh2Δ double deletion is epistatic to an ure2 deletion. We conclude Bmh1/2 are absolutely required for nuclear Gln3 localization and likely function downstream of Ure2, heretofore the most downstream regulator of the nitrogen‐sensitive regulon.Support or Funding InformationNational Institute of Health General Medical Sciences GM‐35642‐27.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of capsicoside G on lipolysis and fatty acid oxidation in 3T3‐L1 adipocytes
Authors: LEE JUNSOO , Kim Younghwa , Sung Jeehye 
Published: June 2021

We have previously demonstrated that capsicoside G, a furostanol saponin, isolated from pepper seeds, inhibited adipogenesis in 3T3‐L1 adipocytes. However, the effects of capsicoside G on lipolysis and fatty acid oxidation remain unclear. The aim of this study was to extend the anti‐obesity investigation of capsicoside G by examining its effect on lipolysis and fatty acid oxidation in mature 3T3‐L1 adipocytes. Capsicoside G promoted lipolysis of adipocytes, which effectively decreased the intracellular triglyceride concentration of adipocytes and increased glycerol released into the medium. The increased lipolysis was accompanied by an augmentation in the expression of adipose triglyceride lipase and hormone‐sensitive lipase. Furthermore, capsicoside G also induced the expression of fatty acid oxidative genes such as peroxisome proliferator activated receptor α and carnitine palmitoyltransferase‐1 in mature 3T3‐L1 adipocytes. These results suggest that capsicoside G exerts anti‐obesity property by stimulating lipolysis and fatty acid oxidation in adipocytes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Glucocorticoid Receptor Polymorphisms in Obese Populations
Authors: Bonnet Claude‐Luvier , Dowdye Ayanah , Cohen Brian D. 
Published: June 2021

The glucocorticoid receptor (GR) is part of a family of nuclear receptors that control gene expression. In the presence of the steroid hormone cortisol certain genes are expressed; the products of which control, among other things, blood pressure, serum triglycerides, and blood sugar which are often elevated in patients with obesity. Specific polymorphisms of the GR and other regulators of either GR (or the closely related mineralocorticoid receptor) such as heat shock protein 90 (HSP90) and 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) have been found in our labs and others to be present at a higher frequency in obese populations. Our current research is investigating polymorphisms in these genes as well as the gene encoding the FK506 binding protein (FKBP) which works in conjunction with HSP90 to negatively regulate GR activity. It is hypothesized that polymorphisms may exist in the FKBP51 gene that would result in decreased activity of FKBP and subsequent overactivity of GR resulting in phenotypic hypercortisolemia. Analysis of the FKBP51 single nucleotide polymorphism RS1360780 showed a correlation with serum triglycerides, blood pressure, BMI, and weight regain in bariatric surgery patients. This finding in combination with previous results will lead to a better understanding of the genetic underpinnings of obesity and may reveal novel therapeutic approaches.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In vivo Effects of Adipose‐Specific Deletion of Jagged 1 in Mice Administered a High Fat Diet
Authors: Langlais Audrie 
Published: June 2021

Adipose tissue is a major site of lipid metabolism that works synergistically with other organs to regulate whole body energy homeostasis. Adipose tissue also modulates metabolism by secretion of adipokines such as leptin and adiponectin. Abnormalities in the differentiation and function of mature fat cells (adipocytes) are linked to metabolic disease. The evolutionarily‐conserved Notch signaling pathway is necessary for the differentiation of all cell types including adipocytes. Both inhibition and activation of Notch 1 interfere with adipose remodeling in response to nutritional stress. Although activation of the canonical Notch pathway requires binding to one of its transmembrane ligands such as Jagged‐1, the importance of the ligand itself in regulating adipose development and function has been poorly studied. Furthermore, the ability of the Jagged1 ligand to regulate adipocyte cell fate decisions directly through translocation of its intracellular domain (ICD) into the nucleus is unknown. To address these questions regarding the role of Jagged1 in maintaining metabolically healthy adipose, we generated a novel Cre‐mediated conditional knockout murine strain (ap2‐Jag1tm2Grid) to study the impact that jagged1 gene dosage has on visceral adipose depots under conditions of dietary stress. The ap2‐Jag1tm2Grid line produces progeny with three possible Jagged‐1 genotypes that result in either wild type, haploinsufficient or complete loss of Jagged1 expression in mature adipocytes. Physiological and biochemical measurements conducted on young mice representing all three genotypes after administration of either a high fat (“Western”) or standard diet for 5 weeks support our hypothesis that complete or partial loss of Jagged1 in adipocytes is detrimental to the ability of adipose to remodel and function normally in response to the intake of dietary lipids. Differences among the cohorts regarding weight gain, % visceral fat mass, fasting blood glucose, serum lipids and expression of adipokines leptin and adiponectin support a role for Jagged‐1 in maintaining proper adipose growth and metabolism. Data from this study may lead to a better understanding of how the expression level of Jagged1 influences an individual's susceptibility to obesity and metabolic disease.Support or Funding InformationDeena J. Small, University of New England Department of Chemistry & PhysicsWoon Yuen Koh, University of New England Department of Mathematical SciencesThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.