bluuebunny/crossref_metadata_2025_split huggingface dataset:
A computational evaluation of the steric and electronic contributions to the stability of the structures of α− and β−D‐glucopyanose Part 1: conformational analysis of monosubstituted cyclohexanes and pyrans (593.2)
Authors: Brutofsky Christian , Abdulsalam Maryam , Baello Rofe , Balilli Rahih , Kasner Marc 
Published: June 2021

Geometry optimizations at a variety of computational levels were carried out on a series of substituted cyclohexanes (‐CH3 ‐OH and ‐CH2OH) and similarly 2‐substituted tetrahydro‐2H‐pyrans. By comparing the relationship between structure and energy of the cyclohexane series to the tetrahydro‐2H‐pyran series, it is possible to isolate the steric factors that are the principle contributor to the energy in the cyclohexanes from the combined steric and electronic factors that are present in pyrans.The optimized geometries and minimum energies for each compound were determined using HF, DFT and MP2 methods with increasingly complex basis sets. Thermal correction factors were applied to the conformational energies and the conformational free energy from the B3LYP/6‐311G++(d,p) optimizations closely reflected the available experimental values. Relaxed scans (also at the B3LYP/6‐311G++(d,p) level) of rotations of the exocyclic substituents helped to identifiy the steric contributions for different orientations of the rotamers. Analysis of the optimized geometries non‐bonded distances bond lengths, and bond angles contributed to an understanding of the steric contributions. Additionally, analysis of bond lengths and bond angles showed changes that reflected possible hyper‐conjugation (electronic) contributions to the stability of some configurations. The series of compounds selected in this study provide a series of model compounds that lead to modeling the steric and electronic contributions to the free energies energies of D‐glucopyranose.
Photocleavable dimerizer for the rapid reversal of molecular trap antagonists (597.3)
Authors: Ahmed Shubbir , Xie Jun , Horne David , Williams John 
Published: June 2021

Developing a molecular tool that can rapidly turn on and off an acute antagonist will be immensely helpful to understand the initiation of a cellular program. Herein, we report the development of a photocleavable analog of AP20187, a cell permeable molecule used to dimerize FKBP fusion proteins and initiate biological signaling cascades, gene expression or disrupt protein‐protein interactions. We demonstrate that this reagent in conjugation with a molecular trap permits the ability to rapidly and specifically antagonize a molecular interaction in vitro and follow a biological process due to this acute antagonism (e.g., endosome dispersion). More importantly, photocleavage of the analog permits the unique ability to follow the cell’s return to homeostasis. Herein, we demonstrated the fine temporal control of endosome dispersion and restoration using an LC8 trap. We posit that this photocleavable AP20187 analog can be used in other systems where the dimerization of FKBP has been used to initiate signaling pathways (e.g., kinases). In toto, the combination of a molecular trap and the photocleavable analog opens up new avenues to study signaling events within a single cell in a whole organism.Grant Funding Source: Supported by the generous support of R21NS071166 and also by P30 CA033572 from the NCI
CAR‐like membrane protein negatively regulates the intestinal barrier and promotes neutrophil transepithelial migration (60.6)
Authors: Luissint Anny‐Claude , Bennett Ashley , McCall Ingrid , Nusrat Asma , Parkos Charles 
Published: June 2021

CAR‐Like Membrane Protein (CLMP) is expressed in the intestinal epithelium and has been shown to be essential for embryonic intestinal development in Humans and Zebrafish; however, the role of CLMP in adult intestinal homeostasis is unknown. In analyses of CLMP expression in the gut, we observed upregulation of CLMP under inflammatory conditions in human and murine colonic epithelium. In human intestinal epithelial cell lines (IEC), CLMP localized at contacts between adjacent cells, and soluble recombinant proteins consisting of the CLMP extracellular domain bound specifically to IEC expressing CLMP, suggesting that CLMP may engage in homophilic interactions. Interestingly, permeability as assessed by transepithelial resistance, was decreased after siRNA‐mediated loss of CLMP, while its overexpression enhanced permeability. CLMP overexpression in IECs also resulted in delayed wound healing, suggesting that CLMP may alter epithelial repair. Further expression analyses revealed surface expression of CLMP in human and murine neutrophils (PMN). Therefore, we evaluated whether CLMP may play a role in mediating PMN migration during inflammation and observed that anti‐CLMP antibodies inhibited PMN transepithelial migration in vitro. Altogether, our findings suggest that CLMP is an adhesion molecule that negatively controls intestinal epithelial homeostasis and promotes immune cells response.Grant Funding Source: Supported by PR121194 Department of Defense, Peer Reviewed Medical Research Program
Pilin SpaC‐mediated epithelial adhesion is required for Lactobacillus rhamnosus GG‐induced probiotic effects (60.9)
Authors: Ardita Courtney , Mercante Jeffery , Kwon Young Man , Jones Rheinallt , Powell Domonica , Neish Andrew 
Published: June 2021

Lactobacillus rhamnosus GG (LGG) is a widely used probiotic, and the strain’s salutary effects on the intestine have been extensively documented. We have demonstrated this strain can modulate inflammatory signaling, as well as epithelial migration and proliferation via stimulation of Nox1 dependent reactive oxygen species. However, how this strain mediates these effects is largely unknown. Here, we report that LGG’s probiotic benefits are dependent on the SpaC pilin subunit. By comparing LGG to an isogenic mutant that lacks SpaC (LGGΩSpaC) in a variety of in vitro and in vivo assays, we establish that SpaC is necessary for LGG to adhere to gut mucosa, that SpaC contributes to LGG’s ability to induce the epithelial generation of ROS, and that SpaC plays a role in LGG’s capacity to stimulate the phosphorylation of ERK in enterocytes. In addition, we show that SpaC is required for LGG‐mediated stimulation of cell proliferation, and protection against radiologically‐inflicted intestinal injury. The identification of a critical surface protein required for LGG to mediate its probiotic influence furthers our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat inflammatory bowel diseases and other intestinal disorders.Grant Funding Source: Supported by NIH RO1 AI 64462
cAMP stimulates exocytic delivery of vesicle‐associated membrane protein 2 in renal epithelial cells (604.6)
Authors: Caceres Paulo , Ortiz Pablo 
Published: June 2021

Soluble NSF Attachment Protein Receptors (SNAREs) drive membrane fusion in eukaryotic cells. SNAREs of the Vesicle Associated Membrane Protein (VAMP) family reside in vesicles and mediate vesicle fusion. After fusion, VAMP remains inserted in the plasma membrane until retrieved for another round of fusion. Thus, exocytic delivery of VAMP could be used to measure VAMP‐mediated exocytosis. In renal epithelia like the collecting duct and the thick ascending limb (TAL), cAMP stimulates exocytic delivery of apical transporters (aquaporin‐2 and NKCC2) and this is mediated by VAMP2. However, the effect of cAMP in VAMP2 exocytic delivery has not been studied. We hypothesize that cAMP stimulates VAMP2 exocytic delivery in renal epithelia. We studied cultured TALs and LLC‐PK1 cells transduced with VAMP2‐GFP where GFP faces the vesicle lumen and the extracellular space after exocytosis. We labeled surface VAMP2‐GFP with an extracellular GFP antibody to differentiate it from intracellular VAMP2‐GFP. We observed a clustered distribution of VAMP2 at the surface by confocal microscopy (n = 5). Stimulation of cAMP with forskolin+IBMX enhanced the rate of VAMP2‐GFP exocytic delivery from 4.8 ± 0.9 to 8.7 ± 1.4 exocytic events/ min (81% increase, p<0.05, n = 4) as measured by surface biotinylation. To monitor VAMP2‐GFP exocytic delivery in real time, we quantified the number of exocytic events by total internal reflection fluorescence (TIRF) microscopy. We observed that cAMP increased VAMP2‐GFP exocytic events from 10 ± 4 to 87 ± 8 events/min/area (p<0.01, n = 3). We conclude that VAMP2 is distributed in surface clusters in renal epithelial cells and cAMP stimulates VAMP2 exocytic delivery to the plasma membrane.Grant Funding Source: Supported by National Institutes of Health and American Heart Association
Depicting a novel regulatory Bcl2/SHP/lncRNA H19 network in bile acid homeostasis (605.12)
Authors: Zhang Yuxia , Tsuchiya Hiroyuki , Smalling Rana , Cox James , Delker Don , Hagedorn Curt , Wang Li 
Published: June 2021

Objective: The aim of this study is to investigate a novel function of the anti‐apoptotic protein Bcl2 in bile acid (BA) homeostasis. Methods: Bcl2 was overexpressed using adenoviruses in male C57BL6 livers. After two weeks, BA homeostasis, liver morphology, fibrosis, and inflammation were analyzed. RNA‐seq and GC/MS were used to identify changes in gene expression and metabolites, respectively. Results: Hepatic overexpression of Bcl2 largely induced serum BA and FGF15 levels and decreased total BA pool size and fecal BA output. Severe hepatocyte necrosis, fibrosis, and Kupffer cell activation were observed, which was accompanied by a marked induction of long non‐coding RNA H19. RNA‐seq identified significant downregulation of genes in bile acid synthesis and transport, but upregulation of genes in collagen formation and inflammatory response by Bcl2. The increases in TCA cycle and lipid metabolites were the most drastic metabolomics changes. Using a series of cell based biochemistry and molecular biology approaches, we found that Bcl2 induced rapid nuclear receptor SHP protein degradation via activation of the caspase 8‐caspase 3 pathway. Furthermore, bile acid directly induced H19 expression in cultured primary hepatocytes that was independent of Bcl2 activation by bile acid. Importantly, Bcl2 and H19 were both dramatically increased in human cirrhotic livers and in mouse cholestatic liver fibrosis models. The dysregulation of the Bcl2/SHP/H19 network resulted in the disruption of bile acid homeostasis which contributed to the development of cholestatic liver fibrosis. Conclusions: Our results uncovered a unique metabolic regulatory network that integrated Bcl2 and SHP with lncRNA H19 to control BA homeostasis.Grant Funding Source: Supported by DK080440 (L.W).
Stearoyl‐CoA desaturase‐2 deficiency protects mice against high fat diet‐induced adiposity (605.16)
Authors: O'Neill Lucas , Ntambi James 
Published: June 2021

Stearoyl‐CoA desaturase‐2 (SCD2) is the second most abundant Δ9 desaturase in mouse and is strongly induced in adipose tissue by high fat feeding. Remarkably, SCD2 deficient mice (SCD2KO) are protected against high fat diet induced adiposity. Compared to wild‐type controls, SCD2KO mice fed a high fat diet exhibit reduced adipose triglycerides, smaller adipocytes, and improved glucose tolerance. Currently, it is unclear why deletion of SCD2 plays such an important role in the development of adiposity given the presence of SCD1. Both SCD2 and SCD1 synthesize monounsaturated fatty acids (MUFAs), predominately oleate and palmitoleate, from saturated fatty acids, predominately stearate and palmitate. We hypothesize that in adipose tissue, SCD2 plays a differential role from SCD1 and that either the SCD2 protein itself or SCD2 derived MUFAs regulate the biosynthesis of triglycerides, the main form of energy storage in adipose tissue.Grant Funding Source: NIH, ADA, MBTG, and SciMed GRS
cAMP stimulates diacylglycerol kinase‐θ phosphorylation in adrenocortical cells (605.18)
Authors: Cai Kai , Sewer Marion 
Published: June 2021

Diacylglycerol kinase theta (DGKθ) is an intercellular lipid kinase that coordinates nuclear phospholipid metabolism and cortisol production in the adrenal cortex by generating phosphatidic acid (PA), an agonist of the nuclear receptor steroidogenic factor 1 (SF1). We previously found that cAMP signaling induces the transcription of DGKθ and increases nuclear DGK activity. Moreover, mass spectrometric analysis of DGKθ isolated from H295R human adrenocortical cells has revealed that DGKθ is a phosphoprotein. However, the functional significance of DGKθ phosphorylation is unclear. Additionally, the mechanism by which cAMP signaling regulates DGKθ activity is not known. We show that dibutyryl cAMP increases the phosphorylation of DGKθ in H295R human adrenocortical cells. Alanine substitution at Ser‐354 reduces cAMP‐stimulated phosphorylation impairs the ability of DGKθ to activate SF1‐dependent transcription of CYP17. Cycloheximide sensitivity assays revealed that the S354A mutant is more stable than the wild type. Notably, we also identify Ser‐241 as a second site that is phosphorylated, where the S241A mutant abrogates DGKθ‐stimulated activation of a CYP17 reporter gene. Since Ser‐241 lies near to two proline residues, Pro‐244 and Pro‐245, that have been identified as key for optimal catalytic activity, we postulate that mutation of these prolines also alters the ability of DGKθ to be phosphorylated at this site. Indeed, P244L and P245L impair SF1‐dependent CYP17 luciferase activity. In summary, we identify DGKθ as an effector in the cAMP signaling pathway and a target of protein kinase A.Grant Funding Source: Supported by NIH DK084178.
Deletion of the cardiolipin‐specific phospholipase Cld1 rescues growth and lifespan defects in the tafazzin mutant: implications for Barth syndrome (605.4)
Authors: Ye Cunqi , Lou Wenjia , Li Yiran , Chatzispyrou Iliana , Hüttemann Maik , Lee Icksoo , Houtkooper Riekelt , Vaz Frédéric , Chen Shuliang , Greenberg Miriam 
Published: June 2021

Cardiolipin (CL) that is synthesized de novo is deacylated to monolysocardiolipin (MLCL), which is reacylated by tafazzin. Remodeled CL contains mostly unsaturated fatty acids. In eukaryotes, loss of tafazzin leads to growth and respiration defects and, in humans, results in the life‐threatening disorder Barth syndrome (BTHS). Tafazzin deficiency causes a decrease in the CL/MLCL ratio and decreased unsaturated CL species. Which of these biochemical outcomes contributes to the physiological defects is not known. Yeast cells have a single CL‐specific phospholipase, Cld1, which can be exploited to distinguish between these outcomes. The cld1Δ mutant has decreased unsaturated CL, but the CL/MLCL ratio is similar to that of wild type cells. We show that cld1Δ rescues growth, life span, and respiratory defects of the taz1Δ mutant. This suggests that defective growth and respiration in tafazzin‐deficient cells is caused by the decreased CL/MLCL ratio, not by a deficiency in unsaturated CL. CLD1 expression is increased during respiratory growth and regulated by the HAP transcriptional activation complex. Overexpression of CLD1 leads to decreased mitochondrial respiration and growth, and instability of mitochondrial DNA. However, ATP concentrations are maintained by increasing glycolysis. We conclude that transcriptional regulation of Cld1‐mediated deacylation of CL influences energy metabolism by modulating the relative contribution of glycolysis and respiration.Grant Funding Source: the Barth Syndrome Foundation
A novel protein family controls trafficking and metabolism of glycosphingolipids (606.9)
Authors: Jovic Marko , Kim Yeun Ju , Toth Balazs , Tuymetova Galina , Tsiomenko Arnold , Balla Tamas 
Published: June 2021

Glycosphingolipid (GSL) trafficking and metabolism are closely linked with cell fate determination, balancing signaling cascades controlling proliferation, apoptosis and autophagy. Altered cellular levels of complex glycosphingolipids often correlate with cancer progression, yet little is known about the principles that guide trafficking of these lipids.Here we identified a family of proteins, consisting of five mammalian homologues of the yeast Sfk1 protein that are involved in ganglioside transport. These six‐transmembrane domain proteins are widely expressed, each showing a unique cellular localization spanning the ER, Golgi, lysosomes and plasma membrane. One of these proteins, DNA‐damage regulated autophagy modulator 1 (DRAM1), was recently described as a p53‐inducible lysosomal regulator of autophagy. However, the cellular function(s) of the Sfk1 homologues remains unknown. Here we describe that mammalian Sfk1 proteins control the cellular distribution and metabolism of complex GSLs and one of them, DRAM2, specifically regulates transport of ganglioside GM3 from the plasma membrane to the Golgi and lysosomes. DRAM2‐mediated trafficking of GM3, in turn, was found to be critical for processing GM3 into higher ganglioside species.We propose that the Sfk1/DRAM family of proteins recognize GSLs within membranes and facilitate their sorting and hence their metabolic fate within the cell.Grant Funding Source: Supported by the NICHD Intramural research program
O‐GlcNAcomic profiling reveals altered O‐GlcNAcylation of mitochondrial proteins in diabetes (608.4)
Authors: Ma Junfeng , Liu Ting , Wei An‐Chi , O'Rourke Brian , Hart Gerald 
Published: June 2021

As a dynamically regulated post‐translational modification, O‐linked β‐D‐N‐acetylglucosamine addition (O‐GlcNAcylation) plays critical roles in various physiological events [1]. Aberrant O‐GlcNAcylation is involved in numerous diseases including diabetes and diabetic complications [2]. However, mitochondrial O‐GlcNAc proteins have far from being well characterized and their functions are largely unknown.To this end, a refined β‐Elimination followed by Michael Addition with Dithiothreitol approach was combined with reversed phase liquid chromatography (RPLC) fractionation followed by tandem mass spectrometry, yielding dozens of O‐GlcNAcylated proteins from cardiac mitochondria. The identified O‐GlcNAc proteins are involved not only in energy metabolism (i.e., oxidative phosphorylation, the Krebs cycle, and beta‐oxidation) but also in many other processes (e.g., heme biosynthesis, and amino acid metabolism). Moreover, by incorporating isotopic dithiothreitol reagents, the relative site‐specific O‐GlcNAc occupancy was also uncovered in control and diabetic hearts.Of note, multiple respiratory chain proteins have been found differentially O‐GlcNAcylated, as confirmed by western blots with O‐GlcNAc‐specific antibodies. Moreover, the O‐GlcNAcylation of respiratory chain proteins appears to be closely related to the alteration of mitochondrial dysfunctions including respiratory rate and production of reactive oxygen species. These results suggest that mitochondrial protein O‐GlcNAcylation underlies the etiology and progression of diabetic cardiomyopathy, providing a novel insight and, more importantly, new treatments to diabetes.Grant Funding Source: Supported by NIH R01CA42486, R01DK61671, N01‐HV‐00240, P01HL107153.
AKAP13 coordinates cAMP signaling and glucocorticoid production in H295R human adrenocortical cells (612.2)
Authors: Escajadillo Tamara , Sewer Marion 
Published: June 2021

The cAMP pathway activates steroid hormone biosynthesis in the adrenal cortex by regulating the activity of various proteins. We have previously identified a macromolecular protein complex that modulates cortisol biosynthesis by facilitating communication between the endoplasmic reticulum (ER) and mitochondria. One of the proteins in this complex is A‐kinase anchoring protein (AKAP)‐13. AKAPs play a critical role in the spatiotemporal compartmentalization of cAMP signaling, with AKAP13 being implicated in integrating Rho and cAMP‐dependent protein kinase A (PKA) signaling. AKAP13 has also been shown to regulate extracellular‐signal regulated kinase (ERK) and protein kinase D (PKD) activity in other cell types. We hypothesized that AKAP13 regulates cortisol production by coordinating RhoA and PKA signaling at the mitochondrial‐ER interface, and that disruption of AKAP13 leads to mislocalized cAMP signaling. We show that silencing AKAP13 in H295R human adrenocortical cells impairs cAMP‐stimulated RhoA, PKA and PKD phosphorylation and decreases cortisol and DHEA production. Consistent with previous findings, silencing AKAP13 had no effect on ERK activation or on the activation of hormone sensitive lipase. We conclude that AKAP13 is critical for the relay of cAMP signaling for cortisol production.Grant Funding Source: Supported by NIH DK094151.
Modulation of ERK and breast cancer cell growth by estrogen receptors (613.3)
Authors: Rofelty Angela , Schmitt John 
Published: June 2021

Estrogen Receptors participate in both genomic and non‐genomic signaling pathways that affect breast cancer cell growth and proliferation. The growth‐enhancing actions of estrogen (E2) in MCF‐7 breast cancer cells are mediated through Estrogen Receptor alpha (ERα), activating a kinase pathway involving CaM KK, CaM KI, and ERK. Conversely, overexpression of Estrogen Receptor beta (ERβ) has been shown to restrict the transcriptional activity of ERα and inhibit breast cancer cell growth. Our goal was to explore the roles of ERα and ERβ in ERK activation and cell growth in MCF‐7 and MDA‐MB‐231 breast cancer cells. E2 treatment of MCF‐7 cells led to a significant rise in phosphorylation of CaM KI and its downstream target, ERK, and this effect was blocked by pretreatment with the ERα antagonist MPP and siRNA directed against ERα. Inhibition of ERβ with its antagonist PHTPP did not reduce ERK activity following stimulation with E2. Interestingly, pretreatment of MCF‐7 cells with the ERβ agonist, FERb, prevented the activation of ERK by E2. Treatment of MCF‐7 cells with E2 stimulated an increase in cell growth that was blocked by pretreatment with MPP, but not by pretreatment with PHTPP. In both MCF‐7 and MDA‐MB‐231 cells, stimulation with FERb reduced cell growth slightly below control levels. Treatment of MDA‐MB‐231 cells with FERb rapidly increased ERK phosphorylation and this effect was blocked by pretreatment with PHTPP. Our data suggest ERβ differentially regulates ERK in MCF‐7 and MDA‐MB‐231 cells. While activation of ERβ inhibits the growth of both cell lines, ERβ prevents ERK phosphorylation in MCF‐7 cells, but enhances ERK phosphorylation in MDA‐MB‐231 cells.Grant Funding Source: Supported by M.J.Murdock Charitable Trust #2011267
Teaching resources for undergraduate educators in biochemistry and molecular biology: TRUE BMB (618.28)
Authors: Tansey John , Bailey Cheryl , Zhao Weiyi , Benore Marilee , Cox Michael , Elliott Samantha , Wright Ann , Bell Ellis 
Published: June 2021

Through a grant from the NSF, the ASBMB Implementing Vision and Change: developing concept driven teaching strategies in biochemistry and molecular biology project has held 14 regional workshops around the country and at 4 national meetings starting in 2011.The workshops focused on identifying foundational concepts and assessment tools for biochemistry and molecular biology educators. These were highlighted in the September/October 2013 edition of Biochemistry and Molecular Biology Education. In the next phase of this project, the ASBMB is developing an online repository of teaching resources for the scientific community; the BMB Online Instructional Resources. These will include test questions, laboratory exercises, and syllabi of entire courses submitted by biochemistry and molecular biology educators. These artifacts will be mapped back to broader principles, concepts, skills, and learning objectives proposed earlier in the project, curated and peer reviewed. Resources will also be open to the community for comments and reviews. We anticipate a beta version of this site launching in the early summer of 2014. This work was supported by NSF‐ 0957205 RCN‐UBE to EB.Grant Funding Source: Supported by NSF‐ 0957205 RCN‐UBE