bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of mouse UDP‐glucuronosyltransferase homologs of human isozymes that inactivate mutagenizing catechol estrogens
Authors: Chakraborty Kushal , Banerjee Rajat , Basu Mousumi , Basu Nikhil , Owens Ida S
Published: June 2021

The UDP‐glucuronosyltransferase system plays an important role in the pharmacokinetics of clearance of endogenous metabolites, therapeutic drugs, and xenobiotics. Among this superfamily of isozymes human UGT2B7and UGT1A10 plays an important role in the detoxification of several estrogen metabolites, including 4‐OH estrone, by converting chemicals to water‐soluble derivatives. Recently we showed that UGT2B7 requires Src‐dependent phosphorylation for activity. In the cellular milieu, the lack of Src kinase caused a marked decrease in UGT 2B7 activity demonstrated by expressing the isozyme in the Src‐knockout, SYF‐/‐, cell line. To extrapolate these findings to an animal model to explore clinical relevance, computer‐based sequence‐homology identified 2 mouse homologs of 2B7. Importantly, RT‐PCR analysis of mRNA expression demonstrated both isozymes, like 2B7 and 1A10, are expressed in mammary gland. Biochemical evidence indicates that separate mouse isozymes metabolize preferentially 2‐hydroxy and 4‐hydroxy estrogen metabolites, whereas both UGT2B7 and UGT1A10 prefer 4‐hydroxy estrogen derivatives. Evidence also suggests that, like 2B7, both isozymes are dependent upon Src kinase activity. Based on these observations, combined studies of the two mouse isozymes can be used as a model for clinical relevance.
Videos as teaching tools: a demonstration
Authors: Zigmond Michael
Published: June 2021

If a picture is worth a thousand words, how many words does a film clip merit? In our experience that number is very high when videos are used as teaching tools. The medium of video is able to convey complex issues more quickly and comprehensively than text, speech, or static images. And the use of film ‐ particularly popular film ‐ can be a powerful way to capture students' attention and thus promote learning and retention. Indeed, participants often rate a 5‐min video clip as the best part of a 1‐hr workshop or class! Here we will demonstrate the effectiveness of video by showing several clips and then asking symposium participants to simulate a class discussion. Included will be scenes from "And the Band Played On," a movie about the search for the AIDS virus. These scenes will provide examples of the use of film to show clinical symptoms, both positive and negative aspects of competition in science, methods of conflict resolution, and the impact that a single, dedicated individual can have.
Regulation of Hepatic Gene Expression by Thyroid Hormone
Authors: Attia Ramy Raafat , Connaughton Sara , Cook George A , Elam Marshall B , Park Edwards A
Published: June 2021

Thyroid hormone (T3) is a key regulator of several processes including development and metabolic rate. Our laboratory has investigated the regulation of hepatic genes by T3. Here, we examined the mechanisms by which T3 activates the pyruvate dehydrogenase kinase (PDK4) gene. PDK4 regulates pyruvate oxidation through the phosphorylation and inhibition of the pyruvate dehydrogenase complex (PDC). PDC catalyzes the conversion of pyruvate to acetyl‐CoA and is an important control point in the metabolism of glucose and pyruvate. We identified a T3 response element in the PDK4 gene and demonstrated that the T3 receptor binds this element. The peroxisome proliferator activated receptor gamma coactivator (PGC‐1a) is a transcriptional coactivator that promotes hepatic gluconeogenesis and fatty acid oxidation. We found that PGC‐1a is associated with the PDK4 gene following T3 administration and that the abundance of PGC‐1a is increased by T3. We explored the role of several transcription factors including estrogen related receptor (ERRa), CCAAT/enhancer binding protein beta (C/EBPß), forkhead transcription factor (FOXO1) in the activation of the PDK4 gene by T3. Moreover, we identified a number of factors including ERRa that are induced by T3. Our results suggest that the hepatic actions of T3 are mediated in part by the induction of transcription factors as well as the recruitment of coactivators to T3 responsive genes.
Regulation of lipoprotein lipase in rat adipose tissue ‐ a study of the role of angiopoietin‐like protein 4
Authors: Kroupa Olessia , Olivecrona Thomas , Olivecrona Gunilla
Published: June 2021

Lipoprotein lipase (LPL) plays a central role in hydrolysis of triglyceride‐rich lipoproteins. Rapid regulation of LPL activity by the nutritional status occurs mainly at the post‐translational level. Recent studies have demonstrated that angiopoietin‐like protein 4 (angptl4) controls lipoprotein metabolism via its ability to inhibit LPL. This occurs by direct binding of angptl4 to LPL resulting in conversion of active LPL dimers to inactive monomers. To further characterize the relationship between angptl4 and LPL, we studied the response of angptl4 mRNA and LPL activity in rat adipose tissue to different experimental conditions. Food withdrawal caused significant down‐regulation of LPL activity after 3 h, whereas angptl4 mRNA levels started to increase only after 4 h. During re‐feeding angptl4 mRNA expression was significantly decreased 1 h after feeding while a significant increase of LPL activity was not seen until after 2 h. Intraperitoneal injection of insulin caused a rapid increase in adipose tissue LPL activity after 30 min whereas the total mass of LPL protein remained the same. At this time (30 min) angptl4 mRNA levels had not changed significantly. We conclude that regulation of LPL activity in adipose tissue does not exactly correspond to changes in angptl4 mRNA levels. Thus, other mechanisms than transcriptional regulation of angptl4 must be involved in the control of LPL. Sponser: Swedich MRC.
The role of His349 in receptor‐stimulated iron release from the C‐lobe of transferrin
Authors: Steere Ashley N. , Byrne Shaina L. , Mason Anne B.
Published: June 2021

The primary uptake and delivery pathway for iron in mammalian systems involves serum transferrin (Tf), a bilobal protein, which releases iron within the acidic environment of the endosome (pH ~5.6) in a receptor mediated manner. Following iron release, apo Tf remains bound to the Tf receptor (TfR) until the basic extracellular pH (~7.4) is reached, and Tf is released to continue the cycle. Previous structural and mutagenesis studies suggest that residue His349 in the C‐lobe of Tf makes essential contacts with the TfR to simulate iron release and stabilize the apo complex. Due to the physiologically relevant pKa of the histidine side chain (~6.5), His349 presumably undergoes a change in protonation state during endo‐and exocytosis. We sought to further investigate the role of His349 in iron release from the C‐lobe of Tf in the presence of the TfR using intrinsic tryptophan fluorescence and urea gel analysis of various His349 mutants in a monoferric C‐lobe construct. Mutation of His349 to a lysine causes a loss of receptor stimulated iron release, indicating that not only the charge, but the orientation of residue 349 is important for stabilizing the apo form of Tf in complex with the TfR. Likewise, mutation of His349 to an alanine, aspartic acid, leucine, tryptophan, or tyrosine residue also decreased iron release rates in the presence of the TfR as compared to the control. These results indicate an essential role of His349 in acceleration of iron release from Tf as stimulated by the TfR.

This work was supported by USPHS Grant R01 DK 21739 (ABM) and NRSA Grant 5T32HL007594.
Fluorescence Correlation Spectroscopy study of the diffusion of biomolecules in Ficoll solutions
Authors: Boukari Hacene , Zerari Fatima
Published: June 2021

Probing and understanding the dynamics of biomolecules in crowded media is challenging, requiring the development and application of appropriate tools. We have exploited the advantages of fluorescence correlation spectroscopy (FCS) to examine the movement of various fluorescent biomolecules (GFP, phycoerythrin, BSA, clathrin, dextran, polystyrene beads..) in Ficoll70 solutions prepared at different concentrations. These biomolecules have not only different size and weight but also shape, from globular (GFP, BSA, .) to branched (dextran) to triskelia (clathrin). Changes in the apparent diffusion of these biomolecules with Ficoll concentration appears to be adequately described by a single exponential [~exp(‐αc)] with α being dependent on the biomolecule. The changes cannot be attributed to changes of the bulk viscosity of Ficoll, as would be expected from the Stokes‐Einstein relation derived for a structureless, continuum viscous fluid. Instead, we use de‐Gennes‐Langevin‐Rondeles model, which takes into account the nanoscopic porous‐like structures of the Ficoll solutions. Here, the exponential behavior can be interpreted as Ficoll‐biomolecule system being under theta condition.
RGDSK/K rosette nanotubes induce inflammation and apoptosis through phosphorylation of p38 MAPK in human lung adenocarcinoma (Calu‐3) cells
Authors: Singh (Suri) Sarabjeet , Myles Andrew J , Fenniri Hicham , Singh Baljit
Published: June 2021

Rosette nanotubes (RNTs), a novel class of biologically inspired nanotubes, hold tremendous potential as targeted drug delivery shuttle. We synthesized hybrid RNTs that composed of RGDSK‐ and K‐functionalized RNTs in a ratio of 1:10 M (RGDSK/K‐RNTs). We noticed the expression of avb3 integrin on Calu‐3 cells and believe that these RNTs may bind to integrin. We investigated cell signaling events caused by these RNTs for induction of inflammation and apoptosis in Calu‐3 cells. RGDSK/K‐RNTs rapidly induced phosphorylation of p38 MAPK. RGDSK/K‐RNTs (>10:100 μM) promoted p38 MAPK‐dependent secretion of TNF‐α. All the tested concentrations of RGDSK/K‐RNTs resulted in an increase in caspase‐3 activity and DNA fragmentation in Calu‐3 cells at 18 hours of the exposure. Blocking phosphorylation of p38 MAPK strongly inhibited caspase‐3 activity and DNA fragmentation. Pro‐apoptotic properties of RGDSK/K‐RNTs were also supported by over‐expression of pro‐apoptotic genes. We therefore conclude that RGDSK/K‐RNTs induce phosphorylation of p38 MAPK, which regulates secretion of TNF‐α, activation of caspase‐3 and apoptosis in Calu‐3 cells. These results suggest that the RGDSK/K‐RNTs could be used as a drug to induce apoptosis in cancer cells or as a versatile platform to deliver a variety of biologically active molecules for cancer therapy. Support: NSERC, AARI.

Grant Funding Source

NSERC discovery, AARI
Chemical interaction between the immobilized enzymes on Cu‐MCM41 determinated by EPR
Authors: Rosales‐hernandez Martha cecilia , Terres‐Rojas Eduardo , Ramirez‐Rosales Daniel , Kispert Lowell
Published: June 2021

MCM‐41 is a member of a family of mesoporous molecular sieves, it has been used for the proteins immobilizations such as cytochrome c, papina, tripsina, cytochrome P‐450 and peroxidase. The incorporation of different transition metal ions such as Al+3, Cu+2, Ti+4 y V into the framework of MCM‐41 changes their physico‐chemical properties such as the electron transfer efficiency between embedded molecules and the material MCM‐41 However, the importance of these metals over the material surface during the enzyme immobilization process has been not studied enough which could be of great importance. Therefore, the main of this work is to immobilize peroxidase, acetilcholinesterase and cytocrome P‐450 2B4 on Cu‐MCM‐41 in order to evaluate their catalytic activity and the interaction between the enzymes and Cu+2 by using EPR studies. The EPR results showed that copper reacts with NH3 groups from aminoacids of the enzyme such as Lysin and Arginine which are found in its surface and this allow to immobilize the enzyme as an uni‐directional manner allow to the optimal substrate access and product egress.
Hybrid Structural Model of the Complete Human ESCRT‐0 Complex
Authors: Ren Xuefeng , Kloer Daniel , Kim Youngchan , Ghirlando Rodolfo , Saidi Layla F , Hummer Gerhard , Hurley James H
Published: June 2021

The human Hrs and STAM proteins comprise the ESCRT‐0 complex, which has a pivotal role in directing ubiquitinated cell surface receptors to the lysosomal degradation pathway. Here we report a structural model for the complete ESCRT‐0 complex based on the high resolution structure of the Hrs‐STAM core complex, previously solved crystal and solution structures, hydrodynamic measurements, and coarse‐grained Monte Carlo simulations. Recombinant ESCRT‐0 expressed in insect cells has a hydrodynamic radius of RH = 7.9 nm and is a 1:1 heterodimer. The 2.3 Åcrystal structure of the ESCRT‐0 core complex reveals two domain‐swapped GAT domains and an antiparallel twostranded coiled‐coil, similar to the core of yeast ESCRT‐0. Coarse‐grained Monte Carlo simulations constrained by experimental RH values for full‐length ESCRT‐0 reveal a dynamic ensemble of conformations well‐suited for the diverse functions of the complex.ESCRT‐0 typifies a class of biomolecular assemblies that combine both structured andunstructured elements, and have overall dynamic and open conformations to ensure versatility in target recognition and function. The multipronged approach used here, combining experiments and simulations at different levels of resolution, should be useful whenever the complexes are only partially structured. This research was supported by NIH intramural support, NIDDK (J.H.H. and G.H.) and IATAP (J.H.H.).
Vps45p—a paradigm for Sec1p/Munc18 protein function
Authors: Munson Mary , Furgason Melonnie L.M. , Bryant Nia J.
Published: June 2021

The compartmentalization of eukaryotic cells into functionally distinct membrane‐bound organelles requires a network of vesicles to transport cargo between these compartments, and to the plasma membrane for growth and secretion. The fusion of vesicles with the target organelle requires specific complex formation between v‐SNAREs on the vesicle, and t‐SNAREs on the target membrane, and regulation by the Sec1/Munc‐18 (SM) family proteins. SM proteins are conserved throughout evolution, yet their precise function(s) are unclear. Although SM proteins bind to their cognate t‐SNARE (syntaxin) proteins, seemingly conflicting data from several experimental systems indicates strikingly different modes of interaction for different SM/syntaxin pairs. The SM protein Vps45p (which controls endocytic traffic in conjunction with its cognate syntaxin Tlg2p) uses distinct mechanisms to interact with uncomplexed Tlg2p, the assembled Tlg2p‐containing SNARE complex, and its cognate v‐SNARE Snc2p. We discovered and biochemically characterized a hitherto uncharacterized Vps45p‐Tlg2p interaction‐Vps45p binds to the closed conformation of Tlg2p. Thus, we demonstrate that Vps45p uses all of the binding modes reported between SM proteins and their cognate syntaxins, providing a unifying model for studying SM‐SNARE interactions during distinct stages in the SNARE assembly/disassembly cycle.
Rab9 regulation of the Rab GTPase activating protein, RUTBC1
Authors: Nottingham Ryan M , Ganley Ian G , Barr Francis A , Lambright David G , Pfeffer Suzanne R.
Published: June 2021

Rab GTPases are master regulators of membrane trafficking. Rab GTPase activating proteins (GAPs) enhance a Rab's slow, intrinsic rate of GTP hydrolysis, thereby inactivating them. A so‐called TBC domain is the catalytic domain found in most Rab GAPs. The goal of this study is to determine the functions of RUTBC1 and RUTBC2, which were identified by two hybrid screen as TBC domain‐containing proteins that bind Rab9. Rab9 is required for recycling of mannose 6‐phosphate receptors (MPRs) from endosomes to the Golgi complex. Over‐expression of RUTBC1 and RUTBC2 destabilized MPRs and increased lysosomal enzyme secretion, consistent with Rab9 regulation. Biochemical screening of 33 Rab GTPases revealed that RUTBC1 is not a GAP for Rab9 but rather acts on the Golgi‐localized, Rab33B with a kcat/Km value of ~2800 M−1 s−1; RUTBC2 is most active on Rab17. Moreover, Rab9 binds RUTBC1 and RUTBC2 at a site far from their catalytic domains and does not competitively inhibit RUTBC1 GAP activity on Rab33B. These results suggest a novel mode of regulation where Rab9 in some way regulates a GAP for another Rab protein. This may help maintain the identity of a Rab‐specific membrane microdomain to inactivate other Rabs should they migrate into a Rab9‐enriched compartment. Further work will explore the significance of this cross‐talk between Rab proteins that mediate distinct membrane traffic events. Research was supported by NIH R37 DK37332.
Modulation of Human Cytochrome P450 by Momordica charantia (Bitter Melon)
Authors: Shah Pranjal A , Eck Philip , Nerurkar Pratibha V
Published: June 2021

Momordica charantia (bitter melon, BM) is traditionally used to treat diabetes and its complications. However, the safety and interaction of BM with other therapeutic drugs is unknown. The cytochrome (CYP) P450 enzymes metabolize variety of xenobiotics. Since BM modulates CYP in diabetic rats, we hypothesized that BM will also differentially modulate human P450 enzymes. Our aim was to investigate the effects of BM on CYP1A2 and CYP3A4, using cDNA‐expressed human CYP450, human hepatoma cells, HepG2 and primary human hepatocytes. BMJ had no effect on cDNA‐expressed human CYP1A2 but significantly inhibited CYP3A4 activity, up to 85%. BMJ was non‐toxic to both, HepG2 and primary human hepatocytes as measured by cellular ATP levels and had no effect on CYP1A2 induction in HepG2 cells, for up to 72 h, but significantly inhibited the 3‐methylcholanthrene induced CYP1A2 activity by 48%. BMJ exhibited a dose‐ and time‐dependent inhibition of CYP3A4 activity by 64%. BMJ for 4 h demonstrated a significant reduction of CYP1A2 activity by 30% and CYP3A4 by 20% respectively in primary human hepatocytes. CYP3A4 is an important drug metabolizer for majority of clinically relevant therapeutic drugs. Identifying the BMJ‐associated modulation of P450 is important to determine its safety for future clinical trials and to minimize drug‐drug interactions. [NCCAM (R21AT003719) and NCMHD (P20MD000173), NIH].
Long‐chain acyl‐acyl carrier protein activates transcriptional regulation of fatty acid biosynthesis in Streptococcus pneumoniae
Authors: Jerga Agoston , Rock Charles O
Published: June 2021

In the human pathogen S. pneumoniae, transcription of the fab genes encoding the enzymes of type II fatty acid biosynthesis is controlled by FabT, a transcriptional repressor. However, FabT overexpression fails to suppress fab gene expression indicating that a metabolic signal in addition to FabT is required for transcriptional regulation. A screen of metabolic intermediates for their effect on FabT DNA binding revealed that long‐chain acyl‐acyl carrier protein (ACP) end‐products of fatty acid synthesis increased FabT binding to specific sites within the promoters of the fab genes. Activation of FabT DNA binding was most prominent in the presence of acyl‐ACPs carrying either saturated or unsaturated fatty acids of 16 to 18 carbon chain length. Electrophoresis mobility shift assay confirmed that acyl‐ACP binds to FabT and forms a FabT‐acyl‐ACP‐DNA complex. Thus, the transcription of the fab genes is controlled by a feedback regulatory loop involving the binding of acyl‐ACP end products of fatty acid biosynthesis to FabT that increases its affinity for specific sites within the fab promoters thereby repressing fab gene expression. (Supported by NIH GM34496 and ALSAC)
Whole Genome Expression Profile of Human Peripheral Blood Mononuclear cells Exposed to Bacillus anthracis in vitro
Authors: Miller Stacy ‐ Ann , Hammamieh Rasha , Barmada Moshen , Hellman Matthew , Muhie Seid , Chatmon Samuel , Koterski James , Jett Marti
Published: June 2021

Dependable and efficient diagnosis of exposures to Bacillus anthracis has long been a major concern for clinicians because effective treatment requires intervention early in the course of disease. Thus, treatment of B. anthracis infection has relied on much earlier identification of the pathogen before the onset of symptoms. We used peripheral blood mononuclear cells (PBMCs) as early sentinels of anthrax exposure. We identified a number of host biomarkers for B. anthracis infection by profiling the expression of mRNA transcripts in infected PBMCs. Exposure to B. anthracis down regulated p38 MAP kinase, NF‐kB, fibronectin, IL‐2 and IL‐8, matrix metallopeptidases, T‐cell receptors. On the other hand, B. anthracis induced genes involved in COX‐2, PKC, JAK/STAT, IFNG pathways, TLR4, MYD88, CASP1, IL‐1alpha, IL‐6 and the B‐cell CD markers: CD79, CD38 and CD72. Identification of definite gene expression profiles and patterns in infected cells may suggest suitable strategies for prevention or treatment that would lead to alteration of the disease progression.
Identification and characterization of the cholesterol 25‐hydroxylase gene promoter
Authors: Watters Dianne Josephine , Nourse Jamie
Published: June 2021

25‐Hydroxycholesterol (25HC) is the product of the cholesterol 25‐hydroxylase (CH25H) gene, and is one of the most potent inhibitors of cholesterol synthesis, through the suppression of SREBP activation. 25HC has also been implicated in neurodegenerative disease and shown to be toxic to primary cultured neuronal cells. Very little is known about the transcriptional regulation of the CH25H gene. Using in silico analysis of the mouse and human promoters, potential binding sites for several transcription factors were identified including p53, CREB and Sp1. The functionality of the putative promoter elements of the mouse promoter was investigated using an in vitro reporter assay with deletion constructs. The results suggest the proximal CREB binding site may be involved in driving expression from the CH25H promoter. While a high proportion TATA‐less promoters contain CREB binding sites, these promoters appear to be generally unresponsive to cAMP, suggesting activation of these promoters depends on co‐stimulatory signals. In this study we observed co‐activation of the CH25H promoter by Sp1 and CREB in a synergistic manner. A negative feedback mechanism involving 25HC was also observed in the in vitro reporter assays and this effect appeared to be mediated by an element within the proximal 250 bp of the promoter. The region containing the p53 consensus site was demonstrated to repress transcription.
Adult Stem Cells and Nanomaterials for Skeletal Tissue Engineering and Regeneration
Authors: Tuan Rocky S
Published: June 2021

Understanding nanobiology and application of nanotechnology is important in regenerative medicine, since nanoscale materials are the basic functional subunits of cells and tissues. Stem/progenitor cells are a promising candidate cell type in tissue engineering and regeneration, because of their expandability and multi‐differentiation potential. A key requirement in tissue engineering is the three‐dimensionality of the "regenerate tissue", particularly for weight‐bearing musculoskeletal tissues. The challenges in skeletal tissue engineering and regeneration and the application of adult stem cells and nanomaterial scaffolds will be presented. The biology of human mesenchymal stem cells, e.g., proliferation vs differentiation, is intricately regulated by cell‐cell interactions, signaling by extracellular biofactors, and transcriptional and epigenetic events. Architectural and structure‐dependent cues provided by the matrix also guide cell‐based tissue morphogenesis. We have developed biomimetic, biodegradable nanofibrous biomaterial scaffolds for cell‐based tissue engineering. The fabrication and biological basis of the scale‐dependent bioactivities of nanofibrous scaffolds will be presented, as well as their application for cartilage tissue engineering, including articular cartilage and intervertebral disc. (Support: NIH AR Z01 41131)
Membrane binding and insertion of the GRP1 PH and Epsin ENTH domains
Authors: Kutateladze Tatiana G , He Ju , Vora Mohsin , Zhang Honglu , Verkhusha Vladislav V , Prestwich Glenn D , Stahelin Robert V
Published: June 2021

Phosphoinositide (PI)‐binding domains recruit a variety of signaling and trafficking proteins to the distinct intracellular membranes. In the plasma membrane, PtdIns(4,5)P2 and PtdIns(3,4,5)P3 lipids are recognized by the PH (pleckstrin homology) and ENTH (Epsin N‐terminal homology) domains. Here, the molecular basis of membrane anchoring and penetration by the PH domain of general receptor for phosphoinositides isoform 1 (GRP1) and Epsin ENTH domain is detailed. The specificities and binding affinities for PI‐containing SUVs and soluble metabolically‐stabilized analogues of PIs, insertion into monolayers and bilayers, and regulation of the lipid binding and membrane penetration by pH are investigated using nuclear magnetic resonance spectroscopy, surface plasmon resonance, liposome‐binding and surface tension experiments. The significance of the pH sensitivity and the effect of the acidic and basic media on the PI recognition, hydrophobic insertion and non‐specific electrostatic contacts is established.
Regulation of SH2 domain‐containing inositol 5′ phosphatase by the cyclic AMP dependent protein kinase
Authors: Zhang Jun , Garrison James C.
Published: June 2021

The SH2 domain‐containing inositol 5' phosphatase, SHIP1, regulates hematopoietic cell function by opposing the action of phosphatidylinositol 3‐kinase (PI3K) and maintains low levels of phosphatidylinositol‐3,4,5‐trisphosphate (PIP3). The activation of the cyclic AMP dependent protein kinase (PKA) opposes many of the pro‐inflammatory responses of hematopoietic cells. Thus, we tested to see if the activity of SHIP1 was regulated via phosphorylation by PKA. We prepared highly pure SHIP1 from HEK‐293 cells by immunoaffinity chromatography. Purified SHIP1 can be rapidly phosphorylated by PKA to a stoichiometry of over 0.6 mole PO4/mole SHIP1. In 32P labeled HEK‐293 cells transfected with SHIP1, stimulation with Sp‐cAMPS or activation of the endogenous β‐adrenergic receptor caused an increase in the phosphorylation state of SHIP1. Inhibition of protein phosphatase activity with okadaic acid also increased the phosphorylation of SHIP1. The 5' phosphatase activity of SHIP1 was measured directly using an improved Malachite Green assay. Phosphorylation of the protein in vitro or in cells increased the activity of SHIP1 two fold. Thus, activation of G‐protein coupled receptors which raise cyclic AMP cause SHIP1 to be phosphorylated and stimulate its inositol phosphatase activity. These results outline a novel mechanism of SHIP1 activation by PKA phosphorylation. Supported by NIH Grant R01GM076236.
Activity of Fixed Lectin Beads, A Drug Testing Model
Authors: Sosa Juan R. , Sepidehdam Sahar , Oppenheimer Steven B.
Published: June 2021

Lectin derivatized agarose beads are widely used for glycan purification, cell surface analysis, and in the development of anti‐infection and anti‐cancer drugs. These beads have limited shelf life, primarily because lectins, like other proteins, can degrade over time. Here we ask, can fixed lectin beads maintain their binding properties, thereby probably extending their shelf lives? We have already shown that fixed yeast display the same binding properties as live yeast. Concanavalin A (Con A) derivatized agarose beads were fixed with 3.7% formaldehyde in distilled water, washed three times, and their binding to mannose (a Con A binding sugar) rich yeast (Saccharomyces cerevisiae) was compared with unfixed washed beads, in the presence and absence of alpha methyl mannose. In scores of experiments, examining and counting thousands of yeast bound to both bead types, it was found that bead fixation did not inhibit yeast binding, while alpha methyl mannose reduced binding. The results suggest that formaldehyde fixed beads are as active as unfixed beads, offering promise that the shelf life of the beads could be greatly extended. The yeast‐lectin bead system is a useful model for assessing lectin‐glycan interactions, because unlike free glycan‐containing molecules, yeast binding is quickly, precisely and easily assessed by eye, without the need for expensive assay systems. Assays are carried out in 0.2 ml droplets on glass slides, using the flat ends of toothpicks to add beads and yeast and mix samples, plus and minus sugar solutions at varying concentrations. See Zem, et al. (Acta Histochem 108: 311‐317, 2006) for a detailed description of this assay system (Supported by NIH NIGMS SCORE (S0648680), MARC, RISE, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Hybrid hydrogels for use in vocal fold tissue engineering
Authors: Woods Meghan Danielle , Grieshaber Sarah , Jia Xinqiao
Published: June 2021

Elastin is an extracellular matrix (ECM) protein abundant in vocal folds and other mechanically active tissues. This protein provides these tissues with elastic recoil and strength. Due to difficulties with natural elastin, there is a need to develop scaffolds for vocal fold tissue regeneration that mimics this elasticity and also provides tunability in a range of morphological, biological and mechanical properties. Hybrid polymer‐peptide materials are attractive candidates for tissue engineering scaffolds because their synthesis allows for this tunability. In this work, synthetic polymers of poly(ethylene glycol) (PEG) and peptides with the amino acid repeat unit AKAAAKA found in natural elastin were synthesized and cross‐linked to form hybrid hydrogels. PEG was functionalized with azide end groups and cross‐linked through a copper‐catalyzed azide‐alkyne cycloaddition reaction with peptides functionalized with multiple alkyne groups. Both linear and 4‐arm star PEG polymers with varying molecular weights were used in these trials, and the peptide length and functionality was varied by the number of repeat units. It was found that the swelling ratio and mechanical properties of the hybrid hydrogels can be tuned according to the molecular weight and geometry of each component. This project was funded by HHMI, NIH/NIDCD (1R01DC008965‐01), and NSF/DMR (Career: 0643226).
Multimeric heparan sulfate modulates FGF signaling in zebrafish development
Authors: Nguyen Thao Kim Nu , Tran Vy M. , Veien Eric S. , Dorsky Richard I. , Chien Chi‐Bin , Kuberan Balagurunathan
Published: June 2021

Heparan sulfate proteoglycans (HSPGs) play crucial roles in a number of signaling pathways during development through binding to various secreted molecules such as FGF, WNT, SHH and BMP. Most of the studies examined the interactions between these secreted proteins with monomeric heparin sequences. However, almost all endogenous proteoglycans (PGs) possess two or more glycosaminoglycan (GAG) chains. This study is the first attempt to stimulate the expressions of endogenous PG mimetics with two or more HS chains connected covalently in an animal model to investigate the roles of these molecules in various signaling events. A library of monomeric and multimeric xylosides, which prime GAG chains, was examined in zebrafish embryos. We found that only clustered HS chains primed by multimeric xylosides hyperactivated the FGF/FGFR mediated signaling pathways leading to elongation of zebrafish embryos. Based on our findings, we propose that multimeric HS is required for the formation of biologically relevant HS/FGF/FGFR ternary complexes, leading to receptor dimerization in vivo and subsequent cell signaling events.
Histone‐folds: A novel membrane‐anchoring domain in Son of Sevenless
Authors: Yadav Kamlesh Kumar , Bar‐Sagi Dafna
Published: June 2021

The RAS‐MAPK pathway controls a variety of cellular functions such as survival, proliferation and differentiation. The guanine nucleotide exchange factor SOS couples receptor tyrosine kinases to the RAS‐MAPK pathway by mediating a signal dependent activation of RAS. The activity of SOS is tightly regulated by multiple inter‐ and intramolecular interactions and the loss of this regulation due to germ line mutations leads to gingival fibramatosis type‐I and Noonan Syndrome (NS). SOS has a unique Histone Folds (HF) domain similar to H2A/H2B nuclear histones of as yet unknown function. Electrostatic modeling of the HF suggested a membrane binding function. Indeed, the isolated HF shows signal‐dependent membrane localization. A screen of 15 common membrane‐lipids, using recombinant HF, identified Phosphatidic acid (PA) as the specific lipid ligand. The motif for the PA binding on HF has been identified by mutagenesis studies and shown to be essential for the membrane localization of the HF and SOS‐mediated RAS activation. The gain of function NS mutation E108K, which lies in the PA binding motif, increases PA binding and membrane localization of the HF and enhances SOS‐mediated RAS activation. These findings identify a new membrane anchoring modality and provide insights into the spatial regulation of RAS activation.

This work was supported by research grants from the National Institutes of Health to D.B.‐S. (CA55360 and GM078266).
The START domain Coq10 polypeptide binds Coenzyme Q and is required for mitochondrial respiratory electron transport
Authors: Morvaridi Susan , Saiki Ryoichi , Liau Wei‐Siang , Kawashima Tadashi , Clarke Cathy
Published: June 2021

Coenzyme Q (Q) is a redox active lipid that is an essential component of the electron transport chain in the mitochondrial membrane. In Saccharomyces cerevisiae, ten COQ genes are required for Q biosynthesis and function in respiratory electron transport. Nine genes are required for synthesis of Q, COQ1 through COQ9. However, unlike the other coq mutants, each of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. The defect in oxidation of NADH and succinate in coq10 mutant mitochondria can be rescued by the addition of Q2, a water soluble analog.

Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic‐rich protein family" Pfam03654. The structure of the Caulobacter crescentus Coq10 homolog, CC1736, identified it as a member of the START domain superfamily. The START domain containes a hydrophobic cavity that binds lipophilic molecules such as cholesterol or polyketides.

Our data show that expression of C. crescentus CC1736 is able to rescue the respiratory deficiency of the yeast coq10 null mutant. The rescue depends on the presence of a mitochondrial leader sequence. An assay of Q binding shows that purified CC1736 is able to bind Q10, Q6 and Q2 in molar ratio near to one.

Site directed mutagenesis shows that Valine 70 of CC1736 is crucial for both rescue of the coq10 mutant and the coenzyme Q10 binding. The results suggest that the Coq10 polypeptide:coenzyme Q, protein:ligand complex may serve an essential function in the delivery of Q from its site of synthesis in the inner mitochondrial membrane to the respiratory electron transport chain.
Auto‐inhibition of dynamin GTPase activity is regulated by PH domain interactions
Authors: Kenniston Jon A. , Lemmon Mark A.
Published: June 2021

The dynamin GTPase drives receptor‐mediated endocytosis in addition to other membrane vesicle scission events. We aim to understand how vesicle interactions of the dynamin pleckstrin homology (PH) domain contribute to the conversion of basal, auto‐inhibited dynamin tetramers to an activated, multi‐oligomeric state with a >200‐fold increased rate of GTP hydrolysis. Although dynamin self‐assembly alone has been used to explain this activation, emerging evidence suggests that dynamin may have a more complex conformational interplay between its PH and GTPase domains. Through selective mutagenesis of the dynamin PH domain based on common PH‐GTPase interaction motifs in biology, we have identified an interface of the dynamin PH domain that regulates auto‐inhibition of dynamin GTP hydrolysis rates. Specifically, we find that mutations within this interface fall into two distinct groups: a "weak" class with modestly elevated basal and lipid‐stimulated GTPase activity, as well as an "uncoupled" class whose GTPase rates are elevated ~70‐fold even in the absence of lipid.

Funding provided by The Jane Coffin Childs Memorial Fund for Medical Research (JAK) and NIH R01‐GM‐078345 (MAL)
Crystal structure of the murine Voltage Dependent Anion Channel 1 (mVDAC1) at 2.3 Å resolution
Authors: Ujwal Rachna , Cascio Duilio , Colletier Jacques‐Philippe , Faham Salem , Zhang Jun , Toro Ligia , Ping Peipei , Abramson Jeff
Published: June 2021

VDAC constitutes the primary pathway for the exchange of metabolites across the outer mitochondrial membrane. It acts as a scaffold for modulator proteins and is implicated in mitochondria‐dependent cell death. Because of its critical role in mitochondrial biology, we sought to obtain the crystal structure of VDAC as a first step to understanding its gating mechanism, in vivo regulation and interaction with other proteins. mVDAC1 was expressed in E. coli, purified from inclusion body and refolded. The purified protein was biochemically characterized to ensure that its identity, proper fold and electrophysiological properties are the same as the native protein.

We present the structure of mVDAC1 at 2.3 Åresolution crystallized in lipidic bicelles to preserve the native conformation. mVDAC1 adopts a β‐barrel architecture composed of 19 β‐strands, thus displaying a new fold for β‐barrel membrane proteins resulting from an odd number of strands. The N‐terminus is located inside the barrel and forms an α‐helix halfway down the pore resulting in a partial narrowing at the center. This orientation of the helix is ideally suited to gate metabolite flux through the channel. The mVDAC1 structure presented here represents the first high resolution crystal structure of a eukaryotic β‐barrel membrane protein providing critical information for our understanding of its role in mitochondrial physiology. Funding: NIH and AHA.
The development of a quick screen in yeast for functional epithelial sodium channels (ENaC)
Authors: Ybanez Raquel V , Boswell Mikki G , Booth Rachell E
Published: June 2021

The epithelial sodium channel (ENaC) is essential for sodium re‐absorption in the distal nephron of the kidney, thus, plays a critical role in fluid maintenance. In an effort to identify critical regions within the channel subunits, we have developed a quick screen for functional ENaC in yeast. In this system, ENaC genes were co‐transformed into Saccharomyces cerevisiae strain S1InsE4A on individual yeast expression vectors with different selectable markers. Western blot analysis was used to confirm ENaC production, immunocytochemistry was used to demonstrate membrane localization of subunits, and survival pronging dilutions were used to assay salt sensitivity. Five different αENaC mutants with various levels of expected function were then screened for salt sensitivity. Our results demonstrated various levels of salt sensitivity indicating that when expressed in yeast, survival pronging can be used to detect variations in ENaC mutant function. Additionally, we found that αENaC with a C‐terminal tag is not functional (i.e. no salt sensitivity) in yeast; however, function seems unaffected by an tag at the N‐terminal. This yeast system will enable quick screening of additional mutations in ENaC subunits that may affect function. Funding has been provided by Research Corporation, the Welch Foundation, and Texas State University‐San Marcos.
Cell culture density affects the susceptibility of human glioma cells to β‐ODAP toxicity
Authors: Hilbrand Ashley , Goltz Abby , Chase Leah
Published: June 2021

β‐ODAP is a neurotoxin that is found in the seeds of the grass pea, Lathyrus satvius, and is reported to cause neurolathyrism. The mechanism by which β‐OD AP causes neurolathyrism is not well understood, but it has been shown to be a transportable inhibitor of the transporter, system xc−. System xc− is involved in the exchange of cystine and glutamate across the plasma membrane in order to supply the cell with the cysteine necessary to maintain cellular glutathione levels. Previous studies in our lab have shown that system xc− cell surface expression is regulated by the redox state of the cell and cell culture density. In this study, we used β‐ODAP to inhibit cystine uptake by system xc− in order to reduce cellular glutathione levels in cultured glioma cells, and we subsequently examined the susceptibility of these cells to oxidative cell death. We have shown that effect of β‐ODAP on cellular glutathione levels and susceptibility to oxidative cell death is also dependent on cell culture density, with confluent cultures demonstrating an increased capacity to maintain cellular gluathione levels and resist oxidative cell death compared to non‐confluent cells. We hypothesize that the observed differences in susceptibility of confluent and non‐confluent cultures to β‐ODAP are a result of reduced capacity of non‐confluent cells to increase cell surface expression of System xc− when the oxidative load of the cell is increased.
CREB‐MEDIATED IL‐6 EXPRESSION IS REQUIRED FOR 15(S)‐HYDROXYEICOSATETRAENOIC ACID‐INDUCED VASCULAR SMOOTH MUSCLE CELL MIGRATION
Authors: Chava Koteswara Rao , Karpurapu Manjula , Wang Dong , Bhanoori Manjula , Sridharan Venkatesh Kundumani , Rao Gadiparthi N
Published: June 2021

Migration of vascular smooth muscle cells (VSMC) from media to intima is a key event in the pathophysiology of atherosclerosis and restenosis. Cyclic AMP response element binding protein (CREB) has been implicated in the regulation of VSMC growth and motility in response to thrombin and angiotensin II. In this study, we tested the role of CREB in an oxidized lipid molecule, 15(S)‐HETE‐induced VSMC migration and injury‐induced restenosis. 15(S)‐HETE stimulated VSMC migration in a CREB‐dependent manner, as measured by the modified Boyden chamber method. Blockade of MEK1, JNK1 or p38MAPK inhibited 15(S)‐HETE‐induced CREB phosphorylation and VSMC migration. 15(S)‐HETE induced expression and secretion of interleukin‐6 (IL‐6), as analyzed by RT‐PCR and ELISA, respectively. Neutralizing anti‐IL‐6 antibodies blocked 15(S)‐HETE‐induced VSMC migration. Adenoviral mediated expression of dominant negative MEK1, JNK1, p38MAPK or CREB suppressed 15(S)‐HETE induced IL‐6 expression in VSMC. Serial 5′‐deletions and site‐directed mutagenesis analysis in an IL‐6 promoter‐dependent luciferase reporter system showed that cAMP response element is essential for 15(S)‐HETE‐induced IL‐6 expression. Dominant negative CREB also suppressed balloon injury‐induced IL‐6 expression and neointima formation. Thus, these results suggest a role for MAPK‐dependent CREB‐mediated IL‐6 expression in 15(S)‐HETE‐induced VSMC migration. These findings also suggest a role for CREB in balloon injury‐induced IL‐6 expression and neointima formation.
Effects of vitamin D on tamoxifen resistant breast cancer cell growth
Authors: Chow Rachel Y. , Siewit Christina L. , Louie Maggie C.
Published: June 2021

According to the Centers for Disease Control and Prevention (CDC), breast cancer is currently the 6th leading cause of death of women in the U.S. (CDC, 2007). Currently, estrogen receptor (ER) positive breast cancers are frequently treated with anti‐estrogens such as tamoxifen. Tamoxifen is a selective ER modulator (SERM) that binds to ER and inhibits the expression of genes associated with cell growth. Although tamoxifen therapy has resulted in some success, resistance often develops with prolonged therapy, in which case the patients no longer respond to tamoxifen. How this resistance develops is unclear. To further understand the mechanism of anti‐estrogen resistance, our lab has developed a tamoxifen resistant breast cancer cell line (MCF7‐TamR). Data from our lab suggests that MCF7‐TamR express lower levels of vitamin D receptor (VDR) than parental MCF7 cells. Additionally, resistant cells also display a greater sensitivity to vitamin D inhibition. Whether this increased inhibition by vitamin D is associated with the VDR levels is unclear. The understanding of how vitamin D inhibits resistant and non‐resistant cells may provide further insight into the development of tamoxifen resistant breast cancer and potentially identify new targets for future therapeutics.
PKG induces the expression of tumor suppressor genes in colon cancer cells
Authors: Sellak Hassan , Wu Songwei , Lincoln Thomas M.
Published: June 2021

The role of cyclic guanosine 3',5′‐monophosphate (cGMP)‐dependent protein kinase (PKG) as an anticancer messenger emerged recently, but there is little known about its action. In this study, we examined the role of PKG‐Iβ on the beta‐catenin pathway and the expression of tumor suppressor proteins in human colon cancer cells. Our results revealed that cells overexpressing PKG‐Iβ showed β‐catenin translocation to the nucleus compared to control cells. This translocation of β‐catenin was accompanied with an increase in T‐cell factor (TCF)‐luciferase activity and β‐catenin binding to TCF oligonucleotide as tested by elecromobility shift assays in both LoVo and SW480 cells. We analyzed the expression of the tumor suppressor retinoblastoma (Rb), p27KIP1, p53, and p21CIP1 at both the protein levels and promoter activities. Cells overexpressing PKG‐Iβ showed an increase in expression of the p21CIP1, p27KIP1, and Rb proteins. Both p21CIP1 and p27KIP1 promoters were activated in LoVo or SW480 cells overexpressing PKG as demonstrated by luciferase reporter assays. Specific PKG inhibitors (DT2/DT3) as well as phosphatase inhibitors (okadaic acid, Calyculin A) reduced β‐catenin accumulation in the nucleus as well as the promoter activities of p21 and Rb luciferase reporter constructs. These results suggest that PKG is stabilizing β‐catenin, probably through activation of a phosphatase. These effects of PKG activation on the expression of tumor suppressor genes may explain, at least in part, the anticancer effects of activation of PKG.
ERK Activation Requires CaM Kinases in MCF‐7 Breast Cancer Cells
Authors: Abell Ellen , Schmitt John M.
Published: June 2021

A key signaling pathway involved in regulating cell growth and proliferation throughout the body is the ERK signaling pathway. ERK is activated via numerous pathways including intracellular calcium release downstream of G‐Protein Coupled Receptors (GPCRs). Carbachol, a GPCR‐agonist, both increases intracellular calcium and ERK activation in MCF‐7 cells. ERK activation and control of cell growth may act through the transcription factor Elk‐1. Our goal was to elucidate the specific proteins and kinases upstream of ERK in MCF‐7 cells treated with carbachol. Secondly, we wanted to investigate the potential involvement of Elk‐1 downstream of ERK. Carbachol treatment of MCF‐7 cells triggered ERK and Elk‐1 phosphorylation within 5 minutes. Interestingly, transfection of cells with shRNAs directed to either CaM KKα or CaM KIγ significantly inhibited carbachol activation of ERK. Phosphorylation of Elk‐1, following carbachol stimulation, was also blocked in siERK2 transfected cells, suggesting that ERK2 is required for carbachol's activation of Elk‐1. Our results suggest that carbachol treatment of MCF‐7 cells activates ERK and cell growth through CaM KK, CaMKI, and an Elk‐1‐dependent pathway.