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Metabolic regulation of CaMKII protein and caspases in Xenopus laevis egg extracts.
McCoy F, Darbandi R, Chen SI, Eckard L, Dodd K, Jones K, Baucum AJ, Gibbons JA, Lin SH, Colbran RJ, Nutt LK
(2013) J Biol Chem 288: 8838-48
MeSH Terms: Animals, Apoptosis, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Caspase 2, Caspase 3, Caspase 7, Cell Death, Cell Proliferation, Gene Expression Regulation, Developmental, Gene Expression Regulation, Enzymologic, Mass Spectrometry, Oocytes, Oxygen, Peptides, Phosphorylation, Protein Phosphatase 1, Recombinant Proteins, Sepharose, Serine, Xenopus, Xenopus laevis
Show Abstract · Added June 21, 2013
The metabolism of the Xenopus laevis egg provides a cell survival signal. We found previously that increased carbon flux from glucose-6-phosphate (G6P) through the pentose phosphate pathway in egg extracts maintains NADPH levels and calcium/calmodulin regulated protein kinase II (CaMKII) activity to phosphorylate caspase 2 and suppress cell death pathways. Here we show that the addition of G6P to oocyte extracts inhibits the dephosphorylation/inactivation of CaMKII bound to caspase 2 by protein phosphatase 1. Thus, G6P sustains the phosphorylation of caspase 2 by CaMKII at Ser-135, preventing the induction of caspase 2-mediated apoptotic pathways. These findings expand our understanding of oocyte biology and clarify mechanisms underlying the metabolic regulation of CaMKII and apoptosis. Furthermore, these findings suggest novel approaches to disrupt the suppressive effects of the abnormal metabolism on cell death pathways.
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21 MeSH Terms
A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology.
Gao Y, Majumdar D, Jovanovic B, Shaifer C, Lin PC, Zijlstra A, Webb DJ, Li D
(2011) Biomed Microdevices 13: 539-48
MeSH Terms: Animals, Cell Communication, Cell Line, Tumor, Cell Movement, Coculture Techniques, Dimethylpolysiloxanes, Endothelial Cells, Humans, Hydrodynamics, Mice, Microfluidic Analytical Techniques, Neurons, Pressure, Sepharose
Show Abstract · Added June 24, 2013
A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.
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14 MeSH Terms
Paracrine signals from the mouse conceptus are not required for the normal progression of decidualization.
Herington JL, Underwood T, McConaha M, Bany BM
(2009) Endocrinology 150: 4404-13
MeSH Terms: Animals, Decidua, Embryo Implantation, Embryo, Mammalian, Female, Mice, Models, Animal, Paracrine Communication, Pregnancy, Pseudopregnancy, Sepharose
Show Abstract · Added April 10, 2018
The purpose of this study was to determine whether the conceptus directs the formation of a tight- and adherens-dependent permeability barrier formed by the primary decidual zone and normal progression of decidual cell differentiation during embryo implantation. Four artificial models of decidualization were used, some apparently more physiological than others. The results show that both the formation of the permeability barrier and decidual cell differentiation of three of the artificial models were quite different from that of pregnant uteri. One artificial model of decidualization, namely pseudopregnant animals receiving concanavalin A-coated Sepharose bead transfers on d 2.5 of pseudopregnancy, better recapitulated the decidual changes that occur in the pregnant uterus undergoing decidualization. This included the formation of a primary decidual zone-like permeability barrier and decidual growth. This model also exhibited similar temporal changes of the expression of genes involved in decidualization that are markers of decidual cell differentiation. Overall, the results of this study indicate that some models of inducing decidualization artificially produce responses that are more similar to those occurring in the pregnant uterus, whereas others are quite different. More importantly, the results suggest that concanavalin A-coated Sepharose beads can provide an equivalent stimulus as the trophectoderm to cause the formation of the primary decidual zone permeability barrier.
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MeSH Terms
Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression.
Chandler KJ, Chandler RL, Broeckelmann EM, Hou Y, Southard-Smith EM, Mortlock DP
(2007) Mamm Genome 18: 693-708
MeSH Terms: Animals, Chromosomes, Artificial, Bacterial, DNA, Gene Expression, Genetic Techniques, Mice, Mice, Transgenic, Models, Genetic, Nucleic Acid Hybridization, Pedigree, Polymorphism, Genetic, Reverse Transcriptase Polymerase Chain Reaction, Sepharose, Transgenes
Show Abstract · Added October 31, 2013
Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.
3 Communities
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14 MeSH Terms
Positive regulation of IkappaB kinase signaling by protein serine/threonine phosphatase 2A.
Kray AE, Carter RS, Pennington KN, Gomez RJ, Sanders LE, Llanes JM, Khan WN, Ballard DW, Wadzinski BE
(2005) J Biol Chem 280: 35974-82
MeSH Terms: Active Transport, Cell Nucleus, Adenosine Triphosphate, Alkenes, Animals, B-Lymphocytes, Catalysis, Cell Line, Chromatography, Liquid, Cytoplasm, Enzyme Activation, Enzyme Inhibitors, Fibroblasts, Gene Deletion, Humans, I-kappa B Kinase, Immunoblotting, Immunoprecipitation, Inflammation, Jurkat Cells, Mice, Mice, Inbred C57BL, Mutation, Okadaic Acid, Phosphoprotein Phosphatases, Phosphorylation, Polyenes, Protein Phosphatase 2, Pyrones, Sepharose, Serine, Signal Transduction, Spleen, Threonine, Time Factors, Transfection, Tumor Necrosis Factor-alpha
Show Abstract · Added December 10, 2013
Transcription factor NF-kappaB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF). In the absence of TNF, NF-kappaB is sequestered in the cytoplasm by inhibitory IkappaB proteins. Phosphorylation of IkappaBby the beta-catalytic subunit of IKK, a multicomponent IkappaB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-kappaB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKbeta, which greatly stimulates IkappaB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IkappaB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKKgamma regulatory subunit. Third, deletion of the PP2A-binding site in IKKgamma attenuates T loop phosphorylation and catalytic activation of IKKbeta in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK.PP2A complexes is required for the proper induction of IkappaB kinase activity in vivo.
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36 MeSH Terms
Multivalent interactions of calcium/calmodulin-dependent protein kinase II with the postsynaptic density proteins NR2B, densin-180, and alpha-actinin-2.
Robison AJ, Bass MA, Jiao Y, MacMillan LB, Carmody LC, Bartlett RK, Colbran RJ
(2005) J Biol Chem 280: 35329-36
MeSH Terms: Actinin, Amino Acid Sequence, Animals, Baculoviridae, Blotting, Western, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Glutathione, Glutathione Transferase, Histidine, Immunoprecipitation, Insecta, Macromolecular Substances, Male, Mice, Molecular Sequence Data, Multiprotein Complexes, Mutation, Neurons, Phosphorylation, Protein Binding, Protein Isoforms, Protein Structure, Tertiary, Rats, Rats, Sprague-Dawley, Receptors, N-Methyl-D-Aspartate, Sepharose, Sialoglycoproteins, Signal Transduction, Swine, Synapses, Threonine, Xenopus
Show Abstract · Added June 21, 2013
Dendritic calcium/calmodulin-dependent protein kinase II (CaMKII) is dynamically targeted to the synapse. We show that CaMKIIalpha is associated with the CaMKII-binding proteins densin-180, the N-methyl-D-aspartate receptor NR2B subunit, and alpha-actinin in postsynaptic density-enriched rat brain fractions. Residues 819-894 within the C-terminal domain of alpha-actinin-2 constitute the minimal CaMKII-binding domain. Similar amounts of Thr286-autophosphorylated CaMKIIalpha holoenzyme [P-T286]CaMKII bind to alpha-actinin-2 as bind to NR2B (residues 1260-1339) or to densin-180 (residues 1247-1495) in glutathione-agarose cosedimentation assays, even though the CaMKII-binding domains share no amino acid sequence similarity. Like NR2B, alpha-actinin-2 binds to representative splice variants of each CaMKII gene (alpha, beta, gamma, and delta), whereas densin-180 binds selectively to CaMKIIalpha. In addition, C-terminal truncated CaMKIIalpha monomers can interact with NR2B and alpha-actinin-2, but not with densin-180. Soluble alpha-actinin-2 does not compete for [P-T286]CaMKII binding to immobilized densin-180 or NR2B. However, soluble densin-180, but not soluble NR2B, increases CaMKII binding to immobilized alpha-actinin-2 by approximately 10-fold in a PDZ domain-dependent manner. A His6-tagged NR2B fragment associates with GST-densin or GST-actinin but only in the presence of [P-T286]CaMKII. Similarly, His6-tagged densin-180 or alpha-actinin fragments associate with GST-NR2B in a [P-T286]CaMKII-dependent manner. In addition, GST-NR2B and His6-tagged alpha-actinin can bind simultaneously to monomeric CaMKII subunits. In combination, these data support a model in which [P-T286]CaMKIIalpha can simultaneously interact with multiple dendritic spine proteins, possibly stabilizing the synaptic localization of CaMKII and/or nucleating a multiprotein synaptic signaling complex.
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33 MeSH Terms
Calmodulin interacts with the V2 vasopressin receptor: elimination of binding to the C terminus also eliminates arginine vasopressin-stimulated elevation of intracellular calcium.
Nickols HH, Shah VN, Chazin WJ, Limbird LE
(2004) J Biol Chem 279: 46969-80
MeSH Terms: Amino Acid Sequence, Animals, Binding, Competitive, COS Cells, Calcium, Calmodulin, Cell Line, Cell Membrane, Cyclic AMP, DNA, Complementary, Detergents, Dogs, Dose-Response Relationship, Drug, Endocytosis, Enzyme-Linked Immunosorbent Assay, Glutathione Transferase, Humans, Hydrogen-Ion Concentration, Kinetics, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Peptides, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Receptors, Vasopressin, Sepharose, Sequence Homology, Amino Acid, Signal Transduction, Time Factors, Transfection, Two-Hybrid System Techniques, Vasopressins
Show Abstract · Added December 10, 2013
To identify molecules that might contribute to V2 vasopressin receptor (V2R) trafficking or signaling, we searched for novel interacting proteins with this receptor. Preliminary data, using the V2R C terminus as bait in a yeast two-hybrid screen, revealed calmodulin as a binding partner. Because calmodulin interacts with other G protein-coupled receptors, we explored this interaction and its possible functional relevance in greater detail. A Ca2+ -dependent interaction occurs between calmodulin-linked agarose and the holo-V2R as well as the V2R C terminus. Truncation and site-directed mutagenesis of the V2R C terminus revealed an involvement of an RGR sequence in this interaction. NMR studies showed that a peptide fragment of the V2R C terminus containing the RGR sequence binds to calmodulin in a Ca2+ -dependent manner with a Kd < or =1.5 microm; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of approximately 200 nm for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells.
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33 MeSH Terms
The preferred pathway of glycosaminoglycan-accelerated inactivation of thrombin by heparin cofactor II.
Verhamme IM, Bock PE, Jackson CM
(2004) J Biol Chem 279: 9785-95
MeSH Terms: Binding Sites, Chromatography, Affinity, Dermatan Sulfate, Dose-Response Relationship, Drug, Glycosaminoglycans, Heparin, Heparin Cofactor II, Humans, Hydrogen-Ion Concentration, Ions, Kinetics, Ligands, Models, Chemical, Mutation, Peptides, Protein Binding, Sepharose, Spectrometry, Fluorescence, Thermodynamics, Thrombin
Show Abstract · Added January 20, 2015
Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength (I) 0.125 m demonstrated that DS and heparin bound much tighter to thrombin (K(T(DS)) 1-5.8 microm; K(T(H)) 0.02-0.2 microm) than to HCII (K(HCII(DS)) 236-291 microm; K(HCII(H)) 25-35 microm), favoring formation of T.GAG over HCII.GAG complexes as intermediates for T.GAG.HCII complex assembly. At [GAG] < K(HCII(GAG)) the GAG and HCII concentration dependences of the first-order inactivation rate constants (k(app)) were hyperbolic, reflecting saturation of T.GAG complex and formation of the T.GAG.HCII complex from T.GAG and free HCII, respectively. At [GAG] > K(HCII(GAG)), HCII.GAG complex formation caused a decrease in k(app). The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T.GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(-F1)) with K(MzT(-F1)(GAG)) 10 and 20 microm, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T.DS and MzT(-F1).DS interactions were saturable at DS concentrations substantially below K(HCII(DS)), consistent with DS bridging T.DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex.
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20 MeSH Terms
Characterization of a heparin binding site on the heavy chain of factor XI.
Zhao M, Abdel-Razek T, Sun MF, Gailani D
(1998) J Biol Chem 273: 31153-9
MeSH Terms: Alanine, Amino Acids, Diamino, Antithrombin III, Binding Sites, Chromatography, Affinity, Complement C1 Inactivator Proteins, Complement C1 Inhibitor Protein, Enzyme Activation, Factor XI, Factor XIa, Glutamic Acid, Heparin, Mutation, Recombinant Proteins, Sepharose
Show Abstract · Added May 19, 2014
The glycosaminoglycan heparin enhances several reactions involving coagulation factor XI (FXI) including activation of FXI by factor XIIa, thrombin, and autoactivation; and inactivation of activated FXI (FXIa) by serine protease inhibitors. We examined the effect of heparin on inhibition of FXIa by the inhibitors C1-inhibitor (C1-INH) and antithrombin III (ATIII). Second order rate constants for inhibition in the absence of heparin were 1.57 x 10(3) and 0.91 x 10(3) M-1 s-1 for C1-INH and ATIII, respectively. Therapeutic heparin concentrations (0.1-1.0 units/ml) enhanced inhibition by ATIII 20-55-fold compared with 0.1-7.0-fold for C1-INH. For both inhibitors, the effect of heparin over a wide range of concentrations (10(-1) to 10(5) units/ml) produced bell-shaped curves, demonstrating that inhibition occurs by a template mechanism requiring both inhibitor and protease to bind to heparin. This implies that FXI/XIa contains structural elements that interact with heparin. Human FXI contains a sequence of amino acids (R250-I-K-K-S-K) in the apple 3 domain of the heavy chain that binds heparin (Ho, D., Badellino, K., Baglia, F., and Walsh, P. (1998) J. Biol. Chem. 273, 16382-16390). To determine the importance of this sequence to heparin-mediated reactions, recombinant FXI molecules with alanine substitutions for basic amino acids were expressed in 293 fibroblasts, and tested in heparin-dependent assays. Inhibition of FXIa by ATIII in the presence of heparin was decreased 4-fold by alanine substitution at Lys253 (A253), with smaller effects noted for mutants A255 and A252. FXI undergoes autoactivation to FXIa in the presence of heparin. The rate of autoactivation was decreased substantially for A253 with modest decreases for A255 and A252. Substituting all four charged residues in the sequence resulted in a profound decrease in autoactivation, significantly greater than for any single substitution. Relative affinity for heparin was tested by determining the concentration of NaCl required to elute FXIa from heparin-Sepharose. Wild type FXIa eluted from the column at 320 mM NaCl, whereas FXIa with multiple substitutions (A252-254 or A250-255) eluted at 230 mM NaCl. All proteins with single substitutions in charged amino acids eluted at intermediate NaCl concentrations. The data indicate that FXI/XIa must bind to heparin for optimal inhibition by ATIII and for autoactivation. Lys253 is the most important amino acid involved in binding, and Lys255 and Lys252 also have roles in interactions with heparin.
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15 MeSH Terms
Demonstration of exosite I-dependent interactions of thrombin with human factor V and factor Va involving the factor Va heavy chain: analysis by affinity chromatography employing a novel method for active-site-selective immobilization of serine proteinases.
Dharmawardana KR, Bock PE
(1998) Biochemistry 37: 13143-52
MeSH Terms: Binding Sites, Chromatography, Affinity, Enzymes, Immobilized, Factor V, Factor Va, Hirudins, Humans, Hydrolysis, Peptide Fragments, Sepharose, Serine Endopeptidases, Substrate Specificity, Thrombin
Show Abstract · Added January 20, 2015
In an essential step of blood coagulation, factor V is proteolytically processed by thrombin to generate the activated protein cofactor, factor Va, and to release the activation fragments E and C1. For the identification and characterization of sites of thrombin binding to factor V and its activation products, a new method was developed for immobilizing thrombin and other serine proteinases specifically (>/=92%) through their active sites and used in affinity chromatography studies of the interactions. Interactions of factor V with exosite I of thrombin were shown to regulate the factor V activation pathway from the 93% +/- 12% inhibition of the rate of activation correlated with specific binding of hirudin54-65 to this exosite. Chromatography of factor V on active-site-immobilized thrombin showed only a weak interaction, while the factor Va heterodimer bound specifically and with apparently higher affinity, in an interaction that was prevented by hirudin54-65. The heavy chain of subunit-dissociated factor Va bound to immobilized thrombin, while the light-chain subunit and fragment E had no detectable affinity. These results demonstrate a previously undescribed, exosite I-dependent interaction of thrombin with factor Va that occurs through the factor Va heavy chain. They support the further conclusion that similar exosite I-dependent binding of thrombin to the heavy-chain region of factor V contributes to recognition of factor V as a specific thrombin substrate and thereby regulates proteolytic activation of the protein cofactor.
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13 MeSH Terms