Higuchi Biosciences Center - University of Kansas

Fall 2006 Science Talks
November 30, 2006

Abstracts 1-15 | Abstracts 16-30 | Abstracts 31-45 | Abstracts 46-54

46. Identification and Structure –Activity Relationships of Arodyn Analogs Exhibiting Inverse Agonist Activity at Kappa Opioid Receptors
Wei-Jie Fang,1 Marco A. Bennett,2 Thomas F. Murray,3 Jane V. Aldrich1*
1Department of Medicinal Chemistry, University of Kansas, Lawrence, KS
2Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD
3Department of Physiology and Pharmacology, University of Georgia, Athens, GA

We are interested in the design and synthesis of peptide antagonists for kappa opioid receptors as pharmacological tools to study ligand-receptor interactions and their physiological consequences. Arodyn (Ac[Phe1,2,3,Arg4,D-Ala8]dynorphin A(1-11)-NH2) is an acetylated dynorphin A analog that is a potent and selective kappa opioid receptor antagonist [1]. Unexpectedly, some analogs of arodyn exhibited inverse agonist activity at kappa opioid receptors. Structural requirements for the inverse agonist activity were explored. Preliminary pharmacological results will be presented for arodyn analogs containing substitutions in positions 1 and 3. A bulky aromatic ring in position 3 (such as Trp or Nal(2’)) was found to be important for this activity, although the residue at position 1 also influences the type of activity observed.
[1] Bennett, M.A., Murray, T.F., Aldrich, J.V. J. Med. Chem. 2002, 45, 5617-5619.

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47. Investigation of the Paclitaxel-Binding Site in Tubulin Using Mutants of Saccharomyces Cerevisiae
R. D. Winefield, T. B. Foland, R. H. Himes
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Tubulin from the budding yeast Saccharomyces cerevisiae does not bind paclitaxel. However, previously we were able to create paclitaxel binding to S. cerevisiae tubulin by mutating five residues in yeast ß-tubulin (purported to play important roles in the putative taxoid-binding site in mammalian ß-tubulin) to those that occur in brain ß-tubulin. We also created a S. cerevisiae strain that is sensitive to paclitaxel by introducing the mutated b-tubulin gene into a yeast strain that has diminished multi-drug transporter activity. To determine the relative importance of the five residues we have now created new strains that contain different combinations of the five mutations; A19K, T23V, G26D, N227H and Y270F, and tested their sensitivities to paclitaxel. Strains containing only the N227H, Y270F, or the three N-terminus (NT) mutations showed greatly reduced sensitivity to paclitaxel. Similarly, the strains containing the N227H+Y270F or NT+N227H mutations were resistant to paclitaxel. The results indicate that one or more of the mutations in the NT region and the Y270F mutation are required to produce maximum sensitivity to the taxanes. On the other hand, the NT+Y270F strain (without the N227H mutation) was as sensitive to paclitaxel as was the strain with all five mutations. This is a surprising result since His227 has been proposed to play a significant role in paclitaxel binding. In contrast, however, the N227H mutation was necessary to produce maximum sensitivity to docetaxel. Tubulin assembly studies confirmed this result. Docetaxel was twice as effective as paclitaxel in inhibiting the growth of the strain with all five mutations, but about 60% as effective as paclitaxel when used against the strain lacking the N227H mutation. The results indicate that His227 plays a greater role in tubulin’s affinity for docetaxel than for paclitaxel.
Supported by NIH grant CA105305

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48. Cloning and Characterization of E(BR)165, a Mutation that Dominantly Enhances br1 in Drosophila
Xiaochen Wang, Elspeth Pearce, Robert Ward
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

The elongation and eversion of leg imaginal discs during metamorphosis in Drosophila is an ideal system for studying hormone-regulated morphogenesis. Broad (br) is a key ecdysone-inducible early gene for leg morphogenesis. Previously we conducted a screen for dominant modifiers of br1, and found that signaling through the Rho1 small GTPase is necessary to direct the cell shape changes that drive morphogenesis of the adult legs. In order to understand how ecdysone might be regulating Rho signaling, we have begun to clone Enhancer of broad (E(br)) mutations that also genetically interact with Rho1. One such mutation, E(br)165, is a completely penetrant embryonic lethal that displays defects in dorsal closure and cuticle deposition. Meiotic mapping placed the mutation in 26D and complementation tests indicated that it is a new allele of Sec61a, which encodes the main subunit of the translocon complex for co-translational import of proteins into the ER. Sequence analysis indicated that the E(br)165 mutation truncates the protein in the amino-terminal half of the protein, suggesting that this is a strong loss of function mutation in Sec61a. We are currently attempting to rescue the mutation by ectopic expression of Sec61a. We are interested in the role of Sec61a during embryogenesis and imaginal disc morphogenesis. Preliminary expression studies indicate that Sec61a is strongly expressed in leg imaginal discs during metamorphosis, but that its expression is not regulated by broad. We therefore suspect that a secreted or transmembrane protein regulated by the ecdysone pathway is playing a critical role in driving imaginal disc morphogenesis.

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49. Genes, Gene Ontologies and Biological Pathways Altered in the Hippocampus of Glud1 Hyper-Glutamate-Activity Transgenic Mice
Xinkun Wang, Xiaodong Bao, Ranu Pal, Elias K Michaelis
Department of Pharmacology and Toxicology, Higuchi Biosciences Center, University of Kansas, Lawrence, KS

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system (CNS). Hyperactivity of this neurotransmitter plays an important role in neurodegeneration caused by aging and neurodegenerative diseases including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). To elucidate the molecular mechanisms of neurodegeneration to glutamate hyperactivity, we have developed a transgenic mouse model featuring intrinsically chronic over-release of glutamate, which mimics in vivo physiological states occurring in human neurodegenerative conditions. Glutamate hyperactivity in the transgenic mice is due to neuron-specific over-expression of glutamate dehydrogenase (Glud1), an enzyme for glutamate biosynthesis. Phenotypically, these mice are characterized by shortened life span and accelerated neurodegeneration during aging. In order to identify genes whose activity is altered by the Glud1 over-expression, Affymetrix GeneChip-based gene expression profiling was employed. Through stringent microarray data analysis, 1018 probe sets were identified to be differentially expressed in the hippocampus of transgenic mice compared to age-matched controls, with 707 up-regulated and 311 down-regulated. To determine functional changes in the transgenic mice, GO (Gene Ontology) and biological pathway analyses were conducted. In the GO analysis, calcium ion transport, synapse activity, protein ubiquitination and cytoskeleton were among the most significant functional alterations. The biological pathway analysis, in addition to supporting some of the GO analysis results, also identified several pathways to be significantly altered, including locomotor behavior, protein kinase activity, cell migration, cell growth and development, cell adhesion and signal transduction. In conclusion, the genomics approach being undertaken in this study uncovers genes, biological processes and molecular functions altered in the transgenic animal model of neurodegeneration caused by glutamate hyperactivity. [Supported by NIH AG 12993, Kansas MRDDRC (NICHD HD-02528), KTEC, and Miller-Hedwig-Wilbur Fund]

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50. Comparative Microarray Study of Selective Neuronal Vulnerability in the Brain
Xinkun Wang1, S. Garrett2, Asma Zaidi1, Mary L. Michaelis1, Elias K. Michaelis1
1 Higuchi Biosciences Center, University of Kansas, Lawrence, KS
2 Information & Telecommunication Technology Center, University of Kansas, Lawrence, KS

Different neuronal populations exhibit enhanced vulnerability to various types of brain insults, a phenomenon that may be associated with the death of specific neuronal cells in neurodegenerative diseases such as Alzheimer’s disease. For example, the CA1 neurons of the hippocampus are much more likely to die following a stroke or oxidative stress than are the CA3 hippocampal cells. We have also found that cerebellar granule cells are much more sensitive to oxidative stress than are cortical neurons. Development of strategies to protect such neurons requires new insights into the basis of the vulnerability. The goal of our studies was to make use of comparative gene expression profiling to begin identifying genes differentially expressed in vulnerable versus non-vulnerable cells. We used paraquat-induced superoxide generation in brain cells to produce oxidative stress in hippocampal, cortical, and cerebellum slice cultures. Cerebellar granule cells and hippocampal CA1 neurons suffered extensive cell death, while neurons in the other regions were relatively resistant. After establishing this selective vulnerability pattern in ex vivo experiments, we dissected both vulnerable (CA1 pyramidal and cerebellum granule) and resistant (CA3 and cerebral cortex pyramidal) neurons with laser capture microdissection (LCM) from frozen sections of rat brains. The rats were not treated with paraquat. Affymetrix Rat Expression 230A GeneChips were used for comparative microarray analyses. We have identified 766 genes that showed significantly different expression patterns between vulnerable and resistant regions. The functional categories of these genes included protein kinases, oxidoreductases, ion and neurotransmitter transporters, receptors and transcription factors. In addition, we identified one EST that was expressed only in the two vulnerable regions, and 7 genes and ESTs expressed only in the two resistant regions. Possible roles of these identified genes in the development of selective vulnerability are currently being explored.

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51. Function of Nuclear Adenomatous Polyposis Coli in Cell Differentiation
Yang Wang, Kristi Neufeld
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Adenomatous Polyposis Coli (APC) is a tumor suppressor, whose mutations are responsible for over 85% of all colorectal cancers. Other than down-regulating b-catenin in Wnt pathway, APC protein contains Nuclear Localization Signals and Nuclear Export Signals that enable APC to shuttle in and out of the nucleus (Neufeld et al., 2000). Loss of APC function in mouse model immediately disturbs cell differentiation in colon crypts (Sansom et al., 2004). In order to determine if nuclear APC has a role in cell differentiation, we study both embryonic stem (ES) cells and mouse model with inactivated NLS. We induced wildtype mouse ES cells to differentiate into different adult cell types, including mesodermal smooth muscle cells, neuralectodermal cells, organ-like ES gut and cardiomyocytes. We will investigate if ES cells lacking nuclear APC display defects in differentiation. We will also observe if intestines of mice lacking nuclear APC are deficient in their populations of differentiated cells. This knowledge will help elucidating the role of APC in the pathogenesis of colorectal cancer.

References Neufeld, K.L., Zhang, F., Cullen, B.R. & White, R.L. (2000). APC-mediated downregulation of beta-catenin activity involves nuclear sequestration and nuclear export. EMBO Rep, 1, 519-23.
Sansom, O.J., Reed, K.R., Hayes, A.J., Ireland, H., Brinkmann, H., Newton, I.P., Batlle, E., Simon-Assmann, P., Clevers, H., Nathke, I.S., Clarke, A.R. & Winton, D.J. (2004). Loss of Apc in vivo immediately perturbs Wnt signaling, differentiation, and migration. Genes Dev, 18, 1385-90.

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52. Solution Structure of Monomeric PrgI, the Type III Secretion Needle Protein of Salmonella
Yu Wang, Chet Egan, Andrew Quellette, Wendy Picking, William Picking, Roberto De Guzman
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

The type III secretion system is used by Gram-negative bacteria to inject virulence factors into their host cells and cause human diseases. More than 20 different proteins coordinately assemble into a macromolecular protein complex and form an external tube-like structure called the needle apparatus. The needle apparatus serves as a conduit for the passage of bacterial effector proteins into the target cells. The needle apparatus is formed by the polymerization of multiple copies of a small, acidic protein that shows sequence conservation among pathogens. We have previously determined the high resolution structure of the needle monomer called BsaL from Burkholderia (formerly known as Pseudomonas) by nuclear magnetic resonance (NMR) spectroscopy. Here, we report the NMR structure of the PrgI needle protein of Salmonella, a human pathogen associated with food poisoning and typhoid fever. These NMR structures reveal that the needle proteins are inherently flexible molecules, probably a feature that allows them to pass through a narrow channel as well. Like the structure of BsaL, the central part of PrgI assumes a helix-turn-helix core domain with a four-residue linker. There are however, subtle differences between the two structures, that may account for the widely different observed thermal stabilities of these proteins. For example, the N-terminal region of PrgI has better convergence compared to BsaL, with a slight bend caused by the presence of a glycine residue. Our results suggest the needle proteins are inherently flexible molecules which probably allow their passage through a narrow needle channel as well.

Key words: type III secretion; PrgI; BsaL; needle protein; NMR

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53. RNAi-Induced Silencing of the Plasma Membrane Ca2+-ATPase 2 Increases the Vulnerability of Neurons to Stimuli that Alter Intracellular Calcium
Denzyl Fernandes, Asma Zaidi, Jennifer L. Bean, Dongwei Hui, Mary L. Michaelis
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS

Intraneuronal calcium ([Ca2+]i) regulation is altered in aging brain, possibly due to changes in critical Ca2+ transporters. We previously reported that the levels of the plasma membrane Ca2+ - ATPase (PMCA) and the Vmax for enzyme activity are significantly reduced in synaptic membranes in aging rat brain. The goal of these studies was to use RNAi techniques to suppress expression of a major neuronal isoform, PMCA2, in neurons in culture to determine the potential functional consequences of a loss of PMCA activity on Ca2+ homeostasis and the sensitivity of neurons to stressful stimuli. Embryonic rat brain neurons and SH-SY5Y neuroblastoma cells were transfected with in vitro - transcribed siRNA or a shRNA-expressing vector, respectively. Immunoblots of the particulate fraction isolated from both cell types showed ~80% suppression of PMCA2 expression within 48 h. Fluorescence ratio imaging of free [Ca2+]i using Fura-2 revealed that primary neurons with reduced PMCA2 had higher basal [Ca2+]i, slower recovery from KCl-induced Ca2+ transients, and incomplete return to pre-stimulation Ca2+ levels. Primary neurons and SH-SY5Y cells with PMCA2 suppression both exhibited significantly greater vulnerability to stimuli that increase [Ca2+]i such as thapsigargin, NMDA, ionomycin, and β amyloid peptide. Our results indicate that a loss of PMCA such as occurs in aging brain likely leads to subtle disruptions in normal Ca2+ signaling and enhanced susceptibility to stresses that can alter the regulation of Ca2+ homeostasis. (Supported by AG12993 and COBRE RR 017708).

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54. Age-dependent Decrease in Synaptic Membrane Ca2+-ATPase Occurs Selectively in Cholesterol Rich Microdomains
A. Zaidi, L. Jiang, J.L. Bean, M.L. Michaelis
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS

Background: Disruption of Ca2+ homeostasis is a major contributing factor in the decline in brain function in aged individuals and their increased risk for developing neurodegenerative diseases. The Ca2+ transporter called the plasma membrane Ca2+-ATPase (PMCA) plays an important role in regulating intracellular Ca2+ levels. We have previously shown a 50% decline in PMCA activity in synaptic membranes (SM) isolated from aged brain although its protein levels were diminished only by 20%. The current study was conducted to determine the mechanisms underlying the discrepancy between the observed decreases in activity vs protein levels in aged brain.
Methods: SMs isolated from young (5 mos) and aged (34 mos) rat brain were solubilized with Triton X-100 followed by sucrose density gradient centrifugation. A total of 10 fractions containing the detergent-resistant low buoyant density fractions (rafts) and detergent-soluble phospholipid-enriched fractions (non-rafts) were collected. PMCA levels were determined by immunoblotting and activity monitored by Ca2+-activated ATP hydrolysis.
Results: PMCA in SMs appeared to have a dual distribution, ~40% was localized in detergent-resistant low density fractions characteristic of lipid rafts while ~60% was present in the high density non-raft domains. PMCA specific activity was 3-fold higher in rafts compared to the non-raft domains. Analysis of PMCA protein levels in aged brain showed a 50% decrease in raft-associated pool but almost no change in non-raft pool. Levels of raft lipid markers such as cholesterol and GM1 ganglioside and protein markers such as flotillin and Thy-1 did not alter with age, indicating that the decrease in PMCA was selective. Ongoing research is outlined to determine the mechanisms underlying the targeting of PMCA to rafts and its selective decrease with aging.

Conclusions: Higher activity of raft-PMCA suggests that the lipid/protein environment of these microdomains may have a stimulatory effect on PMCA function. Selective loss of raft-PMCA may contribute to the disproportional decrease in PMCA activity observed in aged SMs and to the disruption of Ca2+ regulation observed in brain aging.

[Grant support: P20RR 017708, AG12993, and Inez Jay Funds]

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Higuchi Biosciences Center
University of Kansas
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