Biochemistry, Biophysics, and Structural Biology Commons^™

The Leishmania Donovani Lipophosphoglycan Excludes The Vesicular Proton-Atpase From Phagosomes By Impairing The Recruitment Of Synaptotagmin V, Adrien F. Vinet, Mitsunori Fukuda, Salvatore J. Turco, Albert Descoteaux

Molecular and Cellular Biochemistry Faculty Publications

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania …

Suppression Of Rft1 Expression Does Not Impair The Transbilayer Movement Of Man5glcnac2-P-P-Dolichol In Sealed Microsomes From Yeast., Jeffrey S. Rush, Ningguo Gao, Mark A Lehrman, Sergey Matveev, Charles J Waechter

Molecular and Cellular Biochemistry Faculty Publications

To further evaluate the role of Rft1 in the transbilayer movement of Man(5)GlcNAc(2)-P-P-dolichol (M5-DLO), a series of experiments was conducted with intact cells and sealed microsomal vesicles. First, an unexpectedly large accumulation (37-fold) of M5-DLO was observed in Rft1-depleted cells (YG1137) relative to Glc(3)Man(9)GlcNAc(2)-P-P-Dol in wild type (SS328) cells when glycolipid levels were compared by fluorophore-assisted carbohydrate electrophoresis analysis. When sealed microsomes from wild type cells and cells depleted of Rft1 were incubated with GDP-[(3)H]mannose or UDP-[(3)H]GlcNAc in the presence of unlabeled GDP-Man, no difference was observed in the rate of synthesis of [(3)H]Man(9)GlcNAc(2)-P-P-dolichol or Man(9)[(3)H]GlcNAc(2)-P-P-dolichol, respectively. In addition, no …

Conservation Of The Glucan Phosphatase Laforin Is Linked To Rates Of Molecular Evolution And The Glucan Metabolism Of The Organism, Matthew S. Gentry, Rachel M. Pace

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Lafora disease (LD) is a fatal autosomal recessive neurodegenerative disease. A hallmark of LD is cytoplasmic accumulation of insoluble glucans, called Lafora bodies (LBs). Mutations in the gene encoding the phosphatase laforin account for approximately 50% of LD cases, and this gene is conserved in all vertebrates. We recently demonstrated that laforin is the founding member of a unique class of phosphatases that dephosphorylate glucans.

RESULTS: Herein, we identify laforin orthologs in a protist and two invertebrate genomes, and report that laforin is absent in the vast majority of protozoan genomes and it is lacking in all other invertebrate …

Human Cerebral Neuropathology Of Type 2 Diabetes Mellitus, Peter T. Nelson, Charles D. Smith, Erin L. Abner, Frederick A. Schmitt, Stephen W. Scheff, Gregory J. Davis, Jeffrey N. Keller, Gregory A. Jicha, Daron Davis, Wang-Xia Wang, Adria Hartman, Douglas G. Katz, William R. Markesbery

Pathology and Laboratory Medicine Faculty Publications

The cerebral neuropathology of Type 2 diabetes (CNDM2) has not been positively defined. This review includes a description of CNDM2 research from before the ‘Pubmed Era’. Recent neuroimaging studies have focused on cerebrovascular and white matter pathology. These and prior studies about cerebrovascular histopathology in diabetes are reviewed. Evidence is also described for and against the link between CNDM2 and Alzheimer's disease pathogenesis. To study this matter directly, we evaluated data from University of Kentucky Alzheimer's Disease Center (UK ADC) patients recruited while non-demented and followed longitudinally. Of patients who had come to autopsy (N = 234), 139 met …

Polyglutamine Disruption Of The Huntingtin Exon 1 N Terminus Triggers A Complex Aggregation Mechanism, Ashwani K. Thakur, Murali Jayaraman, Rakesh Mishra, Monika Thakur, Veronique M. Chellgren, In-Ja L Byeon, Dalaver H. Anjum, Ravindra Kodali, Trevor P. Creamer, James F. Conway, Angela M. Gronenborn, Ronald Wetzel

Molecular and Cellular Biochemistry Faculty Publications

Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17-amino-acid flanking sequence (HTT^NT) N-terminal to the polyQ in the toxic huntingtin exon 1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation, the HTT^NT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in HTT^NT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with HTT^NT cores and exposed polyQ. In a second …

Substrate And Regulation Of Mitochondrial Μ-Calpain, Aashish Joshi

University of Kentucky Doctoral Dissertations

μ -Calpain is localized to the mitochondrial intermembrane space. Apoptosisinducing factor (AIF), which executes caspase-independent cell death, is also localized to the mitochondrial intermembrane space. Following processing at the N-terminus, AIF becomes truncated (tAIF) and is released from mitochondria. The protease responsible for AIF processing has not been established. The same submitochondrial localization of mitochondrial μ-calpain and AIF gives support to the hypothesis that mitochondrial μ-calpain may be responsible for processing AIF. Atractyloside-induced tAIF release in rat liver mitochondria was inhibited by cysteine protease inhibitor MDL28170, but not by calpain inhibitors PD150606 or calpastatin. Moreover, μ-calpain immunoreactivity was difficult to …

Capturing Hammerhead Ribozyme Structures In Action By Modulating General Base Catalysis, Young-In Chi, Monika Martick, Monica Lares, Rosalind Kim, William G. Scott, Sung-Hou Kim

Center for Structural Biology Faculty Publications

We have obtained precatalytic (enzyme-substrate complex) and postcatalytic (enzyme-product complex) crystal structures of an active full-length hammerhead RNA that cleaves in the crystal. Using the natural satellite tobacco ringspot virus hammerhead RNA sequence, the self-cleavage reaction was modulated by substituting the general base of the ribozyme, G12, with A12, a purine variant with a much lower pKa that does not significantly perturb the ribozyme's atomic structure. The active, but slowly cleaving, ribozyme thus permitted isolation of enzyme-substrate and enzyme-product complexes without modifying the nucleophile or leaving group of the cleavage reaction, nor any other aspect of the substrate. The predissociation …

Sumoylation Regulates Lamin A Function And Is Lost In Lamin A Mutants Associated With Familial Cardiomyopathies, Yu-Qian Zhang, Kevin D. Sarge

Molecular and Cellular Biochemistry Faculty Publications

Lamin A mutations cause many diseases, including cardiomyopathies and Progeria Syndrome. The covalent attachment of small ubiquitin-like modifier (SUMO) polypeptides regulates the function of many proteins. Until now, no examples of human disease-causing mutations that occur within a sumoylation consensus sequence and alter sumoylation were known. We show that lamin A is sumoylated at lysine 201 and that two lamin A mutants associated with familial dilated cardiomyopathy, E203G and E203K, exhibit decreased sumoylation. E203 occupies the conserved +2 position in the sumoylation consensus Psi KXE. Lamin A mutants E203G, E203K, and K201R all exhibit a similar aberrant subcellular localization and …

The Influence Of Citrate, Maltolate And Fluoride On The Gastrointestinal Absorption Of Aluminum At A Drinking Water-Relevant Concentration: A 26Al And 14C Study, Yuzhao Zhou, Wesley R. Harris, Robert A. Yokel

Pharmaceutical Sciences Faculty Publications

The objectives were to test the null hypotheses that (1) citrate, maltolate, and fluoride do not significantly influence oral Al bioavailability, C_max or T_max at an Al dose relevant to drinking water exposure; and (2) Al citrate and maltolate are absorbed intact from the gastrointestinal tract. Male Fisher rats were given 1 ml of solution intra-gastrically containing 1 nCi ²⁶Al (65 nmol total Al) as the Al³⁺ ion, or as complexes with ¹⁴C-citrate, ¹⁴C-maltolate or fluoride, during concurrent ²⁷Al iv infusion. Blood was repeatedly collected for serum ²⁶Al, total Al and ^{14 …}

Recycling Of Dolichyl Monophosphate To The Cytoplasmic Leaflet Of The Endoplasmic Reticulum After The Cleavage Of Dolichyl Pyrophosphate On The Lumenal Monolayer., Jeffrey S Rush, Ningguo Gao, Mark A Lehrman, Charles J Waechter

Molecular and Cellular Biochemistry Faculty Publications

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in …

Pre-Mrna Secondary Structures Influence Exon Recognition, Michael Hiller, Zhaiyi Zhang, Rolf Backofen, Stefan Stamm

Molecular and Cellular Biochemistry Faculty Publications

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of …

Induction Of Insulin Secretion In Engineered Liver Cells By Nitric Oxide, Latha Muniappan, Sabire Özcan

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Type 1 Diabetes Mellitus results from an autoimmune destruction of the pancreatic beta cells, which produce insulin. The lack of insulin leads to chronic hyperglycemia and secondary complications, such as cardiovascular disease. The currently approved clinical treatments for diabetes mellitus often fail to achieve sustained and optimal glycemic control. Therefore, there is a great interest in the development of surrogate beta cells as a treatment for type 1 diabetes. Normally, pancreatic beta cells produce and secrete insulin only in response to increased blood glucose levels. However in many cases, insulin secretion from non-beta cells engineered to produce insulin occurs …

A Simple Array Platform For Microrna Analysis And Its Application In Mouse Tissues, Xiaoqing Tang, Jozsef Gal, Xun Zhuang, Wang-Xia Wang, Haining Zhu, Guiliang Tang

Plant and Soil Sciences Faculty Publications

MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression at the post-transcriptional level and play a critical role in many important biological processes. Most miRNAs are conserved between humans and mice, which makes it possible to analyze their expressions with a set of selected array probes. Here, we report a simple array platform that can detect 553 nonredundant miRNAs encompassing the entire set of miRNAs for humans and mice. The platform features carefully selected and designed probes with optimized hybridization parameters. Potential cross-reaction between mature miRNAs and their precursors was investigated. The array platform was …

Chronic Administration Of R-Flurbiprofen Attenuates Learning Impairments In Transgenic Amyloid Precursor Protein Mice, Thomas Kukar, Sonya Prescott, Jason L. Eriksen, Vallie Holloway, M. Paul Murphy, Edward H. Koo, Todd E. Golde, Michelle M. Nicolle

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a reduced incidence of Alzheimer's disease (AD). We and others have shown that certain NSAIDs reduce secretion of Abeta42 in cell culture and animal models, and that the effect of NSAIDs on Abeta42 is independent of the inhibition of cyclooxygenase by these compounds. Since Abeta42 is hypothesized to be the initiating pathologic molecule in AD, the ability of these compounds to lower Abeta42 selectively may be associated with their protective effect. We have previously identified R-flurbiprofen (tarenflurbil) as a selective Abeta42 lowering agent with greatly reduced cyclooxygenase activity that …

Evidence That Talin Alternative Splice Variants From Ciona Intestinalis Have Different Roles In Cell Adhesion, Richard H. Singiser, Richard O. Mccann

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: Talins are large, modular cytoskeletal proteins found in animals and amoebozoans such as Dictyostelium discoideum. Since the identification of a second talin gene in vertebrates, it has become increasingly clear that vertebrate Talin1 and Talin2 have non-redundant roles as essential links between integrins and the actin cytoskeleton in distinct plasma membrane-associated adhesion complexes. The conserved C-terminal I/LWEQ module is important for talin function. This structural element mediates the interaction of talins with F-actin. The I/LWEQ module also targets mammalian Talin1 to focal adhesion complexes, which are dynamic multicomponent assemblies required for cell adhesion and cell motility. Although Talin1 is …

The Role Of Mapks In B Cell Receptor-Induced Down-Regulation Of Egr-1 In Immature B Lymphoma Cells, Jiyuan Ke, Murali Gururajan, Anupam Kumar, Alan Simmons, Lilia Turcios, Ralph Lakshman Chelvarajan, David M. Cohen, David L. Wiest, John G. Monroe, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Cross-linking of the B cell receptor (BCR) on the immature B lymphoma cell line BKS-2 induces growth inhibition and apoptosis accompanied by rapid down-regulation of the immediate-early gene egr-1. In these lymphoma cells, egr-1 is expressed constitutively and has a prosurvival role, as Egr-1-specific antisense oligonucleotides or expression of a dominant-negative inhibitor of Egr-1 also prevented the growth of BKS-2 cells. Moreover, enhancement of Egr-1 protein with phorbol 12-myristate 13-acetate or an egr-1 expression vector rescued BKS-2 cells from BCR signal-induced growth inhibition. Nuclear run-on and mRNA stability assays indicated that BCR-derived signals act at the transcriptional level to …

Tsc2 Modulates Actin Cytoskeleton And Focal Adhesion Through Tsc1-Binding Domain And The Rac1 Gtpase, Elena Goncharova, Dmitry Goncharov, Daniel J. Noonan, Vera P Krymskaya

Molecular and Cellular Biochemistry Faculty Publications

Tuberous sclerosis complex (TSC) 1 and TSC2 are thought to be involved in protein translational regulation and cell growth, and loss of their function is a cause of TSC and lymphangioleiomyomatosis (LAM). However, TSC1 also activates Rho and regulates cell adhesion. We found that TSC2 modulates actin dynamics and cell adhesion and the TSC1-binding domain (TSC2-HBD) is essential for this function of TSC2. Expression of TSC2 or TSC2-HBD in TSC2-/- cells promoted Rac1 activation, inhibition of Rho, stress fiber disassembly, and focal adhesion remodeling. The down-regulation of TSC1 with TSC1 siRNA in TSC2-/- cells activated Rac1 and induced loss of …

A Subset Of Liver Nk T Cells Is Activated During Leishmania Donovani Infection By Cd1d-Bound Lipophosphoglycan, Joseph L. Amprey, Jin S. Im, Salvatore J. Turco, Henry W. Murray, Petr A. Illarionov, Gurdyal S. Besra, Steven A. Porcelli, Gerald F. Späth

Molecular and Cellular Biochemistry Faculty Publications

Natural killer (NK) T cells are activated by synthetic or self-glycolipids and implicated in innate host resistance to a range of viral, bacterial, and protozoan pathogens. Despite the immunogenicity of microbial lipoglycans and their promiscuous binding to CD1d, no pathogen-derived glycolipid antigen presented by this pathway has been identified to date. In the current work, we show increased susceptibility of NK T cell–deficient CD1d^−/− mice to Leishmania donovani infection and Leishmania-induced CD1d-dependent activation of NK T cells in wild-type animals. The elicited response was Th1 polarized, occurred as early as 2 h after infection, and was independent from …

A Neuronal-Specific Differentiation Protein That Directly Modulates Retinoid Receptor Transcriptional Activation, Kenneth W. Henry Ii, Michael L. Spencer, Maria Theodosiou, Dingyuan Lou, Daniel J. Noonan

Molecular and Cellular Biochemistry Faculty Publications

BACKGROUND: The specificity of a nuclear receptor's ability to modulate gene expression resides in its ability to bind a specific lipophilic ligand, associate with specific dimerization partners and bind specific DNA sequences in the promoter regions of genes. This sequence of events appears to be the basis for targeting an additional regulatory complex composed of a variety of protein and RNA components that deliver signals for facilitation or inhibition of the RNA polymerase complex. Characterization of the tissue and cell-specific components of these coregulatory complexes appear to be integral to our understanding of nuclear receptor regulation of transcription.

RESULTS: A …

Novel Function Of Phosphoinositide 3-Kinase In T Cell Ca2+ Signaling, Ao-Lin Hsu, Tsui-Ting Ching, Goutam Sen, Da-Sheng Wan, Subbarao Bondada, Kalwant S. Authi, Ching-Shih Chen

Pharmaceutical Sciences Faculty Publications

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca²⁺ signaling via a phosphatidylinositol 3,4,5-trisphosphate PI(3,4,5)P₃-sensitive Ca²⁺entry pathway. First, exogenous PI(3,4,5)P₃ at concentrations close to its physiological levels induces Ca²⁺ influx in T cells, whereas PI(3,4)P₂, PI(4,5)P₂, and PI(3)P have no effect on [Ca²⁺]_i. This Ca²⁺ entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P₃ stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the …

The Snare Machinery Is Involved In Apical Plasma Membrane Trafficking In Mdck Cells, Seng Hui Low, Steven J. Chapin, Christian Wimmer, Sidney W. Whiteheart, László G. Kömüves, Keith E. Mostov, Thomas Weimbs

Molecular and Cellular Biochemistry Faculty Publications

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in …

Role Of Egr-1 Gene Expression In B Cell Receptor-Induced Apoptosis In An Immature B Cell Lymphoma, Subramanian Muthukkumar, Seong-Su Han, Sumathi Muthukkumar, Vivek M. Rangnekar, Subbarao Bondada

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Ligation of B cell receptor (BCR) on BKS-2, an immature B cell lymphoma by anti-IgM antibodies (Ab) caused apoptosis. Here we report that signaling through B cell receptor in wild type BKS-2 cells down-regulated the expression of Egr-1, a zinc finger-containing transcription factor. A reduction in the level ofEgr-1 mRNA could be demonstrated as early as 30 min after the ligation of BCR on BKS-2 cells. Immunocytochemical and Western blot analysis revealed that the expression of EGR-1 protein was also inhibited by anti-IgM treatment. Antisense oligonucleotides to Egr-1 caused growth inhibition and apoptosis in BKS-2 cells, suggesting that …

Cystic Fibrosis Transmembrane Conductance Regulator Mutations That Disrupt Nucleotide Binding, James Logan, David Hiestand, Paru Daram, Zhen Huang, Donald D. Muccio, John Hartman, Boyd Haley, William J. Cook, Eric J. Sorscher

Molecular and Cellular Biochemistry Faculty Publications

Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding …

Factors Regulating The Growth Of Algae In Continuous Culture In Diluted Secondary Sewage Treatment Plant Effluent And Subsequent Biodegradability, Edward G. Foree, Caroline P. Wade

KWRRI Research Reports

Heterogeneous algal cultures were grown in laboratory continuous culture in continuous flow, completely mixed chemostats in secondary sewage treatment plant effluent diluted to give an ammonia nitrogen concentration of 10 mg/1. Variables were lighting, pH, carbon dioxide availability, and hydraulic residence time.

Optimum growth occurred under pH 7.0, excess CO₂, and continuous lighting conditions. The availability of artificially supplied excess CO₂ greatly increased the mass (standing crop) at steady-state over that produced under otherwise identical conditions for all residence times studied. For the case of excess CO₂ availability, the nitrogen concentration in the algal cells regulated …