Life Sciences Commons^™

Infection Of Peripancreatic Lymph Nodes But Not Islets Precedes Kilham Rat Virus-Induced Diabetes In Bb/Wor Rats, David Brown, Raymond Welsh, Arthur Like

David C. Brown

A parvovirus serologically identified as Kilham rat virus (KRV) reproducibly induces acute type I diabetes in diabetes-resistant BB/Wor rats. The tissue tropism of KRV was investigated by in situ hybridization with a digoxigenin-labelled plasmid DNA probe containing approximately 1.6 kb of the genome of the UMass isolate of KRV. Partial sequencing of the KRV probe revealed high levels of homology to the sequence of minute virus of mice (89%) and to the sequence of H1 (99%), a parvovirus capable of infecting rats and humans. Of the 444 bases sequenced, 440 were shared by H1. KRV mRNA and DNA were readily …

A Conserved Three-Nucleotide Core Motif Defines Musashi Rna Binding Specificity, Nancy Zearfoss, Laura Deveau, Carina Clingman, Eric Schmidt, Emily Johnson, Francesca Massi, Sean Ryder

Sean P. Ryder

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside …

The Fly Camta Transcription Factor Potentiates Deactivation Of Rhodopsin, A G Protein-Coupled Light Receptor, Junhai Han, Ping Gong, Keith Reddig, Mirna Mitra, Peiyi Guo, Hong-Sheng Li

Peiyi Guo

Control of membrane-receptor activity is required not only for the accuracy of sensory responses, but also to protect cells from excitotoxicity. Here we report the isolation of two noncomplementary fly mutants with slow termination of photoresponses. Genetic and electrophysiological analyses of the mutants revealed a defect in the deactivation of rhodopsin, a visual G protein-coupled receptor (GPCR). The mutant gene was identified as the calmodulin-binding transcription activator (dCAMTA). The known rhodopsin regulator Arr2 does not mediate this visual function of dCAMTA. A genome-wide screen identified five dCAMTA target genes. Of these, overexpression of the F box gene dFbxl4 rescued the …

Quaking Regulates Hnrnpa1 Expression Through Its 3' Utr In Oligodendrocyte Precursor Cells, Nancy Zearfoss, Carina Clingman, Brian Farley, Lisa Mccoig, Sean Ryder

Sean P. Ryder

In mice, Quaking (Qk) is required for myelin formation; in humans, it has been associated with psychiatric disease. QK regulates the stability, subcellular localization, and alternative splicing of several myelin-related transcripts, yet little is known about how QK governs these activities. Here, we show that QK enhances Hnrnpa1 mRNA stability by binding a conserved 3' UTR sequence with high affinity and specificity. A single nucleotide mutation in the binding site eliminates QK-dependent regulation, as does reduction of QK by RNAi. Analysis of exon expression across the transcriptome reveals that QK and hnRNP A1 regulate an overlapping subset of transcripts. Thus, …

Rna Target Specificity Of The Embryonic Cell Fate Determinant Pos-1, Brian Farley, John Pagano, Sean Ryder

Sean P. Ryder

Specification of Caenorhabditis elegans body axes and cell fates occurs prior to the activation of zygotic transcription. Several CCCH-type tandem zinc finger (TZF) proteins coordinate local activation of quiescent maternal mRNAs after fertilization, leading to asymmetric expression of factors required for patterning. The primary determinant of posterior fate is the TZF protein POS-1. Mutants of pos-1 are maternal effect lethal with a terminal phenotype that includes excess pharyngeal tissue and no endoderm or germline. Here, we delineate the consensus POS-1 recognition element (PRE) required for specific recognition of its target mRNAs. The PRE is necessary but not sufficient to pattern …

Argonaute Protein Identity And Pairing Geometry Determine Cooperativity In Mammalian Rna Silencing, Jennifer Broderick, William Salomon, Sean Ryder, Neil Aronin, Phillip Zamore

Sean P. Ryder

Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs …

A Lipopolysaccharide-Induced Dna-Binding Protein For A Class Ii Gene In B Cells Is Distinct From Nf-Kappa B, Ellen M. Gravallese, Mark R. Boothby, Cynthia M. Smas, Laurie H. Glimcher

Ellen M. Gravallese

Class II (Ia) major histocompatibility complex molecules are cell surface proteins normally expressed by a limited subset of cells of the immune system. These molecules regulate the activation of T cells and are required for the presentation of antigens and the initiation of immune responses. The expression of Ia in B cells is determined by both the developmental stage of the B cell and by certain external stimuli. It has been demonstrated previously that treatment of B cells with lipopolysaccharide (LPS) results in increased surface expression of Ia protein. However, we have confirmed that LPS treatment results in a significant …

Subnuclear Distribution Of The Vitamin D Receptor, Joseph Bidwell, Andre Van Wijnen, Edward Fey, Harold Merriman, Sheldon Penman, Janet Stein, Gary Stein, Jane Lian

Harold L. Merriman

The subnuclear distribution of the vitamin D receptor was investigated to begin addressing the contribution of nuclear architecture to vitamin D-responsive control of gene expression in ROS 17/2.8 rat osteosarcoma cells. The nuclear matrix is an anastomosing network of filaments that is functionally associated with DNA replication, transcription, and RNA processing. The representation of vitamin D receptor in the nuclear matrix and nonmatrix nuclear fractions was determined by the combined application of 1) sequence-specific interactions with the vitamin D receptor binding element of the rat bone-specific osteocalcin gene promoter and 2) Western blot analysis. Both methods confirmed the presence of …

The Tissue-Specific Nuclear Matrix Protein, Nmp-2, Is A Member Of The Aml/Cbf/Pebp2/Runt Domain Transcription Factor Family: Interactions With The Osteocalcin Gene Promoter, Harold Merriman, Andre Van Wijnen, Scott Hiebert, Joseph Bidwell, Edward Fey, Jane Lian, Janet Stein, Gary Stein

Harold L. Merriman

The nuclear matrix protein, NMP-2, was originally identified as an osteoblast-specific DNA-binding complex localized exclusively to the nuclear matrix. NMP-2 was shown to recognize two binding sites, site A (nt-605 to -599) and site B (nt -441 to -435), in the rat bone-specific osteocalcin gene promoter. This study shows that the NMP-2 binding sites A and B as well as a third NMP-2 binding site (nt -135 to -130) constitute a consensus sequence, ATGCTGGT, and represent an AML-1 recognition motif. AML-1 is a member of the AML transcription factor family which is associated with acute myelogenous leukemia and binds to …

Bacterial Rna:Dna Hybrids Are Activators Of The Nlrp3 Inflammasome, Sivapriya Kailasan Vanaja, Vijay A. K. Rathinam, Maninjay K. Atianand, Parisa Kalantari, Brian M. Skehan, Katherine A. Fitzgerald, John M. Leong

Katherine A. Fitzgerald

Enterohemorrhagic Escherichia coli (EHEC) is an extracellular pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The proinflammatory cytokine, interleukin-1beta, has been linked to hemolytic uremic syndrome. Here we identify the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome as an essential mediator of EHEC-induced IL-1beta. Whereas EHEC-specific virulence factors were dispensable for NLRP3 activation, bacterial nucleic acids such as RNA:DNA hybrids and RNA gained cytosolic access and mediated inflammasome-dependent responses. Consistent with a direct role for RNA:DNA hybrids in inflammasome activation, delivery of synthetic EHEC RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, …

Developmental Expression Of A Candidate Mullerian Inhibiting Substance Type Ii Receptor, Jose Teixeira, Wei He, Paresh Shah, Nobuyuki Morikawa, Mary Lee, Elizabeth Catlin, Peter Hudson, John Wing, David Maclaughlin, Patricia Donahoe

Mary M. Lee

We have isolated a candidate Mullerian inhibiting substance (MIS) type II receptor complementary DNA from an embryonic rat urogenital ridge library and have studied its binding to MIS, its developmental pattern of expression and tissue distribution. By in situ hybridization with a full-length riboprobe, the receptor is expressed in the mesenchymal cells surrounding the Mullerian duct at embryonic days 14, 15, and 16 and in tubular and follicular structures of the rat fetal gonads. Expression of the messenger RNA was also seen in the granules cells and seminiferous tubules of pubertal gonads. Northern analysis revealed that the MIS type II …

Developmentally Regulated Polyadenylation Of Two Discrete Messenger Ribonucleic Acids For Mullerian Inhibiting Substance, Mary Lee, Richard Cate, Patricia Donahoe, Gerald Waneck

Mary M. Lee

Mullerian inhibiting substance (MIS) is a 140-kilodalton homodimeric glycoprotein that causes regression of the Mullerian ducts in male embryos, and may also have a role in both males and females in the regulation of germ cell maturation. We examined the ontogeny of MIS messenger RNA (mRNA) in rat testes from midgestation through adulthood and found two discrete MIS mRNA species that are developmentally regulated. The larger 2.0-kilobase species is abundant at embryonic day 14, then decreases in late gestation, and is barely detectable after birth. The smaller 1.8-kilobase species is first noted at embryonic day 18 and is the major …

Mullerian Inhibiting Substance Inhibits Testosterone Synthesis In Adult Rats, V. Sriraman, E. Niu, J. Matias, Patricia Donahoe, David Maclaughlin, Matthew Hardy, Mary Lee

Mary M. Lee

Mullerian inhibiting substance (MIS) is a gonadal hormone that causes regression of the Mullerian ducts during male sexual differentiation. Postnatally, MIS inhibits the proliferation and differentiation of immature Leydig cells, and transgenic mice that overexpress MIS have decreased serum testosterone concentrations. To elucidate the effects of MIS on androgen regulation in the postnatal testis, we examined testosterone synthesis in adult Sprague-Dawley rats following intratesticular and intraperitoneal injections of MIS. Intratesticular MIS injection achieved high local concentrations of MIS (574.0 +/- 60.0 ng/mL) at 4 hours, with a corresponding decline in serum testosterone concentrations to 0.7 +/- 0.1 ng/mL, compared to …

A Single Base Pair Mutation Encoding A Premature Stop Codon In The Mis Type Ii Receptor Is Responsible For Canine Persistent Mullerian Duct Syndrome, Wenfang Wu, Shengqin Wan, Pujar Shashikant, Mark Haskins, Donald Schlafer, Mary Lee, Vicki Meyers-Wallen

Mary M. Lee

Mullerian inhibiting substance (MIS), a secreted glycoprotein in the transforming growth factor-beta family of growth factors, mediates regression of the Mullerian ducts during embryonic sex differentiation in males. In persistent Mullerian duct syndrome (PMDS), rather than undergoing involution, the Mullerian ducts persist in males, giving rise to the uterus, fallopian tubes, and upper vagina. Genetic defects in MIS or its receptor (MISRII) have been identified in patients with PMDS. The phenotype in the canine model of PMDS derived from the miniature schnauzer breed is strikingly similar to that of human patients. In this model, PMDS is inherited as a sex-limited …

Isolation Of The Rat Gene For Mullerian Inhibiting Substance, Christopher Haqq, Mary Lee, Richard Tizard, Mark Wysk, Janice Demarinis, Patricia Donahoe, Richard Cate

Mary M. Lee

Mullerian inhibiting substance (MIS), a testicular glycoprotein also known as anti-Mullerian hormone, plays a key role in male sexual development by causing regression of the Mullerian duct, the anlagen of the uterus, the Fallopian tubes, and part of the vagina. MIS is also expressed in the postnatal ovary, but its precise function is still not known. We report here the complete nucleotide sequence of the rat MIS gene. Rat MIS is encoded in five exons and is synthesized as a precursor of 553 amino acids, containing a 24-amino-acid leader. Based on homology with human MIS, we predict that the rat …

Gel Shift Analysis Of The Empa Promoter Region In Vibrio Anguillarum, Steven Denkin, Pedja Sekaric, David Nelson

David R. Nelson

BACKGROUND: The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements.

RESULTS: Two strains of V. anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region. Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like …

Acute Modulation Of Sugar Transport In Brain Capillary Endothelial Cell Cultures During Activation Of The Metabolic Stress Pathway, Anthony Cura, Anthony Carruthers

Anthony J. Cura

GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete intracellular ATP within 15 min. This results in a 1.7-7-fold increase in V(max) for zero-trans 3-O-methylglucose uptake (sugar uptake into sugar-free cells) and a 3-10-fold increase in V(max) for equilibrium exchange transport (intracellular [sugar] = extracellular [sugar]). …

Persistence Of Episomal Hiv-1 Infection Intermediates In Patients On Highly Active Anti-Retroviral Therapy, Mark Sharkey, Ian Teo, Thomas Greenough, Natalia Sharova, Katherine Luzuriaga, John Sullivan, R. Bucy, Leondios Kostrikis, Ashley Haase, Claire Veryard, Raul Davaro, Sarah Cheeseman, Jennifer Daly, Carol Bova, Richard Ellison, Brian Mady, Kwan Lai, Graeme Moyle, Mark Nelson, Brian Gazzard, Sunil Shaunak, Mario Stevenson

Associate Professor Mark Nelson

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication …

Guanosine Diphosphatase Is Required For Protein And Sphingolipid Glycosylation In The Golgi Lumen Of Saccharomyces Cerevisiae, Claudia Abeijon, Ken Yanagisawa, Elisabet Mandon, Alex Hausler, Kelley Moremen, Carlos Hirschberg, Phillips Robbins

Elisabet Mandon

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn …

N88d Facilitates The Co-Occurrence Of D30n And L90m And The Development Of Multidrug Resistance In Hiv Type 1 Protease Following Nelfinavir Treatment Failure, Yumi Mitsuya, Mark Winters, W. Jeffrey Fessel, Soo-Yon Rhee, Leo Hurley, Michael Horberg, Celia Schiffer, Andrew Zolopa, Robert Shafer

Celia A. Schiffer

Nelfinavir was once one of the most commonly used protease inhibitors (PIs). To investigate the genetic mechanisms of multidrug resistance in protease isolates with the primary nelfinavir resistance mutation D30N, we analyzed patterns of protease mutations in 582 viruses with D30N from 460 persons undergoing HIV-1 genotypic resistance testing at Stanford University Hospital from 1997 to 2005. Three patterns of mutational associations were identified. First, D30N was positively associated with N88D but negatively associated with N88S. Second, D30N and L90M were negatively associated except in the presence of N88D, which facilitated the co-occurrence of D30N and L90M. Third, D30N+N88D+L90M formed …

Endothelial Cells Assemble Two Distinct Alpha6beta4-Containing Vimentin-Associated Structures: Roles For Ligand Binding And The Beta4 Cytoplasmic Tail, Suzanne M. Homan, Arthur M. Mercurio, Susan E. Laflamme

Arthur M. Mercurio

The alpha6beta4 laminin binding integrin functions in the assembly of type I hemidesmosomes, which are specialized cell-matrix adhesion sites found in stratified epithelial cells. Although endothelial cells do not express all the components of type I hemidesmosomes, endothelial cells can express the alpha6beta4 integrin. Because endothelial cells lose expression of alpha6beta4 in culture, we expressed recombinant alpha6beta4 in the dermal microvascular endothelial cell line, HMEC-1, to test whether endothelial cells can assemble adhesion structures containing alpha6beta4. Using immunofluorescence microscopy, we found that recombinant alpha6beta4 concentrates specifically in a novel fibrillar structure on the basal surface of endothelial cells in the …

Regulation Of Alpha 6 Beta 1 Integrin Laminin Receptor Function By The Cytoplasmic Domain Of The Alpha 6 Subunit, Leslie M. Shaw, Arthur M. Mercurio

Arthur M. Mercurio

The alpha 6 beta 1 integrin is expressed on the macrophage surface in an inactive state and requires cellular activation with PMA or cytokines to function as a laminin receptor (Shaw, L. M., J. M. Messier, and A. M. Mercurio. 1990. J. Cell Biol. 110:2167-2174). In the present study, the role of the alpha 6 subunit cytoplasmic domain in alpha 6 beta 1 integrin activation was examined. The use of P388D1 cells, an alpha 6-integrin deficient macrophage cell line, facilitated this analysis because expression of either the alpha 6A or alpha 6B subunit cDNAs restores their activation responsive laminin adhesion …

Presence Of A Polyadenylated Rna Fragment Encoding The Membrane Domain For Immunoglobulin Alpha Chain Indicates That Mrnas For Both Secreted And Membrane-Bound Alpha Chains Can Be Produced From The Same Rna Transcript, Janet Stavnezer

Janet M. Stavnezer

RNA blotting was employed to examine polyadenylated immunoglobulin alpha chain RNAs in a B lymphoma synthesizing membrane-bound and secretory IgA and in a hybridoma which synthesizes predominantly secretory IgA. Both cell lines were derived from the I.29 lymphoma and expressed the identical heavy chain variable region gene. In addition to the predicted mRNA precursors, four novel species of polyadenylated alpha RNAs were detected. The presence of a RNA species which was too large to have the same 3' end as the largest mRNA for membrane-bound alpha chain (alpha m) implied that transcription continued past the alpha m poly(A) site, and …

Domain Analysis Of Supervillin, An F-Actin Bundling Plasma Membrane Protein With Functional Nuclear Localization Signals, J. D. Wulfkuhle, I. E. Donina, N. H. Stark, Robert K. Pope, Kersi N. Pestonjamasp, M. L. Niswonger, Elizabeth J. Luna

Elizabeth J. Luna

A growing number of actin-associated membrane proteins have been implicated in motile processes, adhesive interactions, and signal transduction to the cell nucleus. We report here that supervillin, an F-actin binding protein originally isolated from bovine neutrophil plasma membranes, contains functional nuclear targeting signals and localizes at or near vinculin-containing focal adhesion plaques in COS7-2 and CV1 cells. Overexpression of full-length supervillin in these cells disrupts the integrity of focal adhesion plaques and results in increased levels of F-actin and vinculin. Localization studies of chimeric proteins containing supervillin sequences fused with the enhanced green fluorescent protein indicate that: (1) the amino …