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Multiple Mechanisms Regulate Numa Dynamics At Spindle Poles, Olga Kisurina-Evgenieva, Gary Mack, Quansheng Du, Ian Macara, Alexey Khodjakov, Duane A. Compton

Dartmouth Scholarship

The large coiled-coil protein NuMA plays an essential role in organizing microtubule minus ends at spindle poles in vertebrate cells. Here, we use both in vivo and in vitro methods to examine NuMA dynamics at mitotic spindle poles. Using fluorescence recovery after photobleaching, we show that an exogenously expressed green-fluorescent-protein/NuMA fusion undergoes continuous exchange between soluble and spindle-associated pools in living cells. These dynamics require cellular energy and display an average half-time for fluorescence recovery of approximately 3 minutes. To explore how NuMA dynamics at spindle poles is regulated, we exploited the association of NuMA with microtubule asters formed in …

Esophageal Muscle Physiology And Morphogenesis Require Assembly Of A Collagen Xix-Rich Basement Membrane Zone., Hideaki Sumiyoshi, Niv Mor, Sui Y Lee, Stephen Doty, Scott Henderson, Shizuko Tanaka, Hidekatsu Yoshioka, Satish Rattan, Francesco Ramirez

Department of Medicine Faculty Papers

Collagen XIX is an extremely rare extracellular matrix component that localizes to basement membrane zones and is transiently expressed by differentiating muscle cells. Characterization of mice harboring null and structural mutations of the collagen XIX (Col19a1) gene has revealed the critical contribution of this matrix protein to muscle physiology and differentiation. The phenotype includes smooth muscle motor dysfunction and hypertensive sphincter resulting from impaired swallowing-induced, nitric oxide-dependent relaxation of the sphincteric muscle. Muscle dysfunction was correlated with a disorganized matrix and a normal complement of enteric neurons and interstitial cells of Cajal. Mice without collagen XIX exhibit an additional defect, …

From Biomedicine To Natural History Research: Est Resources For Ambystomatid Aalamanders, Srikrishna Putta, Jeramiah J. Smith, John A. Walker, Mathieu Rondet, David W. Weisrock, James Monaghan, Amy K. Samuels, D. Kevin Kump, David C. King, Nicholas J. Maness, Bianca Habermann, Elly Tanaka, Susan V. Bryant, David M. Gardiner, David M. Parichy, S. Randal Voss

Biology Faculty Publications

BACKGROUND: Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories.

RESULTS: Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the …

The Kini Kinesin Kif2a Is Required For Bipolar Spindle Assembly Through A Functional Relationship With Mcak, Neil J. Ganem, Duane A. Compton

Dartmouth Scholarship

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore–microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle …

Identification Of Sarv (Sa2062), A New Transcriptional Regulator, Is Repressed By Sara And Mgra (Sa0641) And Involved In The Regulation Of Autolysis In Staphylococcus Aureus, Adhar C. Manna, Susham S. Ingavale, Marybeth Maloney, Willem Van Wamel, Ambrose L. Cheung

Dartmouth Scholarship

The expression of genes involved in the pathogenesis of Staphylococcus aureus is known to be controlled by global regulatory loci, including agr, sarA, sae, arlRS, lytSR, and sarA-like genes. Here we described a novel transcriptional regulator called sarV of the SarA protein family. The transcription of sarV is low or undetectable under in vitro conditions but is significantly augmented in sarA and mgrA (norR or rat) (SA0641) mutants. The sarA and mgrA genes act as repressors of sarV expression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of the …

Pf15p Is The Chlamydomonas Homologue Of The Katanin P80 Subunit And Is Required For Assembly Of Flagellar Central Microtubules, Erin E. Dymek, Paul A. Lefebvre, Elizabeth F. Smith

Dartmouth Scholarship

Numerous studies have indicated that the central apparatus plays a significant role in regulating flagellar motility, yet little is known about how the central pair of microtubules or their associated projections assemble. Several Chlamydomonas mutants are defective in central apparatus assembly. For example, mutant pf15 cells have paralyzed flagella that completely lack the central pair of microtubules. We have cloned the wild-type PF15 gene and confirmed its identity by rescuing the motility and ultrastructural defects in two pf15 alleles, the original pf15a mutant and a mutant generated by insertional mutagenesis. Database searches using the 798-amino-acid polypeptide predicted from the complete …

Pv1 Is A Key Structural Component For The Formation Of The Stomatal And Fenestral Diaphragms, Radu V. Stan, Eugene Tkachenko, Ingrid R. Niesman

Dartmouth Scholarship

PV1 is an endothelial-specific integral membrane glycoprotein associated with the stomatal diaphragms of caveolae, transendothelial channels, and vesiculo-vacuolar organelles and the diaphragms of endothelial fenestrae. Multiple PV1 homodimers are found within each stomatal and fenestral diaphragm. We investigated the function of PV1 within these diaphragms and their regulation and found that treatment of endothelial cells in culture with phorbol myristate acetate (PMA) led to upregulation of PV1. This correlated with de novo formation of stomatal diaphragms of caveolae and transendothelial channels as well as fenestrae upon PMA treatment. The newly formed diaphragms could be labeled with anti-PV1 antibodies. The upregulation …

Endogenous No Regulates Superoxide Production At Low Oxygen Concentrations By Modifying The Redox State Of Cytochrome C Oxidase, Miriam Palacios-Callender, Marisol Quintero, Veronica S. Hollis, Roger J. Springett, Salvador Moncada

Dartmouth Scholarship

We have investigated in whole cells whether, at low oxygen concentrations ([O(2)]), endogenous nitric oxide (NO) modulates the redox state of the mitochondrial electron transport chain (ETC), and whether such an action has any signaling consequences. Using a polarographic-and-spectroscopic-coupled system, we monitored redox changes in the ETC cytochromes b(H), cc(1), and aa(3) during cellular respiration. The rate of O(2) consumption (VO(2)) remained constant until [O(2)] fell below 15 microM, whereas the onset of reduction of cytochromes aa(3), part of the terminal ETC enzyme cytochrome c oxidase, occurred at approximately 50 microM O(2). Incubation of the cells with an inhibitor of …

The Eagleville Times,Volume 2, Issue 5, Debbie Ryan

Eagleville Times

No abstract provided.

Estimating Metazoan Divergence Times With A Molecular Clock, Kevin J. Peterson, Jessica B. Lyons, Kristin S. Nowak, Carter M. Takacs, Matthew J. Wargo, Mark A. Mcpeek

Dartmouth Scholarship

Accurately dating when the first bilaterally symmetrical animals arose is crucial to our understanding of early animal evolution. The earliest unequivocally bilaterian fossils are 555 million years old. In contrast, molecular-clock analyses calibrated by using the fossil record of vertebrates estimate that vertebrates split from dipterans (Drosophila) 900 million years ago (Ma). Nonetheless, comparative genomic analyses suggest that a significant rate difference exists between vertebrates and dipterans, because the percentage difference between the genomes of mosquito and fly is greater than between fish and mouse, even though the vertebrate divergence is almost twice that of the dipteran. Here we show …

A Subgroup Algorithm To Identify Cross-Rotation Peaks Consistent With Non-Crystallographic Symmetry, Ryan H. Lilien, Chris Bailey-Kellogg, Amy C. Anderson, Bruce R. Donald

Dartmouth Scholarship

Molecular replacement (MR) often plays a prominent role in determining initial phase angles for structure determination by X-ray crystallography. In this paper, an efficient quaternion-based algorithm is presented for analyzing peaks from a cross-rotation function in order to identify model orientations consistent with proper non-crystallographic symmetry (NCS) and to generate proper NCS-consistent orientations missing from the list of cross-rotation peaks. The algorithm, CRANS, analyzes the rotation differences between each pair of cross-rotation peaks to identify finite subgroups. Sets of rotation differences satisfying the subgroup axioms correspond to orientations compatible with the correct proper NCS. The CRANS algorithm was first …

Hearing Loss And Retarded Cochlear Development In Mice Lacking Type 2 Iodothyronine Deiodinase, Lily Ng, Richard J. Goodyear, Chad A. Woods, Mark J. Schneider

Dartmouth Scholarship

The later stages of cochlear differentiation and the developmental onset of hearing require thyroid hormone. Although thyroid hormone receptors (TRs) are a prerequisite for this process, it is likely that other factors modify TR activity during cochlear development. The mouse cochlea expresses type 2 deiodinase (D2), an enzyme that converts thyroxine, the main form of thyroid hormone in the circulation, into 3,5,3'-triiodothyronine (T3) the major ligand for TRs. Here, we show that D2-deficient mice have circulating thyroid hormone levels that would normally be adequate to allow hearing to develop but they exhibit an auditory phenotype similar to that caused by …

The Cohesion Protein Ord Is Required For Homologue Bias During Meiotic Recombination, Hayley A. Webber, Louisa Howard, Sharon E. Bickel

Dartmouth Scholarship

During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear …

Muc4 Mucin Expression In Human Pancreatic Tumours Is Affected By Organ Environment: The Possible Role Of Tgfbeta2., A. Choudhury, N. Moniaux, A. B. Ulrich, B. M. Schmied, J. Standop, Parviz M. Pour, S. J. Gendler, Michael A. Hollingsworth, J-P Aubert, Surinder K. Batra

Journal Articles: Biochemistry & Molecular Biology

MUC4 is highly expressed in human pancreatic tumours and pancreatic tumour cell lines, but is minimally or not expressed in normal pancreas or chronic pancreatitis. Here, we investigated the aberrant regulation of MUC4 expression in vivo using clonal human pancreatic tumour cells (CD18/HPAF) grown either orthotopically in the pancreas (OT) or ectopically in subcutaneous tissue (SC) in the nude mice. Histological examination of the OT and SC tumours showed moderately differentiated and anaplastic morphology, respectively. The OT tumour cells showed metastases to distant lymph nodes and faster tumour growth (P

Signaling Switches And Bistability Arising From Multisite Phosphorylation In Protein Kinase Cascades., Nick I Markevich, Jan B. Hoek, Boris N. Kholodenko

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal-regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, …

Quantitative Analysis Of Hemocyte Morphological Abnormalities Associated With Campoletis Sonorensis Parasitization, Matthew W. Turnbull, Stacy B. Martin, Bruce A. Webb

Entomology Faculty Publications

Endoparasitoids of arthropods evoke host cellular immune responses that result in hemocytic encapsulation of the endoparasitoid, unless these responses are disrupted by the parasite. Our interest has focused on mutualistic viruses found in some hymenopteran endoparasitoids that disrupt hemocyte function and prevent encapsulation. Specifically, the Campoletis sonorensis polydnavirus interacts with wasp factors to suppress immunity via expression of intracellular and secreted viral proteins. To study the roles of specific parasitization-associated factors on immunocyte morphology, fluorescence microscopy was used to visualize the actin cytoskeleton in infected and uninfected cells, or after treatment with C. sonorensis ovarian proteins or plasma from infected …

The Eagleville Times, Volume 2, Issue 7, Debbie Ryan

Eagleville Times

No abstract provided.

The Eagleville Times, Volume 2, Issue 2, Debbie Ryan

Eagleville Times

No abstract provided.

In Vitro Analog Of Classical Conditioning Of Feeding Behavior In Aplysia, Riccardo Mozzachiodi, Hilde A. Lechner, Douglas A. Baxter, John H. Byrne

Faculty, Staff and Student Publications

The feeding behavior of Aplysia californica can be classically conditioned using tactile stimulation of the lips as a conditioned stimulus (CS) and food as an unconditioned stimulus (US). Moreover, several neural correlates of classical conditioning have been identified. The present study extended previous work by developing an in vitro analog of classical conditioning and by investigating pairing-specific changes in neuronal and synaptic properties. The preparation consisted of the isolated cerebral and buccal ganglia. Electrical stimulation of a lip nerve (AT4) and a branch of the esophageal nerve (En2) served as the CS and US, respectively. Three protocols were used: paired, …

Transcriptional Inhibition Of Type I Collagen Gene Expression In Scleroderma Fibroblasts By The Antineoplastic Drug Ecteinascidin 743., Natalia Louneva, Biagio Saitta, David J Herrick, Sergio A. Jimenez

Department of Medicine Faculty Papers

We previously showed that COL1A1 expression is up-regulated at the transcriptional level in systemic sclerosis (SSc) fibroblasts and that the CCAAT-binding factor (CBF) is involved in this increased expression. Ecteinascidin 743 (ET-743) is a chemotherapeutic agent that binds with sequence specificity to the minor groove of DNA and inhibits CBF-mediated transcriptional activation of numerous genes. Therefore, we examined the effects of ET-743 on the increased COL1A1 expression in SSc fibroblasts. The drug caused a potent and dose-dependent inhibition of type I collagen biosynthesis, which reached 70-90% at 700 pM without affecting cell viability. The same drug concentration caused 60-80% reduction …

Il-7 Enhances Peripheral T Cell Reconstitution After Allogeneic Hematopoietic Stem Cell Transplantation., Onder Alpdogan, Stephanie J Muriglan, Jeffrey M Eng, Lucy M Willis, Andrew S Greenberg, Barry J Kappel, Marcel R M Van Den Brink

Department of Medical Oncology Faculty Papers

We used clinically relevant murine allogeneic bone marrow transplantation (BMT) models to study the mechanisms by which IL-7 administration can improve posttransplant peripheral T cell reconstitution. After transplant we could distinguish two populations of mature donor T cells: (a) alloreactive T cells with decreased expression of CD127 (IL-7 receptor alpha chain) and (b) nonalloreactive T cells, which express CD127 and undergo homeostatic proliferation. IL-7 administration increased the homeostatic proliferation of nonalloreactive T cells, but had no effect on alloreactive T cells and the development of graft-versus-host disease. Allogeneic transplant of purified hematopoietic stem cells and adoptive transfer of thymocytes into …

A Null Mutation For Tissue Inhibitor Of Metalloproteinases-3 (Timp-3) Impairs Murine Bronchiole Branching Morphogenesis., Sean E Gill, M Cynthia Pape, Rama Khokha, Andrew J Watson, Kevin J Leco

Obstetrics & Gynaecology Publications

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs). We have examined the role of TIMP-3 on ECM homeostasis and bronchiole branching morphogenesis during murine embryogenesis. Employing an in vitro organ culture system, we found decreased bronchiolar branching in null lungs when compared with wild type (WT) counterparts after 2 days in culture. When a synthetic inhibitor of MMPs at low dose was added to the culture system, branching was augmented regardless of genotype. Gelatin and in situ zymography revealed that null lungs exhibited enhanced activation of MMPs throughout lung development. We analysed the impact …

Probucol Prevents Early Coronary Heart Disease And Death In The High-Density Lipoprotein Receptor Sr-Bi/Apolipoprotein E Double Knockout Mouse, Anne Braun, Songwen Zhang, Helena E. Miettinen, Shamsah Ebrahim, Teresa M. Holm, Eliza Vasile, Mark J. Post

Dartmouth Scholarship

Mice with homozygous null mutations in the high-density lipoprotein receptor SR-BI (scavenger receptor class B, type I) and apolipoprotein E genes fed a low-fat diet exhibit a constellation of pathologies shared with human atherosclerotic coronary heart disease (CHD): hypercholesterolemia, occlusive coronary atherosclerosis, myocardial infarctions, cardiac dysfunction (heart enlargement, reduced systolic function and ejection fraction, and ECG abnormalities), and premature death (mean age 6 weeks). They also exhibit a block in RBC maturation and abnormally high plasma unesterified-to-total cholesterol ratio (0.8) with associated abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in lecithin/cholesterol acyltransferase deficiency and cholestasis). Treatment …

Human Acyl-Coenzyme A:Cholesterol Acyltransferase Expressed In Chinese Hamster Ovary Cells: Membrane Topology And Active Site Location, Song Lin, Xiaohui Lu, Catherine C.Y. Chang, Ta-Yuan Chang

Dartmouth Scholarship

Acyl-CoA:cholesterol acyltransferase (ACAT) is a membrane-bound enzyme that produces cholesteryl esters intracellularly. Two ACAT genes (ACAT1 and ACAT2) have been identified. The expression of ACAT1 is ubiquitous, whereas that of ACAT2 is tissue restricted. Previous research indicates that ACAT1 may contain seven transmembrane domains (TMDs). To study ACAT2 topology, we inserted two different antigenic tags (hemagglutinin, monoclonal antibody Mab1) at various hydrophilic regions flanking each of its predicted TMDs, and expressed the recombinant proteins in mutant Chinese hamster ovary cells lacking endogenous ACAT. Each tagged ACAT2 was expressed in the endoplasmic reticulum as a single undegraded protein band …

Minus-End Capture Of Preformed Kinetochore Fibers Contributes To Spindle Morphogenesis, Alexey Khodjakov, Lily Copenagle, Michael B. Gordon, Duane A. Compton, Tarun M. Kapoor

Dartmouth Scholarship

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient …

Promoters Of The Murine Embryonic Β-Like Globin Genes Ey And Βh1 Do Not Compete For Interaction With The Β-Globin Locus Control Region, Xiao Hu, Michael Bulger, Julia N. Roach, Susan K. Eszterhas, Emmanuel Olivier, Eric Bouhassira, Mark Groudine, Steven Fiering

Dartmouth Scholarship

Mammalian β-globin loci contain multiple β-like genes that are expressed at different times during development. The murine β-globin locus contains two genes expressed during the embryo stage, Ey and βh1, and two genes expressed at both the fetal and postnatal stages, β-major and β-minor. Studies of transgenic human β-like globin loci in mice have suggested that expression of one gene at the locus will suppress expression of other genes at the locus. To test this hypothesis we produced mouse lines with deletions of either the Ey or βh1 promoter in the endogenous murine β-globin locus. Promoter deletion eliminated expression of …

Regulation Of Human Col9a1 Gene Expression. Activation Of The Proximal Promoter Region By Sox9., Ping Zhang, Sergio A. Jimenez, David G Stokes

Department of Medicine Faculty Papers

The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter …

Separation, Custody, And Estate Planning Issues Relating To Companion Animals, Rebecca J. Huss

Law Faculty Publications

This article first discusses the domestication of companion animals, including the impact of anthropomorphism and neoteny on how animals are viewed in U.S. society. Second, it reviews the current legal status of animals. Third, it considers the voluntary and involuntary separation of companion animals from their human families. Fourth, it examines custody issues in the context of the placement of animals after the divorce of the human family members. Finally, it analyzes estate planning issues relating to companion animals.

G03-1515 Sampling And Analyzing Feed For Fungal (Mold) Toxins (Mycotoxins), Michael P. Carlson, Steven M. Ensley

University of Nebraska-Lincoln Extension: Historical Materials

Mycotoxins are chemicals produced by fungi (molds) under certain conditions. They are not essential for fungal growth or reproduction, and are toxic to animals or humans. They are naturally present in feed and cannot always be avoided.

The purposes of this NebGuide are to provide information about sampling feeds to detect the presence of mycotoxins in them and about how feeds may be analyzed for mycotoxins.

G03-1501 Prudent Use Of Antibiotics In Companion Animals, Katherine Irwin, David R. Smith, Grasso M. Ebako, Steve Ensley, Dicky D. Griffin, Arden Wohlers

University of Nebraska-Lincoln Extension: Historical Materials

This NebGuide explains why antibiotics must be used with care and how to prudently use antibiotics to treat companion animals.

Antibiotics are used to improve animal health and performance. You, as the animal owner or caregiver, make important decisions about how antibiotics are finally used in companion animals. Antibiotics should be used prudently to ensure they are effective and will continue to benefit man and animals in the future. Are you prepared to make the best decisions about using antibiotics in animals?