Cell and Developmental Biology Commons^™

Transport Of Er Vesicles On Actin Filaments In Neurons By Myosin V, George M. Langford, Joel S. Tabb, Bradley J. Molyneaux, Darien L. Cohen, Sergei A. Kuznetsov

Biology - All Scholarship

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was …

Transport Of Er Vesicles On Actin Filaments In Neurons By Myosin V, Joel S. Tabb, Bradley J. Molyneaux, Darien L. Cohen, Sergei A. Kuznetsov, George M. Langford

Dartmouth Scholarship

Axoplasmic organelles in the giant axon of the squid have been shown to move on both actin filaments and microtubules and to switch between actin filaments and microtubules during fast axonal transport. The objectives of this investigation were to identify the specific classes of axoplasmic organelles that move on actin filaments and the myosin motors involved. We developed a procedure to isolate endoplasmic reticulum (ER) from extruded axoplasm and to reconstitute its movement in vitro. The isolated ER vesicles moved on exogenous actin filaments adsorbed to coverslips in an ATP-dependent manner without the addition of soluble factors. Therefore myosin was …

Transport Of Axl2p Depends On Erv14p, An Er–Vesicle Protein Related To The Drosophila Cornichon Gene Product, Jacqueline Powers, Charles Barlowe

Dartmouth Scholarship

COPII-coated ER-derived transport vesicles from Saccharomyces cerevisiae contain a distinct set of membrane-bound polypeptides. One of these polypeptides, termed Erv14p (ER-vesicle protein of 14 kD), corresponds to an open reading frame on yeast chromosome VII that is predicted to encode an integral membrane protein and shares sequence identity with the Drosophila cornichon gene product. Experiments with an epitope-tagged version of Erv14p indicate that this protein localizes to the ER and is selectively packaged into COPII-coated vesicles. Haploid cells that lack Erv14p are viable but display a modest defect in bud site selection because a transmembrane secretory protein, Axl2p, is not …

Developmental Regulation Of A Cyclin-Dependent Kinase Inhibitor Controls Postembryonic Cell Cycle Progression In Caenorhabditis Elegans, Yang Hong, Richard Roy, Victor Ambros

Dartmouth Scholarship

C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of

INTRODUCTION

The proper development of a multicellular organism requires the precise orchestration of cell proliferation and differentiation. Despite considerable progress toward …

Efficacy Of AllowashTm Solution In Solubilization Of Bone Marrow, Alyce Marie Linthurst

Biological Sciences Theses & Dissertations

LifeNet currently cleans allograft bone utilizing Allowash^TM Technology, which encompasses the use of a closed continuous circulation chamber to remove bone marrow by flushing it with Allowash^TM Solution for a prescribed time and temperature after which, it is rinsed with various solutions to remove residual detergent that could be toxic to the recipient. A portion of this process employs Allowash^TM Solution to improve the solubilization and removal of protein and cellular elements in the red bone marrow. This study examines the effectiveness of Allowash^TM Solution in comparison to its component detergents Nonidet P40, Nonoxynol 9 and …

Leukemia Inhibitory Factor (Lif): Murine Preimplantation Embryo Development, Implantation Rates, And Skeletal Development, Michael Hayes Mitchell

Theses and Dissertations in Biomedical Sciences

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine which demonstrates perplexing physiological effects. It has been demonstrated that LIF is essential for implantation in mice. Little is known relating to the manner by which LIF effects pre-implantation and post-implantation development. The objectives of this project were to determine the effects LIF on pre-implantation development, to determine the effects that it may have on implantation rates, successful pregnancy rates, and resorption rates, and to determine the effects that LIF has on the skeletal development of mice. For the embryo transfer experiments, embryos were exposed to test compounds in the transfer medium …

Sec35p, A Novel Peripheral Membrane Protein, Is Required For Er To Golgi Vesicle Docking, Susan M. Vanrheenen, Xiaochun Cao, Vladimir V. Lupashin, Charles Barlowe, M. Gerard Waters

Dartmouth Scholarship

SEC35 was identified in a novel screen for temperature-sensitive mutants in the secretory pathway of the yeast Saccharomyces cerevisiae (Wuestehube et al., 1996. Genetics. 142:393–406). At the restrictive temperature, the sec35-1 strain exhibits a transport block between the ER and the Golgi apparatus and accumulates numerous vesicles. SEC35 encodes a novel cytosolic protein of 32 kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec35-1 is efficiently suppressed by YPT1, which encodes the rab-like GTPase required early in the secretory pathway, or by SLY1-20, which encodes a dominant form of the ER to Golgi target …

Confocal Microscopy: A Powerful Tool For Biological Research, Amit Singh, K. P. Gopinathan

Biology Faculty Publications

Conventional light microscopy allows the observation of living as well as fixed cells and tissues to generate two-dimensional images. The out-of-focus information often obscures the ultrastructural details, especially in thick specimens with overlapping structures. The earliest available light microscopy visualized the objects in hydrated state in two-dimensions during their temporal development. The emergence of electron microscopy (EM) provided superb resolution of ultrastructural details, but it was applicable only for objects in the dehydrated state and thereby potentially introducing handling artifacts. The usefulness of optical methods, however, has been limited by the poor depth discrimination. Often, the fluorescence and reflectance images …

The Biology Of Xenopus By R. C. Tinsley And H. C. Kobel, Rafael O. De Sá

Biology Faculty Publications

The Biology of Xenopus presents a summary of current knowledge about a single genus resulting from a symposium held at the Zoological Society of London in September 1992. This approach to summarizing available information has also been taken for other taxa, such as Atelopus (Lotters, 1996). However, the task of compiling data for Xenopus is enormous relative to any other amphibian group, because Xenopus laevis has become a model system for molecular and development research (Cannatella and de Sa, 1993). Unfortunately, most of our knowledge of Xenopus is biased toward this single species. There are about 20 recognized species of …

Molecular Staging Of Colorectal Cancer: K-Ras Mutation Analysis Of Lymph Nodes Upstages Dukes’ B2 Patients, Jennifer S. Thebo

Dissertations

The successful treatment of colorectal cancer depends upon the ability of staging systems to identify patients who are at risk for recurrence. The currently used systems fail in a significant number of patients. Multiple attempts have been made to improve upon these systems in order to provide better treatment for at-risk patients.

A variety of oncogenes and tumor suppressor genes have been implicated in colorectal cancer. Mutations in one of these, K-ras, occur in 40-60% of colorectal carcinomas. However, the relationship between specific mutations and clinical outcome is unclear.

The purpose of this study was to determine whether or not …

Pressure-Induced Intracellular Signaling In Isolated Arteries, Victor A. Miriel

Theses and Dissertations in Biomedical Sciences

The ability of cells to respond to mechanical stimuli has been studied through a variety of techniques in numerous cell types. The cells of the vascular wall have adapted to specific mechanical stresses through the activation of intracellular signaling pathways which result in cell-specific responses such as hypertrophy, hyperplasia, proliferation, and migration. Vascular smooth muscle of the arteries have been shown to be sensitive to mechanical stimuli such as stretch, and pressure.

This study attempts to add to the current knowledge of mechanotransduction by utilizing the isolated artery preparation. This preparation allows for the study of vascular smooth muscle signal …

The Role Of Glycoconjugates In Mediating Human Fertilization And Induction Of Fetomaternal Tolerance, Manish S. Patankar

Theses and Dissertations in Biomedical Sciences

Using the hemizona assay (HZA), a in vitro sperm-egg binding assay, we show that specific glycoconjugates known to inhibit immune cell interactions mediated by the selectins, potently block human sperm-egg binding. The selectin ligand sialyl Lewis^x inhibits sperm binding in the HZA by 60% at a concentration of 1 mg/ml. Our data indicates that glycodelin-A, a endometrial glycoprotein known to block sperm-egg binding in the HZA at low concentrations expresses unusual fucosylated lacdiNAc type glycans. The fucosylated lacdiNAc type sugars have been previously shown to be 15-20 fold more potent ligands of E-selectin. Glycodelin-S a seminal plasma glycoform of …

Reaction-Diffusion Models Of Cancer Dispersion, Kim Yvette Ward

Mathematics & Statistics Theses & Dissertations

The phenomenological modeling of the spatial distribution and temporal evolution of one-dimensional models of cancer dispersion are studied. The models discussed pertain primarily to the transition of a tumor from an initial neoplasm to the dormant avascular state, i.e. just prior to the vascular state, whenever that may occur. Initiating the study is the mathematical analysis of a reaction-diffusion model describing the interaction between cancer cells, normal cells and growth inhibitor. The model leads to several predictions, some of which are supported by experimental data and clinical observations $\lbrack25\rbrack$. We will examine the effects of additional terms on these characteristics. …

Fission Yeast Mitotic Regulator Dsk1 Is An Sr Protein-Specific Kinase, Zhaohua Irene Tang, Mitsuhiro Yanagida, Ren-Jang Lin

WM Keck Science Faculty Papers

Intricate interplay may exist between pre-mRNA splicing and the cell division cycle, and fission yeast Dsk1 appears to play a role in such a connection. Previous genetic analyses have implicated Dsk1 in the regulation of chromosome segregation at the metaphase/anaphase transition. Yet, its protein sequence suggests that Dsk1 may function as a kinase specific for SR proteins, a family of pre-mRNA splicing factors containing arginine-serine repeats. Using an in vitro system with purified components, we showed that Dsk1 phosphorylated human and yeast SR proteins with high specificity. The Dsk1-phosphorylated SF2/ASF protein was recognized strongly by a monoclonal antibody (mAb104) known …

The Role Of Domain I In Laminin Chain Assembly, George D. Kamphaus

Theses, Dissertations and Capstones

Laminin, a major component of basement membrane, is a trimeric glycoprotein comprised of three chains - α, β and γ (Burgeson et al., 1994). An order for trimer assembly has been deduced: first, the β and γ chains bind to form a dimer and subsequently α is added to complete the trimer (I. Hunter et al., 1990 & 1992; Utani et al., 1994 & 1995). The C-terminal portions, found within the protein structural domain I of the p and y chains, are implicated in dimer and trimer formation by biochemical studies performed extracellularly (Utani, et al., 1994 & 1995; Nomizu …

Molecular Mapping Of The Fatty (F) (Formerly Corpulent) Gene In The La/N Rat Obesity Model, Kenneth G. Butz

Theses, Dissertations and Capstones

The LA/N-corpulent (cp) rat obesity model was a congenic rat strain developed at the National Institutes of Health by C. T. Hansen . The LA/N-cp rat originated from breeding the Koletsky rat, possessing the corpulent gene, with the normotensive LA/N inbred rat. The autosomal recessive single gene mutation, cp, produces obesity with hyperphagia, hyperinsulinemia and elevated activities of liver and skeletal muscle lipogenic enzymes in the LA/N-cp rat model. The Zucker-fatty mutation, which was mapped to rat chromosome , resulted in a comparable, but not identical obesity syndrome. Breeding experiments by Yen et al …

Clones Help Develop Ewe Feeding Strategy, Myra Yelland, Rob Kelly, John Davies, Johan Greeff

Journal of the Department of Agriculture, Western Australia, Series 4

Cloned sheep are not a new animal to the researchers of Agriculture WA, but the method that produced Dolly is.

Cloned sheep have been produced at the great Southern Research Institute as early as the mid 1980s. Myra Yelland, Rob Kelly, John Davies and Johan Greef outline how clones are used in experimental studies on wool production

Type I Camp-Dependent Protein Kinase Delays Apoptosis In Human Neutrophils At A Site Upstream Of Caspase-3, Lav K. Parvathenani, E. Stephen Buescher, Enrique Chacon-Cruz, Stephen J. Beebe

Bioelectrics Publications

Current data suggest that apoptosis controls neutrophil numbers in tissues. We analyzed roles for and the sites of action for the cAMP-dependent protein kinases (cAPKs) in apoptosis induced in human neutrophils by in vitro storage, cycloheximide (CHX) exposure, and anti-Fas exposure. Treatment with 8-chlorophenylthio-cAMP (8-CPT-cAMP) prolonged the time required for 50% of the cells to exhibit apoptotic morphology (t 50) from 16.3 to 41.8 h (in vitro culture), from 2.4 to 7.8 h (CHX), and from 4.8 to 6.5 h (anti-Fas). CHX ± 8-CPT-cAMP did not significantly alter resting intracellular calcium levels and H-89, a selective inhibitor of cAPK, had …

Acceleration Of Amphibian Forelimb Regeneration By Polypeptide Growth Factors, Gehan H. Fahmy, Raymond E. Sicard

Journal of the Minnesota Academy of Science

Growth factors are potentially important modulators of epimorphic regeneration. This study examined effects of intraperitoneal administration of selected growth factors on limb regeneration of adult newts, Notophthalmus viridescens. These agents stimulated regeneration, producing overlapping but nonidentical effects. Fibroblast growth factor-2 (FGF-2) and insulin-like growth factor I (IGF-I) stimulated bud emergence (8.3 ± 0.6 and 8.3 ± 0.7 days, respectively, vs 11.4 ± 1.1 days for controls). Progression to the cone stage was enhanced by both FGF-2 and transforming growth factor beta 5 ( TGF-~ 5) ; 14.6 ± 0.5 and 15.4 ± 0.4 days with FGF-2 and TGF-~5 , respectively, …

Chondrocranial Morphology Of Leptodactylus Larvae (Leptodactylidae: Leptodactylinae): Its Utility In Phylogenetic Reconstruction, Peter M. Larson, Rafael O. De Sá

Biology Faculty Publications

Chondrocranial morphology of leptodactylid frogs is scarcely known and has not been completely described for any species of Leptodactylus. We describe the diversity of chondrocranial morphology in the genus Leptodactylus based on the analysis of 22 species, representing the four species groups: the fuscus Group, ocellatus Group, melanonotus Group, and pentadactylus Group. Furthermore, 26 characters are identified and used in a phylogenetic analysis. The phylogenetic analysis using Physalaemus, Crossodactylus, and Hylodes as outgroups suggests two monophyletic clades within Leptodactylus: the melanonotus-ocellatus clade and the pentadactylusfuscus clade. However, it does not support the monophyly of the species groups as currently recognized …

Polyadenylation Of Vesicular Stomatitis Virus Mrna Dictates Efficient Transcription Termination At The Intercistronic Gene Junctions, Leroy N. Hwang, Nathan Englund, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The intercistronic gene junctions of vesicular stomatitis virus (VSV) contain conserved sequence elements that are important for polyadenylation and transcription termination of upstream transcript as well as reinitiation of transcription of downstream transcript. To examine the role of the putative polyadenylation signal 3' AUACU₇5' at the gene junctions in polyadenylation and transcription termination, we constructed plasmids encoding antigenomic minireplicons containing one or two transcription units. In plasmid-transfected cells, analyses of the bicistronic minireplicon containing the wild-type or mutant intercistronic gene junctions for the ability to direct synthesis of polyadenylated upstream, downstream, and readthrough mRNAs showed that the AUACU …

Diphtheria Toxin Fusion Proteins, Frank Foss, Mansoor Saleh, James Krueger, John Nichols, John Murphy

Haematology and Oncology, East Africa

Two different approaches have been undertaken to develop targeted biomolecules for therapeutics. The first was the construction of immunotoxins consisting of monoclonal antibodies chemically linked through a disulfide bond to a plant or bacterial toxin or radionuclide. Instability of the chemical conjugation of some of the earlier immunotoxins led to the concept of using protein engineering and recombinant DNA to assemble fusion genes combining the sequences for the enzymatically active and translocation domains of a toxin with those of a specific targeting ligand. From the outset, the prospect of using recombinant DNA methods to assemble the structural genes encoding bacterial …

Alterations In Calcium Homeostasis And The Insulin-Like Growth Factor Signaling Pathway Induced By Carcinogenic Polycyclic And Halogenated Aromatic Hydrocarbons In Human Mammary Epithelial Cells, Stacey L. Tannheimer

Pharmaceutical Sciences ETDs

Breast cancer is a major health concern for women, with only a small percent of the risk factors currently identified. It has been estimated that environmental factors may contribute to up to 80% of breast cancer cases. Many environmental carcinogens, such as polycyclic and halogenated aromatic hydrocarbons (PAHs and HAHs), are proven mammary carcinogens in animal models. Therefore, these studies were conducted to elucidate potential roles of PAHs and HAHs in alterations in known signaling pathways in human mammary epithelial cells (HMEC). Carcinogenic PAHs have previously been shown to produce sustained alterations in the calcium (Ca2') homeostasis of lymphocytes Therefore, …

Effects Of Oxidized Low Density Lipoprotein On Nitric Oxide Production In Macrophages, Annong Huang

Electronic Theses and Dissertations

The effects of oxidatively modified low density lipoprotein (oxLDL) on atherogenesis may be partly mediated by alterations in nitric oxide (NO) production by macrophages. A major goal of this study was to identify the lipid components in oxLDL modulating NO production. The effect of a water soluble antioxidants (N-acetylcysteine) and lipid soluble antioxidant (alpha-tocopherol) on NO production in macrophages was also determined. A second goal was to determine if the effects of oxLDL occurred at the transcriptional level. Human LDL was oxidized using an azo-initiator 2,2$\sp\prime$-azobis (2-amidinopropane) HCI (ABAP). OxLDL markedly decreased the production of NO in LPS stimulated RAW264.7 …

Comparison Of Beta-Adrenoceptor Coupled Camp Production In Cultured Human Mononuclear Leukocytes And Myometrial Cells, Yu-Li Liu

Electronic Theses and Dissertations

$\beta\sb2$-Adrenoceptor ($\beta\sb2$-AR) agonists, such as terbutaline, are used as tocolytic agents in the treatment of preterm labor. $\beta$-Adrenoceptor stimulation relaxes myometrium through specific receptors coupled through Gs to adenylyl cyclase (AC) that catalyzes the conversion of ATP to cAMP. The purpose of this study was to compare $\beta$-adrenoceptors and cAMP production in cultured human leukocytes and myometrial cells, and to determine the importance of $\beta$-adrenoceptors and cAMP production in isoproterenol-induced myometrial relaxation. $\sp{125}$I-iodopindolol was used to assess $\beta$-adrenoceptor affinity and number cAMP levels were analyzed before and after stimulation by isoproterenol, AlF$\sb4\sp-$, forskolin, and PGE$\sb1$. Isometric recording was used to …

Coupled Er To Golgi Transport Reconstituted With Purified Cytosolic Proteins, Charles Barlowe

Dartmouth Scholarship

A cell-free vesicle fusion assay that reproduces a subreaction in transport of pro-alpha-factor from the ER to the Golgi complex has been used to fractionate yeast cytosol. Purified Sec18p, Uso1p, and LMA1 in the presence of ATP and GTP satisfies the requirement for cytosol in fusion of ER-derived vesicles with Golgi membranes. Although these purified factors are sufficient for vesicle docking and fusion, overall ER to Golgi transport in yeast semi-intact cells depends on COPII proteins (components of a membrane coat that drive vesicle budding from the ER). Thus, membrane fusion is coupled to vesicle formation in ER to Golgi …

Altered Trafficking Of Mutant Connexin32, Suzanne M. Deschênes, Jessica L. Walcott, Tamara L. Wexler, Steven S. Scherer, Kenneth H. Fischbeck

Biology Faculty Publications

We examined the cellular localization of nine different connexin32 (Cx32) mutants associated with X-linked Charcot–Marie–Tooth disease (CMTX) in communication-incompetent mammalian cells. Cx32 mRNA was made, but little or no protein was detected in one class of mutants. In another class of mutants, Cx32 protein was detectable in the cytoplasm and at the cell surface, where it appeared as plaques and punctate staining. Cx32 immunoreactivity in a third class of mutants was restricted to the cytoplasm, where it often colocalized with the Golgi apparatus. Our studies suggest that CMTX mutations have a predominant effect on the trafficking of Cx32 protein, resulting …

Inhibition Of Axonal Growth By Brefeldin A In Hippocampal Neurons In Culture, Mark Jareb, Gary Banker

Biology Faculty Publications

The outgrowth of neuronal processes involves a great increase in the surface area of the cell. The supply of membrane material necessarily must be coordinated with the demands for neurite growth. The selective growth of only one or two neurites at any given time during the development of polarity raises the possibility that the production of materials by the soma is limiting for growth (Dotti and Banker, 1987; Dotti et al., 1988; Goslin and Banker, 1990). To examine the role of the availability of membrane components during the development of polarity and axonal elongation, we treated neurons with brefeldin A, …

Recovery Of Rna Polymerase Ii Synthesis Following Dna Damage In Mutants Of Saccharomyces Cerevisiae Defective In Nucleotide Excision Repair, Michael S. Reagan, Errol C. Friedberg

Biology Faculty Publications

We have measured the kinetics of the recovery of mRNA synthesis in the inducible GAL10 and RNR3 genes after exposure of yeast cells to ultraviolet (UV) radiation. Such recovery is abolished in mutant strains defective in nucleotide excision repair (NER) of DNA, including a rad23 mutant. Mutants defective in the RAD7 or RAD16 genes, which are required for the repair of the non-transcribed strand but not the transcribed strand of transcriptionally active genes, show slightly faster recovery of RNA synthesis than wild-type strains. A strain deleted of the RAD26 gene, which is known to be required for strand-specific NER in …

Vocal Response Of Piglets To Weaning: Effect Of Piglet Age, Daniel M. Weary, David Fraser

Ontogeny Collection

Piglets vocalize a great deal during the first few days after weaning. The aim of this experiment was to determine if the calls given by piglets vary in response to one factor thought to influence post-weaning adaptation: weaning age. In 22 litters, each containing a minimum of 9 piglets, 3 piglets were weaned under identical conditions at 3, 4 and 5 weeks of age. Vocalizations and weight gain were monitored for the week after weaning. The number of calls produced by piglets of all three ages declined from an average of 8.2 calls/min per piglet on the day of weaning …