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 …

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 …

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 …

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 …

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 …

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 …

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 …

Isolation Of A Cdna Encoding A Kex2-Like Endoprotease With Homology To Furin From The Nematode Caenorhabditis Elegans, Eduardo Gomez-Saladin, Anne E. Luebke, David L. Wilson, Ian M. Dickerson

Biology Articles and Papers

A cDNA was isolated from the nematode Caenorhabditis elegans that encodes an endoprotease which is a member of the Kex2 family of serine endoproteases. Degenerate oligonucleotide primers were designed based on conserved regions within the active sites of known Kex2-like endoproteases, and were used for reverse transcription-polymerase chain reaction (RT-PCR) of poly(A)⁺RNA isolated from C. elegans. A PCR product was isolated that had homology to the active sites of known furin endoproteases, and was used as a probe to screen a C. elegans cDNA library. A Kex2-like endoprotease (CelfurPC) which encoded a 692-amino-acid preproendoprotease, was identified. The …

Endosome To Golgi Retrieval Of The Vacuolar Protein Sorting Receptor, Vps10p, Requires The Function Of The Vps29, Vps30, And Vps35 Gene Products, Matthew N. J. Seaman, Eric G. Marcusson, Joan Lin-Cereghino, Scott D. Emr

College of the Pacific Faculty Articles

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The …

Analysis Of Gene Expression During Embryonic Development In Mulberry Silkworm Bombyx Mori, Amit Singh, K. P. Gopinathan

Biology Faculty Publications

We have developed a method for dechorionation and devitellinization of the silkworm eggs without damage, to facilitate the analysis of gene expression during embryonic development of Bombyx mori. Making use of antibodies available from heterologous systems, the spatio-temporal expression patterns of peroxidase and proliferating cell nuclear antigen have been directly visualized in whole mount embryos at various stages of development without the need for generating transformed ·lines carrying specific reporter constructs. The B. mori system, previously unamenable for such studies, could thus serve as an attractive model for molecular analysis of insect development.

The attention lavished on Drosophila melanogaster as …

Effects Of Creatine Supplementation On Muscular Strength And Power Development In Well-Trained Football Players, Josef D. Mcquain

Theses, Dissertations and Capstones

The purpose of this study was to determine the effects of creatine supplementation on strength and power performance in well-trained football players. Twenty-one subjects were randomly assigned to three treatment groups: 1) creatine monohydrate, 2) creatine monohydrate+dextrose, or 3) placebo utilizing a double-blind design. Prior to assignment, subjects were matched according to lean body mass. After a five day loading dose, a maintenance dose was administered for 12 days. Test protocols included three anaerobic work bouts: l) two 30 s Wingate bike tests interspersed with five minutes recovery were used to ascertain relative and absolute power, 2) two 100 yard …

Regulation Of The Expression Of Hmn-Sod At The Level Of The Mitochondrial Processing Of Precursor Hmn-Sod, Gary Leslie Wright

Theses, Dissertations and Capstones

The mitochondrion imports and processes the vast majority of the proteins which comprise its structural elements and metabolic pathways. Studies in the last decade have been successful at defining the mechanism and protein machinery responsible for recognizing, importing, and processing cytosolic precursor proteins into the mature mitochondrial proteins. Little is known, however, about physiological factors which might influence or compromise mitochondrial protein processing. The objective of this work was to develop a novel approach to study mitochondrial import and assembly of precursor proteins as a process within the context of the cellular environment. To these ends we employed the baculovirus …

In Vivo Incorporation Of Tritium To Measure Lipogenesis In Red Skeletal Muscle: Significance Of A Nonlipogenic Diet In La/N-Fa(K)("Corpulent") And Zucker Fa Rat Strains, Karin Traci Mann

Theses, Dissertations and Capstones

Skeletal muscle lipogenesis in rodents may equal that of liver but its importance in the obese animals is not known. We evaluated the rate of fatty acid and triglyceride synthesis and storage content in red skeletal muscles, liver, inguinal fat, and retroperitoneal fat of the LA/N-fa^k (“corpulent”) and Zucker fa obese rat strains. Obese-lean pairs of both rat strains, weaned at 32 days, fed a nonlipogenic diet (54% starch for 21 days) were either dosed with 2 µCi 3H-H² O per gram of body weight and tested after 60 minutes for whole body 3H-incorporation into fatty acids and …

The Tadpole Of Pseudis Minuta (Anura: Pseudidae), An Apparent Case Of Heterochrony, Rafael O. De Sá, Esteban O. Lavilla

Biology Faculty Publications

The external morphology, oral disc, and coloration pattern of the larva of Pseudis minuta are described. Tadpoles are mostly bottom dwellers that have a small, terminal oral disc with a labial tooth row formula 1(1-1)/(1-1)2. Internal oral anatomy is characterized using scanning electron microscopy, representing the only description available for the family Pseudidae. The coloration pattern of P. minuta tadpoles is compared with that of other pseudids. Pseudis minuta larvae do not exhibit the ontogenetic coloration change reported for P. paradoxa. Considering larval coloration, larval size, and reports of adult size, acceleration or hypermorphosis are suggested as possible heterochronic mechanisms …

The Effects Of Prophylatic Protocols Of Exercise And Tamoxifen On The Induction And Growth Of Estrogen Dependent And Independent Rat Mammary Tumors, Robert H. Doepke Jr.

Morehead State Theses and Dissertations

A thesis presented to the faculty of the College of Science and Technology at Morehead State University in partial fulfillment of the requirements for the Degree of Master of Science in Biology by Robert H. Doepke, Jr. on May 6,1996.

Reversal Of Cell Fate Determination In Caenorhabditis Elegans Vulval Development, Susan Euling, Victor Ambros

Dartmouth Scholarship

In Caenorhabditis elegans, the fates of the multipotent vulval precursor cells (VPCs) are specified by intercellular signals, The VPCs divide in the third larval stage (L3) of the wild type, producing progeny of determined cell types, In lin-28 mutants, vulva development is similar to wild-type vulva development except that it occurs precociously, in the second larval stage (L2), Consequently, when lin-28 hermaphrodites temporarily arrest development at the end of L2 in the dauer larva stage, these otherwise determined VPC progeny become reprogrammed back to the multipotent, signal- sensitive state of VPCs. Our results indicate that VPC fate determination by intercellular …

The Evaluation Of Succinylcholine Chloride For Harmful Effects Upon Early-Stage Mouse Embryos, Francis Warren Prescott

Biological Sciences Theses & Dissertations

Succinylcholine (SCh) chloride is a depolarizing neuromuscular blocking agent used to paralyze patients for surgical procedures. Over 1,800 clinical observations of newborn infants have demonstrated that succinylcholine does not cross the placenta in quantities detrimental to fetuses (Kolstad, et al., 1957, Bakhoum, et al., 1957, Hodges, et al., 1959, Duffield, et al., 1958, Dennis, et al., 1954, 1956, McNab, 1955, Coleman, et al., 1956, Lund, 1953). Later investigations which involved comparison of blood levels of SCh in the mother to those in fetuses bolstered the finding that SCh fails to cross the placenta in significant amounts (Moya and Kvisselgaard, 1961, …

Use Of The (3)H-Tetracycline Rat Model In The Study Of Skeletal Compartmentalization And Metabolism Of Calcium In The Maturing Male And Female Rat And The Role Of The Skeleton In Calcium Homeostasis In The Adolescent Male Rat, Darrin Lee Demoss

Theses, Dissertations and Capstones

The (3)H-tetracycline bone labeling procedure was employed to monitor bone resorption from urinary loss curves in male and female rats of various ages. In addition, whole body dry skeletal mass and the loss of (3)H-tetracycline from individual bones was determined. It was found that the dry skeletal mass/body mass ratio of 24-week-old females was 30-40% greater than that for males. The urinary loss of (3)H-tetracycline was described by a double exponential equation indicating the presence of two distinct and independent exchangeable bone compartments. Both compartments decrease in size with age, but their label loss activities were different. The label loss …

Características De La Osificación Craneal En Phyllomedusa Boliviana (Anura: Hylidae), Rafael O. De Sá, E. O. Lavilla

Biology Faculty Publications

RESUMEN. Dentro de la familia Hylidae disponemos de información sobre las secuencias de osificación craneal para menos del 4.0% de sus miembros. El presente trabajo describe la secuencia de osificación craneal de Phyllomedusa boliviana. El patrón general de osificación es similar a lo reportado para otras especies de la familia, pero existen algunas diferencias importantes tales como la osificación tardía de los próoticos y la osificación premetamórfica de neopalatino, vómer, cuadrado-yugal, pterigoides y columela. El lento desarrollo ulterior de la osificación resulta en juveniles con cráneos extensamente cartilaginosos.

ABSTRACT. Characteristics of the cranial ossification in Phyllomedusa …

Mutations In Rad27 Define A Potential Link Between G₁ Cyclins And Dna Replication, Elizabeth Ann Vallen, Frederick R. Cross , '78

Biology Faculty Works

The yeast Saccharomyces cerevisiae has three G1 cyclin (CLN) genes with overlapping functions. To analyze the functions of the various CLN genes, we examined mutations that result in lethality in conjunction with loss of cln1 and cln2. We have isolated alleles of RAD27/ERC11/YKL510, the yeast homolog of the gene encoding flap endonuclease 1, FEN-1.cln1 cln2 rad27/erc11 cells arrest in S phase; this cell cycle arrest is suppressed by the expression of CLN1 or CLN2 but not by that of CLN3 or the hyperactive CLN3-2. rad27/erc11 mutants are also defective in DNA damage repair, as determined by their increased sensitivity to …

Enhancer Trap Technique: A Novel Tool For Identification And Developmental Characterization Of Genes Of Drosophila, Amit Singh

Biology Faculty Publications

The classical technique of mutational screen for identification of genes controlling early development has now approached saturation. A new era in genetic identification and developmental characterization of genes in Drosophila has commenced with the advent of the enhancer trap technique. This technique involves mobilization of a P-lacZ vector to diverse chromosomal locations in the fruit fly genome to bring it under the regulation of developmentally expressed genes or their enhancer elements. The technique offers a strikingly elegant method of gaining entry into fruit fly genes.

An Improved Method For Chemical Devitellinization Of X-Gal Stained Drosophila Embryos, Amit Singh, Madhuri Kango-Singh, P. Sinha

Biology Faculty Publications

In Drosophila developmental biological studies, X-gal staining is commonly employed to study the spatio-temporal expression of the lacZ reporter gene in the transformed flies or their embryos. Study of the lacZ pattern in embryos often suffers from the lack of an efficient and high yieldirrg technique for devitellinization of X-gal stained embryos. Devitellinization techniques employed during antibody staining, in situ hybridization or embryonic cuticular preparations generally do not give satisfactory results when used for similar purpose in X-gal stained embryos. This results in the flaky appearance of the blue stain. We present here an improved chemical devitellinization technique which gives …

Geographic And Nongeographic Variation In The Genus Microtus (Mammalia: Rodentia) In West Virginia, Tina M. Savage

Theses, Dissertations and Capstones

Geographic and nongeographic variation was assessed on 12 cranial and 4 external characters in approximately 400 specimens of Microtus (M. ochrogaster, M. chrotorrhinus, M. pinetorum, M. pennsylvanicus) in West Virginia. Univariate and multivariate statistical analyses were used to examine age-related variation, sexual dimorphism, elevation and geographic variation and litter size for each species of Microtus in the state. Cranial characters were more beneficial than external in detecting variation. Examination of data for each species of Microtus studied demonstrated that values and trends in variation is a specific rather than a generic trait. Age-related variation was present in some, …

A New Model Species For Cultured Teleost Retinal Cell Studies: Light And Scanning Electron Microscopy, Lori Ann Steinfeldt

Undergraduate Honors Capstone Projects

Purpose: To examine retinal horizontal and bipolar cells cultured from goldfish (Carassius auratus), black crappie (Pomoxis nigromaculatus) and bluegill (Lepomis macrochirus) on the light and electron microscope levels; and to compare them with cells from a more established model, the wild white bass (Roccus chrysops). Methods: Retinas from all species were dissociated by methods developed with wild white bass (Dowling et al., 1985). Cell type was identified within the first 3 hr and cells were cultured for up to 2 wk in L-15 medium. At various intervals, cells were fixed in glutaraldehyde, …