Medical Cell Biology Commons^™

Tyrosine Phosphorylation Of Villin: Effects On Actin Dynamics, Cell Morphology And Cell Migration, Alok Tomar

Theses and Dissertations (ETD)

Cell migration is a key aspect of many normal and abnormal biological processes, including embryonic development, defense against infections, wound healing, and tumor cell metastasis. In this study we demonstrate that an epithelial cell actin-binding protein, villin, plays a crucial role in the process of cell migration. Overexpression of villin in doxycyline-regulated HeLa Tet-off and MDCK Tet-off cells enhanced cell migration. We further demonstrate that tyrosine phosphorylation of villin by c-src is required for villin-induced cell migration. Previously, we identified four tyrosine phosphorylation sites in the amino-terminal domain of villin. I further identified six new sites in the carboxylterminal region …

Combined Effects Of Aldehyde Dehydrogenase Variants And Maternal Mitochondrial Genes On Alcohol Consumption, Yedy Israel, Maria E. Quintanilla, Amalia Sapag, Lutske Tampier

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Two lines of rats bred to differ in their voluntary alcohol consumption — the alcohol-abstaining UChA rats and the alcohol-drinking UChB rats — differ in how effectively toxic acetaldehyde is removed during alcohol metabolism. UChB animals carry efficient variants of the aldehyde dehydrogenase 2 (ALDH2) genes and have active mitochondria, resulting in fast removal of acetaldehyde. UChA animals, in contrast, carry less efficient ALDH2 variants and less active mitochondria, which result in transient elevations of acetaldehyde levels after alcohol ingestion. Cross-breeding studies have demonstrated that the presence of active mitochondria inherited from UChB females can fully abolish the reduction of …

Nucleolin Is Required For Rna Polymerase I Transcription In Vivo, Brendan Rickards, S. Flint, Michael D. Cole, Gary Leroy

Dartmouth Scholarship

Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed …

Soluble Amyloid Oligomers Increase Bilayer Conductance By Altering Dielectric Structure, Yuri Sokolov, J. Ashot Kozak, Rakez Kayed, Alexandr Chanturiya, Charles G. Glabe, James E. Hall

Neuroscience, Cell Biology & Physiology Faculty Publications

The amyloid hypothesis of Alzheimer's toxicity has undergone a resurgence with increasing evidence that it is not amyloid fibrils but a smaller oligomeric species that produces the deleterious results. In this paper we address the mechanism of this toxicity. Only oligomers increase the conductance of lipid bilayers and patch-clamped mammalian cells, producing almost identical current-voltage curves in both preparations. Oligomers increase the conductance of the bare bilayer, the cation conductance induced by nonactin, and the anion conductance induced by tetraphenyl borate. Negative charge reduces the sensitivity of the membrane to amyloid, but cholesterol has little effect. In contrast, the area …

Binding Of Internalized Receptors To The Pdz Domain Of Gipc/Synectin Recruits Myosin Vi To Endocytic Vesicles, Samia N. Naccache, Tama Hasson, Arie Horowitz

Dartmouth Scholarship

Myosin VI (myo6) is the only actin-based molecular motor that translocates along actin filaments toward the minus end. Myo6 participates in two steps of endocytic trafficking; it is recruited to both clathrin-coated pits and to ensuing uncoated endocytic vesicles (UCV). Although there is evidence suggesting that the PDZ adaptor protein GIPC/synectin is involved in the association of myo6 with UCV, the recruitment mechanism is unknown. We show that GIPC/synectin is required for both internalization of cell surface receptors and for coupling of myo6 to UCV. This coupling occurs via a mechanism wherein engagement of the GIPC/synectin PDZ domain by C …

Chronic-Alcohol Exposure Alters Igf1 Signaling In H9c2 Cells Via Changes In Pkc Delta, Richard Ila, Michele Solem

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Previously, we have demonstrated that chronic-alcohol exposure alters insulin-like growth factor 1 (IGF1) signaling in adult rat heart cells. This report examines the effects of alcohol in vitro on the expression of protein kinase C (PKC) alpha, delta, and epsilon using the embryonic heart cell line, H9c2, and how this may be linked to changes in IGF1 signal transduction. Western blot analyses of H9c2 protein preparations demonstrate that there are significant increases in the total protein levels of PKC delta and epsilon after 4 days exposure to alcohol, and similar increases were found after 2 and 6 days exposure. In …

Combining An Autologous Peripheral Nervous System “Bridge” And Matrix Modification By Chondroitinase Allows Robust, Functional Regeneration Beyond A Hemisection Lesion Of The Adult Rat Spinal Cord, John D. Houle, Veronica J. Tom, Debra A. Mayes, Gail Wagoner, Napoleon Phillips, Jerry Silver

Neuroscience, Cell Biology & Physiology Faculty Publications

Chondroitinase-ABC (ChABC) was applied to a cervical level 5 (C5) dorsal quadrant aspiration cavity of the adult rat spinal cord to degrade the local accumulation of inhibitory chondroitin sulfate proteoglycans. The intent was to enhance the extension of regenerated axons from the distal end of a peripheral nerve (PN) graft back into the C5 spinal cord, having bypassed a hemisection lesion at C3. ChABC-treated rats showed (1) gradual improvement in the range of forelimb swing during locomotion, with some animals progressing to the point of raising their forelimb above the nose, (2) an enhanced ability to use the forelimb in …

Antioxidant Enzyme Gene Delivery To Protect From Hiv-1 Gp120-Induced Neuronal Apoptosis, Lokesh Agrawal, Jean-Pierre Louboutin, Beverly A.S. Reyes, Elisabeth J. Van Bockstaele, David S. Strayer

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Human immunodeficiency virus-1 (HIV-1) infection in the central nervous system (CNS) may lead to neuronal loss and progressively deteriorating CNS function: HIV-1 gene products, especially gp120, induce free radical-mediated apoptosis. Reactive oxygen species (ROS), are among the potential mediators of these effects. Neurons readily form ROS after gp120 exposure, and so might be protected from ROS-mediated injury by antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD1) and/or glutathione peroxidase (GPx1). Both enzymes detoxify oxygen free radicals. Because they are highly efficient gene delivery vehicles for neurons, recombinant SV40-derived vectors were used for these studies. Cultured mature neurons derived from NT2 cells …

Quantifying Gene Network Connectivity In Silico: Scalability And Accuracy Of A Modular Approach, Nirupama Yalamanchili, Daniel E. Zak, Babatunde A. Ogunnaike, James S. Schwaber, Andres Kriete, Boris N. Kholodenko

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Large, complex data sets that are generated from microarray experiments, create a need for systematic analysis techniques to unravel the underlying connectivity of gene regulatory networks. A modular approach, previously proposed by Kholodenko and co-workers, helps to scale down the network complexity into more computationally manageable entities called modules. A functional module includes a gene's mRNA, promoter and resulting products, thus encompassing a large set of interacting states. The essential elements of this approach are described in detail for a three-gene model network and later extended to a ten-gene model network, demonstrating scalability. The network architecture is identified by analysing …

Systems Analysis Of Circadian Time-Dependent Neuronal Epidermal Growth Factor Receptor Signaling, Daniel E. Zak, Haiping Hao, Rajanikanth Vadigepalli, Gregory M. Miller, Babatunde A. Ogunnaike, James S. Schwaber

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background

Identifying the gene regulatory networks governing physiological signal integration remains an important challenge in circadian biology. Epidermal growth factor receptor (EGFR) has been implicated in circadian function and is expressed in the suprachiasmatic nuclei (SCN), the core circadian pacemaker. The transcription networks downstream of EGFR in the SCN are unknown but, by analogy to other SCN inputs, we expect the response to EGFR activation to depend on circadian timing.

Results

We have undertaken a systems-level analysis of EGFR circadian time-dependent signaling in the SCN. We collected gene-expression profiles to study how the SCN response to EGFR activation depends on …

Systems Analysis Of Circadian Time-Dependent Neuronal Epidermal Growth Factor Receptor Signaling, Daniel E. Zak, Haiping Hao, Rajanikanth Vadigepalli, Gregory M. Miller, Babatunde A. Ogunnaike, James S. Schwaber

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background

Identifying the gene regulatory networks governing physiological signal integration remains an important challenge in circadian biology. Epidermal growth factor receptor (EGFR) has been implicated in circadian function and is expressed in the suprachiasmatic nuclei (SCN), the core circadian pacemaker. The transcription networks downstream of EGFR in the SCN are unknown but, by analogy to other SCN inputs, we expect the response to EGFR activation to depend on circadian timing.

Results

We have undertaken a systems-level analysis of EGFR circadian time-dependent signaling in the SCN. We collected gene-expression profiles to study how the SCN response to EGFR activation depends on …

A Universal Reference Sample Derived From Clone Vector For Improved Detection Of Differential Gene Expression, Rishi L. Khan, Gregory E. Gonye, Guang Gao, James S. Schwaber

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background

Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs^TM.

Results

We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable …

Mixed Germ Cell Sex Cord-Stromal Tumors Of The Testis And Ovary. Morphological, Immunohistochemical, And Molecular Genetic Study Of Seven Cases, Michal Michal, Tomas Vanacek, Radek Sima, Petr Mukensnabl, Ondrej Hes, Dmitry V. Kazakov, Jozef Matoska, Anna Zuntova, Vladimir Dvorak, Alexander Talerman

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

We present the morphological, immunohistochemical, and molecular genetic features of three cases of testicular and four cases of ovarian mixed germ cell sex cord-stromal tumors (MGSCT). The germ cells in the testicular MGSCTs morphologically differed from those in classical seminomas by lacking the typical "square off" quality of the nuclei. In contrast to the nuclei in classical seminomas, their size in testicular MGSCTs was smaller and nucleoli were inconspicuous and the cytoplasm was Periodic Acid-Schiff(PAS) negative. Quite on the contrary, the variability in the size of the nuclei of the germ cells in the testicular MGSCTs was more similar to …

The Molecular Portraits Of Breast Tumors Are Conserved Acress Microarray Platforms, Zhiyuan Hu, Cheng Fan, Daniel S. Oh, J. S. Marron, Xiaping He, Bahjat F. Qaqish, Chad Livasy, Lisa A. Carey, Evangeline Reynolds, Lynn Dressler, Andrew Nobel, Joel Parker, Matthew G. Ewend, Lynda R. Sawyer, Junyuan Wu, Yudong Liu, Rita Nanda, Maria Tretiakova, Alejandra Ruiz Orrico, Donna Dreher, Juan P. Palazzo, Laurent Perreard, Edward Nelson, Mary Mone, Heidi Hansen, Michael Mullins, John F. Quackenbush, Matthew J. Ellis, Olufunmilayo I. Olopade, Philip S. Bernard, Charles M. Perou

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background

Validation of a novel gene expression signature in independent data sets is a critical step in the development of a clinically useful test for cancer patient risk-stratification. However, validation is often unconvincing because the size of the test set is typically small. To overcome this problem we used publicly available breast cancer gene expression data sets and a novel approach to data fusion, in order to validate a new breast tumor intrinsic list.

Results

A 105-tumor training set containing 26 sample pairs was used to derive a new breast tumor intrinsic gene list. This intrinsic list contained 1300 genes …

Scientific Issues Related To The Cytology Proficiency Testing Regulations, George Birdsong, Lydia Howell, Karen Atkinson, R. Marshall Austin, Marluce Bibbo, Thomas A. Bonfiglio, Diane D. Davey, Catherine Keebler, Dina Mody, Lynnette Savaloja, Jacalyn Papillo, Marianne Prey, Stephen Raab, Brenda L. Schultz, Diane Solomon

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The member organizations of the Cytology Education and Technology Consortium believe there are significant flaws in current cytology proficiency testing regulations. The most immediate needed modifications include lengthening the required testing interval, utilizing stringently validated and continuously monitored slides, changing the grading scheme, and changing the focus of the test from the individual to laboratory level testing. Integration of new computer-assisted and located-guided screening technologies into the testing protocols is necessary for the testing protocol to be compliant with the law.

Voltage-Dependent Gating Rearrangements In The Intracellular T1-T1 Interface Of A K+ Channel., Guangyu Wang, Manuel Covarrubias

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The intracellular tetramerization domain (T1) of most eukaryotic voltage-gated potassium channels (Kv channels) exists as a "hanging gondola" below the transmembrane regions that directly control activation gating via the electromechanical coupling between the S4 voltage sensor and the main S6 gate. However, much less is known about the putative contribution of the T1 domain to Kv channel gating. This possibility is mechanistically intriguing because the T1-S1 linker connects the T1 domain to the voltage-sensing domain. Previously, we demonstrated that thiol-specific reagents inhibit Kv4.1 channels by reacting in a state-dependent manner with native Zn(2+) site thiolate groups in the T1-T1 interface; …

Cell-Signalling Dynamics In Time And Space, Boris N. Kholodenko Phd, Dsci

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

The specificity of cellular responses to receptor stimulation is encoded by the spatial and temporal dynamics of downstream signalling networks. Computational models provide insights into the intricate relationships between stimuli and responses and reveal mechanisms that enable networks to amplify signals, reduce noise and generate discontinuous bistable dynamics or oscillations. These temporal dynamics are coupled to precipitous spatial gradients of signalling activities, which guide pivotal intracellular processes, but also necessitate mechanisms to facilitate signal propagation across a cell.

Trading The Micro-World Of Combinatorial Complexity For The Macro-World Of Protein Interaction Domains, Nikolay M. Borisov, Nick I. Markevitch, Jan B. Hoek, Boris N. Kholodenko

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Membrane receptors and proteins involved in signal transduction display numerous binding domains and operate as molecular scaffolds generating a variety of parallel reactions and protein complexes. The resulting combinatorial explosion of the number of feasible chemical species and, hence, different states of a network greatly impedes mechanistic modeling of signaling systems. Here we present novel general principles and identify kinetic requirements that allow us to replace a mechanistic picture of all possible micro-states and transitions by a macro-description of states of separate binding sites of network proteins. This domain-oriented approach dramatically reduces computational models of cellular signaling networks by dissecting …

A Domain-Oriented Approach To The Reduction Of Combinatorial Complexity In Signal Transduction Networks, Holger Conzelmann, Julio Saez-Rodriguez, Thomas Sauter, Boris N. Kholodenko Phd, Dsci, Ernst D. Gilles

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background:

Receptors and scaffold proteins possess a number of distinct domains and bind multiple partners. A common problem in modeling signaling systems arises from a combinatorial explosion of different states generated by feasible molecular species. The number of possible species grows exponentially with the number of different docking sites and can easily reach several millions. Models accounting for this combinatorial variety become impractical for many applications.

Results:

Our results show that under realistic assumptions on domain interactions, the dynamics of signaling pathways can be exactly described by reduced, hierarchically structured models. The method presented here provides a rigorous way to …

The Inflammatory And Normal Transcriptome Of Mouse Bladder Detrusor And Mucosa, Marcia R. Saban, Helen L. Hellmich, Mary Turner, Ngoc-Bich Nguyen, Rajanikanth Vadigepalli, David W. Dyer, Robert E. Hurst, Michael Centola, Ricardo Saban

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Background

An organ such as the bladder consists of complex, interacting set of tissues and cells. Inflammation has been implicated in every major disease of the bladder, including cancer, interstitial cystitis, and infection. However, scanty is the information about individual detrusor and urothelium transcriptomes in response to inflammation. Here, we used suppression subtractive hybridizations (SSH) to determine bladder tissue- and disease-specific genes and transcriptional regulatory elements (TRE)s. Unique TREs and genes were assembled into putative networks.

Results

It was found that the control bladder mucosa presented regulatory elements driving genes such as myosin light chain phosphatase and calponin 1 that …

Potential Of Vibrational Spectroscopy In The Diagnosis Of Human Tumours., Eoghan O'Faolain

Doctoral

Just fewer than 20,000 people are annually diagnosed with some form of cancer in Ireland and one in three people are likely to contract some form of cancer by age 74. With the number of cases increasing at an annual rate of 2%, the early detection and treatment of cancer is becoming increasingly important. Both IR and Raman spectroscopy offer the potential for real time, quantitative detection of cancer and even precancer. This study investigates the potential of Raman and Fourier transform infrared, both benchtop and synchrotron spectroscopies for the detection of cervical cancer. The tissue was classified and its …

Decreased Intracellular Zinc In Human Tumorigenic Prostate Epithelial Cells: A Possible Role In Prostate Cancer Progression, Liping Huang, Catherine P. Kirschke, Yunfan Zhang

Wayne State University Associated BioMed Central Scholarship

Abstract

Background

Zinc plays important roles in maintaining normal function of the prostate and in development of prostate malignancy. It has been demonstrated that prostate malignant epithelial cells contain much less cellular zinc than the surrounding normal epithelial cells. However, the pathway(s) which leads to lower zinc accumulation in malignant prostate epithelial cells is poorly understood. In this study, the zinc homeostatic features of two human prostate epithelial cell lines (non-tumorigenic, RWPE1, and tumorigenic, RWPE2) were investigated. Effects of over-expression of ZIP1 in RWPE2 on cell proliferation and apoptosis were also studied.

Results

RWPE2 accumulated less intracellular zinc than RWPE1 …

Protein Kinase C Activity In Mouse Eggs Regulates Gamete Membrane Interaction, Hiroto Akabane

Theses, Dissertations and Capstones

Every mammalian life develops from one cell after fertilization of an egg by the sperm. The molecular pathways governing this event are still poorly understood. Numerous reports indicate that mammalian eggs highly express various protein kinase C (PKC) isoforms. Accordingly, we hypothesize that PKC activity in the egg plays an important role during egg-sperm membrane binding and fusion. In this study, we tested our hypothesis in mouse gametes using two types of PKC inhibitors (calphostin c and staurosporine) and the typical PKC activator, phorbol ester 12-tetradecanoylphorbol-13 acetate (PMA). After treatment with the individual drug, eggs were inseminated with sperm. The …

Regulation Of Biofilm Formation Of Pseudomonas Aeruginosa, Nathaniel Edwards Head

Theses, Dissertations and Capstones

Cystic fibrosis (CF) is the most common, autosomal recessive lethal genetic disease in the Caucasian population, resulting from a malfunctioned cystic fibrosis transmembrane conductance regulator (CFTR) and leading to bacterial lung infections. P. aeruginosa, an opportunistic pathogen, establishes a chronic infection in CF with a phenotype of overproduction of an exopolysaccharide (alginate) due to host-directed mutagenesis. While free-floating planktonic bacteria can be properly cleared from the CF lung, P. aeruginosa, along with alginate production, establishes an infection in the form of a biofilm which supports its survival in nature and in vivo. As a result, genomic structure, …

Smc3 Knockdown Triggers Genomic Instability And P53-Dependent Apoptosis In Human And Zebrafish Cells., Giancarlo Ghiselli

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: The structural maintenance of chromosome 3 (SMC3) protein is a constituent of a number of nuclear multimeric protein complexes that are involved in DNA recombination and repair in addition to chromosomal segregation. Overexpression of SMC3 activates a tumorigenic cascade through which mammalian cells acquire a transformed phenotype. This has led us to examine in depth how SMC3 level affects cell growth and genomic stability. In this paper the effect of SMC3 knockdown has been investigated. RESULTS: Mammalian cells that are SMC3 deficient fail to expand in a clonal population. In order to shed light on the underlying mechanism, experiments …

Classification And Risk Stratification Of Invasive Breast Carcinomas Using A Real-Time Quantitative Rt-Pcr Assay., Laurent Perreard, Cheng Fan, John F Quackenbush, Michael Mullins, Nicholas P Gauthier, Edward Nelson, Mary Mone, Heidi Hansen, Saundra S Buys, Karen Rasmussen, Alejandra Ruiz Orrico, Donna Dreher, Rhonda Walters, Joel Parker, Zhiyuan Hu, Xiaping He, Juan P Palazzo, Olufunmilayo I Olopade, Aniko Szabo, Charles M Perou, Philip S Bernard

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

INTRODUCTION: Predicting the clinical course of breast cancer is often difficult because it is a diverse disease comprised of many biological subtypes. Gene expression profiling by microarray analysis has identified breast cancer signatures that are important for prognosis and treatment. In the current article, we use microarray analysis and a real-time quantitative reverse-transcription (qRT)-PCR assay to risk-stratify breast cancers based on biological 'intrinsic' subtypes and proliferation. METHODS: Gene sets were selected from microarray data to assess proliferation and to classify breast cancers into four different molecular subtypes, designated Luminal, Normal-like, HER2+/ER-, and Basal-like. One-hundred and twenty-three breast samples (117 invasive …

No Vaccine Against Hiv Yet--Are We Not Perfectly Equipped?, Mahender Singh

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Enormous effort has been devoted to the development of a vaccine against human immunodeficiency virus (HIV). But it is proving to be an unprecedented challenge to create an effective vaccine mainly due to the high genetic variability of the virus and the necessity of cytotoxic T lymphocytes (CTL) for containing the infection. Currently pursued vaccine strategies appear to induce CTL in nonhuman primate models but in the early clinical trials, these strategies fail to fully control the viral infection. New strategies that can cover the vast genetic diversity of HIV are needed for the development of a potent vaccine.