Medical Cell Biology Commons^™

One-Step Generation Of Multiple Transgenic Mouse Lines Using An Improved Pronuclear Injection-Based Targeted Transgenesis (I-Pitt)., Masato Ohtsuka, Hiromi Miura, Keiji Mochida, Michiko Hirose, Ayumi Hasegawa, Atsuo Ogura, Ryuta Mizutani, Minoru Kimura, Ayako Isotani, Masahito Ikawa, Masahiro Sato, Channabasavaiah B. Gurumurthy

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system …

Assessment Of Artificial Mirna Architectures For Higher Knockdown Efficiencies Without The Undesired Effects In Mice., Hiromi Miura, Hidetoshi Inoko, Masafumi Tanaka, Hirofumi Nakaoka, Minoru Kimura, Channabasavaiah B. Gurumurthy, Masahiro Sato, Masato Ohtsuka

Journal Articles: Genetics, Cell Biology & Anatomy

RNAi-based strategies have been used for hypomorphic analyses. However, there are technical challenges to achieve robust, reproducible knockdown effect. Here we examined the artificial microRNA (amiRNA) architectures that could provide higher knockdown efficiencies. Using transient and stable transfection assays in cells, we found that simple amiRNA-expression cassettes, that did not contain a marker gene (-MG), displayed higher amiRNA expression and more efficient knockdown than those that contained a marker gene (+MG). Further, we tested this phenomenon in vivo, by analyzing amiRNA-expressing mice that were produced by the pronuclear injection-based targeted transgenesis (PITT) method. While we observed significant silencing of the …

Essential Role Of Caveolin-3 In Adiponectin Signalsome Formation And Adiponectin Cardioprotection., Yajing Wang, Xiaoliang Wang, Jean-François Jasmin, Wayne Bond Lau, Rong Li, Yuexin Yuan, Wei Yi, Kurt Chuprun, Michael P. Lisanti, Walter J Koch, Erhe Gao, Xin-Liang Ma

Department of Emergency Medicine Faculty Papers

OBJECTIVE: Adiponectin (APN) system malfunction is causatively related to increased cardiovascular morbidity/mortality in diabetic patients. The aim of the current study was to investigate molecular mechanisms responsible for APN transmembrane signaling and cardioprotection.

METHODS AND RESULTS: Compared with wild-type mice, caveolin-3 knockout (Cav-3KO) mice exhibited modestly increased myocardial ischemia/reperfusion injury (increased infarct size, apoptosis, and poorer cardiac function recovery; P

CONCLUSIONS: Taken together, these results demonstrated for the first time that Cav-3 plays an essential role in APN transmembrane signaling and APN anti-ischemic/cardioprotective actions.

Temporal Changes In Innate Immune Signals In A Rat Model Of Alcohol Withdrawal In Emotional And Cardiorespiratory Homeostatic Nuclei., Kate Freeman, Anthony Brureau, Rajanikanth Vadigepalli, Mary M Staehle, Melanie M Brureau, Gregory E Gonye, Jan B Hoek, D Craig Hooper, James S Schwaber

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: Chronic alcohol use changes the brain's inflammatory state. However, there is little work examining the progression of the cytokine response during alcohol withdrawal, a period of profound autonomic and emotional upset. This study examines the inflammatory response in the central nucleus of the amygdala (CeA) and dorsal vagal complex (DVC), brain regions neuroanatomically associated with affective and cardiorespiratory regulation in an in vivo rat model of withdrawal following a single chronic exposure.

METHODS: For qRT-PCR studies, we measured the expression of TNF-α, NOS-2, Ccl2 (MCP-1), MHC II invariant chain CD74, and the TNF receptor Tnfrsf1a in CeA and DVC …

Wnt Signaling Exerts An Antiproliferative Effect On Adult Cardiac Progenitor Cells Through Igfbp3., Angelos Oikonomopoulos, Konstantina-Ioanna Sereti, Frank Conyers, Michael Bauer, Annette Liao, Jian Guan, Dylan Crapps, Jung-Kyu Han, Hanhua Dong, Ahmad F Bayomy, Gabriel C Fine, Karen Westerman, Travis L Biechele, Randall T Moon, Thomas Force, Ronglih Liao

Center for Translational Medicine Faculty Papers

RATIONALE: Recent work in animal models and humans has demonstrated the presence of organ-specific progenitor cells required for the regenerative capacity of the adult heart. In response to tissue injury, progenitor cells differentiate into specialized cells, while their numbers are maintained through mechanisms of self-renewal. The molecular cues that dictate the self-renewal of adult progenitor cells in the heart, however, remain unclear.

OBJECTIVE: We investigate the role of canonical Wnt signaling on adult cardiac side population (CSP) cells under physiological and disease conditions.

METHODS AND RESULTS: CSP cells isolated from C57BL/6J mice were used to study the effects of canonical …

The Interplay Between Nf-Kappab And E2f1 Coordinately Regulates Inflammation And Metabolism In Human Cardiac Cells., Xavier Palomer, David Álvarez-Guardia, Mercy M Davidson, Tung O Chan, Arthur M Feldman, Manuel Vázquez-Carrera

Department of Medicine Faculty Papers

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, …

Mitostatin Is Down-Regulated In Human Prostate Cancer And Suppresses The Invasive Phenotype Of Prostate Cancer Cells., Matteo Fassan, Domenico D'Arca, Juraj Letko, Andrea Vecchione, Marina P Gardiman, Peter Mccue, Bernadette Wildemore, Massimo Rugge, Dolores Shupp-Byrne, Leonard G Gomella, Andrea Morrione, Renato V Iozzo, Raffaele Baffa

Kimmel Cancer Center Faculty Papers

MITOSTATIN, a novel putative tumor suppressor gene induced by decorin overexpression, is expressed in most normal human tissues but is markedly down-regulated in advanced stages of mammary and bladder carcinomas. Mitostatin negatively affects cell growth, induces cell death and regulates the expression and activation levels of Hsp27. In this study, we demonstrated that ectopic expression of Mitostatin in PC3, DU145, and LNCaP prostate cancer cells not only induced a significant reduction in cell growth, but also inhibited migration and invasion. Moreover, Mitostatin inhibited colony formation in soft-agar of PC3 and LNCaP cells as well as tumorigenicity of LNCaP cells in …

The Variable Presentations Of Anaplastic Spindle Cell Squamous Carcinoma Associated With Tall Cell Variant Of Papillary Thyroid Carcinoma., Pallavi P Gopal, Kathleen T Montone, Zubair Baloch, Madalina Tuluc, Virginia Livolsi

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: In 1976, Hawk and Hazard described the tall cell variant (TCV) of papillary thyroid carcinoma (PTC). While the lesions they described had cytologic features of papillary carcinoma, they showed more aggressive behavior with a greater propensity for extrathyroid extension and lymphovascular invasion than classic PTC. In 1991, Bronner and LiVolsi described a series of patients with TCV that progressed to spindle cell squamous carcinoma (SCSC), a unique form of anaplastic thyroid carcinoma. This study describes the variable clinical and pathologic presentations in 31 patients with anaplastic SCSC arising in association with TCV.

METHODS: The surgical pathology archives as well …

Tdp-43 Potentiates Alpha-Synuclein Toxicity To Dopaminergic Neurons In Transgenic Mice., Tian Tian, Cao Huang, Jianbin Tong, Ming Yang, Hongxia Zhou, Xugang Xia

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

TDP-43 and α-synuclein are two disease proteins involved in a wide range of neurodegenerative diseases. While TDP-43 proteinopathy is considered a pathologic hallmark of sporadic amyotrophic lateral sclerosis and frontotemporal lobe degeneration, α-synuclein is a major component of Lewy body characteristic of Parkinson's disease. Intriguingly, TDP-43 proteinopathy also coexists with Lewy body and with synucleinopathy in certain disease conditions. Here we reported the effects of TDP-43 on α-synuclein neurotoxicity in transgenic mice. Overexpression of mutant TDP-43 (M337V substitution) in mice caused early death in transgenic founders, but overexpression of normal TDP-43 only induced a moderate loss of cortical neurons in …

Acetate Causes Alcohol Hangover Headache In Rats., Christina R Maxwell, Rebecca Jay Spangenberg, Jan B Hoek, Stephen D Silberstein, Michael L Oshinsky

Department of Neurology Faculty Papers

BACKGROUND: The mechanism of veisalgia cephalgia or hangover headache is unknown. Despite a lack of mechanistic studies, there are a number of theories positing congeners, dehydration, or the ethanol metabolite acetaldehyde as causes of hangover headache.

METHODS: We used a chronic headache model to examine how pure ethanol produces increased sensitivity for nociceptive behaviors in normally hydrated rats.

RESULTS: Ethanol initially decreased sensitivity to mechanical stimuli on the face (analgesia), followed 4 to 6 hours later by inflammatory pain. Inhibiting alcohol dehydrogenase extended the analgesia whereas inhibiting aldehyde dehydrogenase decreased analgesia. Neither treatment had nociceptive effects. Direct administration of acetate …

Human Papillomavirus And Survival Of Patients With Oropharyngeal Cancer., K Kian Ang, Jonathan Harris, Richard Wheeler, Randal Weber, David I Rosenthal, Phuc Felix Nguyen-Tân, William H Westra, Christine H Chung, Richard C Jordan, Charles Lu, Harold Kim, Rita S. Axelrod, Md, C Craig Silverman, Kevin P Redmond, Maura L Gillison

Jefferson Hospital Staff Papers and Presentations

BACKGROUND: Oropharyngeal squamous-cell carcinomas caused by human papillomavirus (HPV) are associated with favorable survival, but the independent prognostic significance of tumor HPV status remains unknown.

METHODS: We performed a retrospective analysis of the association between tumor HPV status and survival among patients with stage III or IV oropharyngeal squamous-cell carcinoma who were enrolled in a randomized trial comparing accelerated-fractionation radiotherapy (with acceleration by means of concomitant boost radiotherapy) with standard-fractionation radiotherapy, each combined with cisplatin therapy, in patients with squamous-cell carcinoma of the head and neck. Proportional-hazards models were used to compare the risk of death among patients with HPV-positive …

Transgenic Rat Model Of Neurodegeneration Caused By Mutation In The Tdp Gene., Hongxia Zhou, Cao Huang, Han Chen, Dian Wang, Carlisle P Landel, Pedro Yuxing Xia, Robert Bowser, Yong-Jian Liu, Xu Gang Xia

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

TDP-43 proteinopathies have been observed in a wide range of neurodegenerative diseases. Mutations in the gene encoding TDP-43 (i.e., TDP) have been identified in amyotrophic lateral sclerosis (ALS) and in frontotemporal lobe degeneration associated with motor neuron disease. To study the consequences of TDP mutation in an intact system, we created transgenic rats expressing normal human TDP or a mutant form of human TDP with a M337V substitution. Overexpression of mutant, but not normal, TDP caused widespread neurodegeneration that predominantly affected the motor system. TDP mutation reproduced ALS phenotypes in transgenic rats, as seen by progressive degeneration of motor neurons …

Accurate Molecular Classification Of Cancer Using Simple Rules., Xiaosheng Wang, Osamu Gotoh

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: One intractable problem with using microarray data analysis for cancer classification is how to reduce the extremely high-dimensionality gene feature data to remove the effects of noise. Feature selection is often used to address this problem by selecting informative genes from among thousands or tens of thousands of genes. However, most of the existing methods of microarray-based cancer classification utilize too many genes to achieve accurate classification, which often hampers the interpretability of the models. For a better understanding of the classification results, it is desirable to develop simpler rule-based models with as few marker genes as possible.

METHODS: …

Role Of Mammalian Ecdysoneless In Cell Cycle Regulation., Jun Hyun Kim, Channabasavaiah B. Gurumurthy, Mayumi Naramura, Ying Zhang, Andrew T. Dudley, Lynn Doglio, Hamid Band, Vimla Band

Journal Articles: Genetics, Cell Biology & Anatomy

The Ecdysoneless (Ecd) protein is required for cell-autonomous roles in development and oogenesis in Drosophila, but the function of its evolutionarily conserved mammalian orthologs is not clear. To study the cellular function of Ecd in mammalian cells, we generated Ecd(lox/lox) mouse embryonic fibroblast cells from Ecd floxed mouse embryos. Cre-mediated deletion of Ecd in Ecd(lox/lox) mouse embryonic fibroblasts led to a proliferative block due to a delay in G(1)-S cell cycle progression; this defect was reversed by the introduction of human Ecd. Loss of Ecd led to marked down-regulation of E2F target gene expression. Furthermore, Ecd directly bound to Rb …

Revealing Genes Associated With Vitellogenesis In The Liver Of The Zebrafish (Danio Rerio) By Transcriptome Profiling., Liraz Levi, Irena Pekarski, Ellen Gutman, Paolo Fortina, Terry Hyslop, Jakob Biran, Berta Levavi-Sivan, Esther Lubzens

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: In oviparous vertebrates, including fish, vitellogenesis consists of highly regulated pathways involving 17beta-estradiol (E2). Previous studies focused on a relatively small number of hepatic expressed genes during vitellogenesis. This study aims to identify hepatic genes involved in vitellogenesis and regulated by E2, by using zebrafish microarray gene expression profiling, and to provide information on functional distinctive genes expressed in the liver of a vitellogenic female, using zebrafish as a model fish. RESULTS: Genes associated with vitellogenesis were revealed by the following paired t-tests (SAM) comparisons: a) two-month old vitellogenic (Vit2) females were compared with non-vitellogenic (NV) females, showing 825 …

Demyelinating And Nondemyelinating Strains Of Mouse Hepatitis Virus Differ In Their Neural Cell Tropism., Jayasri Das Sarma, Kathryn Iacono, Lilli Gard, Ryan Marek, Lawrence C. Kenyon, Michael Koval, Susan R. Weiss

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

Some strains of mouse hepatitis virus (MHV) can induce chronic inflammatory demyelination in mice that mimics certain pathological features of multiple sclerosis. We have examined neural cell tropism of demyelinating and nondemyelinating strains of MHV in order to determine whether central nervous system (CNS) cell tropism plays a role in demyelination. Previous studies demonstrated that recombinant MHV strains, isogenic other than for the spike gene, differ in the extent of neurovirulence and the ability to induce demyelination. Here we demonstrate that these strains also differ in their abilities to infect a particular cell type(s) in the brain. Furthermore, there is …

Multiple Forms Of Atypical Rearrangements Generating Supernumerary Derivative Chromosome 15., Nicholas J Wang, Alexander S Parokonny, Karen N Thatcher, Jennette Driscoll, Barbara M Malone, Naghmeh Dorrani, Marian Sigman, Janine M Lasalle, N Carolyn Schanen

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: Maternally-derived duplications that include the imprinted region on the proximal long arm of chromosome 15 underlie a complex neurobehavioral disorder characterized by cognitive impairment, seizures and a substantial risk for autism spectrum disorders1. The duplications most often take the form of a supernumerary pseudodicentric derivative chromosome 15 [der(15)] that has been called inverted duplication 15 or isodicentric 15 [idic(15)], although interstitial rearrangements also occur. Similar to the deletions found in most cases of Angelman and Prader Willi syndrome, the duplications appear to be mediated by unequal homologous recombination involving low copy repeats (LCR) that are found clustered in the …

Role For Akt3/Protein Kinase Bγ In Attainment Of Normal Brain Size, Rachel M. Easton, Han Cho, Kristin Roovers, Diana W. Shineman

Dartmouth Scholarship

Studies of Drosophila and mammals have revealed the importance of insulin signaling through phosphatidylinositol 3-kinase and the serine/threonine kinase Akt/protein kinase B for the regulation of cell, organ, and organismal growth. In mammals, three highly conserved proteins, Akt1, Akt2, and Akt3, comprise the Akt family, of which the first two are required for normal growth and metabolism, respectively. Here we address the function of Akt3. Like Akt1, Akt3 is not required for the maintenance of normal carbohydrate metabolism but is essential for the attainment of normal organ size. However, in contrast to Akt1^−/− mice, which display a …

Palm Is Expressed In Both Developing And Adult Mouse Lens And Retina., Meryl Castellini, Louise V Wolf, Bharesh K Chauhan, Deni S Galileo, Manfred W Kilimann, Ales Cvekl, Melinda K Duncan

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

BACKGROUND: Paralemmin (Palm) is a prenyl-palmitoyl anchored membrane protein that can drive membrane and process formation in neurons. Earlier studies have shown brain preferred Palm expression, although this protein is a major water insoluble protein in chicken lens fiber cells and the Palm gene may be regulated by Pax6. METHODS: The expression profile of Palm protein in the embryonic, newborn and adult mouse eye as well as dissociated retinal neurons was determined by confocal immunofluorescence. The relative mRNA levels of Palm, Palmdelphin (PalmD) and paralemmin2 (Palm2) in the lens and retina were determined by real time rt-PCR. RESULTS: In the …

Cell-Cycle Regulatory Proteins In Podocyte Cell In Idiopathic Nephrotic Syndrome Of Childhood., Tarak Srivastava, Robert E. Garola, J M. Whiting, Uri S. Alon

Manuscripts, Articles, Book Chapters and Other Papers

BACKGROUND: The podocyte cell is believed to play an important role in idiopathic nephrotic syndrome (INS) of childhood. In adults with cellular and collapsing focal segmental glomerulosclerosis (FSGS), the expression of cell-cycle regulatory proteins such as p27, p57, and cyclin D is decreased and expression of cyclin A, Ki-67, and p21 is observed in podocyte cells suggestive of a dysregulated podocyte phenotype. We investigated for alterations in the expression of cyclin kinase inhibitors, p27, p57, p21, and cyclins D and A in the podocyte cell of children with INS.

METHODS: Forty-two kidney biopsies were investigated; 14 with minimal-change disease (MCD), …

Eliciting The Low-Activity Aldehyde Dehydrogenase Asian Phenotype By An Antisense Mechanism Results In An Aversion To Ethanol., E Garver, Tu Gc, Q N Cao, M Aini, F Zhou, Y Israel

Department of Pathology, Anatomy, and Cell Biology Faculty Papers

A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 …

Aldosterone Stimulates K Secretion Across Mammalian Colon Independent Of Na Absorption, Gerhard Rechkemmer, Dan R. Halm

Neuroscience, Cell Biology & Physiology Faculty Publications

K transport across guinea pig (Cavia porcellus) distal colon was measured in vitro using isotopically determined unidirectional fluxes. Aldosterone stimulated electrogenic Na absorption, as measured by amiloride-sensitive short-circuit current (I_sc), and reduced net K absorption from +2.5 ± 0.2 µEq/cm² per hr to +0.8 ± 0.3 µEq/cm² per hr (mean ± SEM). Amiloride addition to the mucosal solution did not enhance net K absorption, as expected if inhibiting active Na absorption would reduce active K secretion as in the distal nephron. The amiloride-insensitive I_sc was -1.0 ± 0.2 µEq/cm² per hr (mean ± SEM) …

Rat Dendritic Cells Function As Accessory Cells And Control The Production Of A Soluble Factor Required For Mitogenic Responses Of T Lymphocytes., Wolfgang E. Klinkert, Jon H. Labadie, James P. O'Brien, Carl F. Beyer, William E. Bowers

Dartmouth Scholarship

Transformation of T lymphocytes, induced by treatment with periodate or with neuraminidase plus galactose oxidase, requires the participation of accessory cells. Procedures were developed for the fractionation of rat lymph node cells, by which most of the lymphocytes can be recovered as a major population of cells that do not respond to mitogenic stimulation unless accessory cells from a separated minor population are added. Further purification led to a 1000-fold overall increase in accessory activity per cell, with a 50-70% yield. The purest preparations were virtually free of macrophages and contained more than 90% typical dendritic cells. Maximum responses occurred …