Fluorescein-labeled tyramide strongly enhances the detection of low bromodeoxyuridine incorporation levels.
AuthorsVan Heusden J, de Jong P, Ramaekers F, Bruwiere H, Borgers M, Smets G
JournalJ Histochem Cytochem
PubMed ID9016320
'Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of ... More
Subcellular and cell-cycle expression profiles of CDK-inhibitors in normal differentiating myeloid cells.
AuthorsYaroslavskiy B, Watkins S, Donnenberg AD, Patton TJ, Steinman RA
JournalBlood
PubMed ID10216085
'A central question in hematopoiesis is how cell-cycling behavior changes during the emergence of the differentiated state. To further understand what genetic regulators might couple proliferation status to differentiation, we studied the expression of the cell-cycle inhibitors p21 and p27 during the in vitro differentiation of normal CD34(+) blast cells ... More
Retinal ganglion cell genesis requires lakritz, a Zebrafish atonal Homolog.
AuthorsKay JN, Finger-Baier KC, Roeser T, Staub W, Baier H
JournalNeuron
PubMed ID11430806
'Mutation of the zebrafish lakritz (lak) locus completely eliminates the earliest-born retinal cells, the ganglion cells (RGCs). Instead, excess amacrine, bipolar, and Müller glial cells are generated in the mutant. The extra amacrines are found at ectopic locations in the ganglion cell layer. Cone photoreceptors appear unaffected by the mutation. ... More
Immunoseparation and immunodetection of nucleic acids labeled with halogenated nucleotides.
AuthorsHaider SR, Juan G, Traganos F, Darzynkiewicz Z
JournalExp Cell Res
PubMed ID9260920
'A novel methodology for labeling, isolation, and detection of nucleic acids is described. Nucleic acid isolation is based on in vivo or in vitro incorporation of BrU or BrdU to either RNA or DNA, respectively, followed by immunoprecipitation of the labeled nucleic acid utilizing anti-BrdU MoAb, which crossreacts with BrU, ... More
Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques.
AuthorsGratzner HG, Pollack A, Ingram DJ, Leif RC
JournalJ Histochem Cytochem
PubMed ID815428
'Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific ... More
Dominant-negative C/EBP disrupts mitotic clonal expansion and differentiation of 3T3-L1 preadipocytes.
AuthorsZhang JW, Tang QQ, Vinson C, Lane MD
JournalProc Natl Acad Sci U S A
PubMed ID14688407
'Hormonal induction of growth-arrested 3T3-L1 preadipocytes rapidly activates expression of CCAAT/enhancer-binding protein (C/EBP) beta. Acquisition of DNA-binding activity by C/EBPbeta, however, is delayed until the cells synchronously enter the S phase of mitotic clonal expansion (MCE). After MCE, C/EBPbeta activates expression of C/EBPalpha and peroxisome proliferator-activated receptor gamma, which then ... More
A sensitive non-isotopic assay specific for HIV-1 associated reverse transcriptase.
AuthorsPorstmann T, Meissner K, Glaser R, Döpel SH, Sydow G
JournalJ Virol Methods
PubMed ID1713913
'A sensitive non-isotopic assay for specific detection of reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is described using 5-bromo-2''-deoxyuridine triphosphate (BrdUTP) instead of tritiated thymidine triphosphate. After the RT reaction the template primer is degraded by alkaline hydrolysis. Single-stranded poly.(BrdU) is detected in an immunoenzymometric assay ... More
A sensitive assay for the quantification of reverse transcriptase activity based on the use of carrier-bound template and non-radioactive-product detection, with special reference to human-immunodeficiency-virus isolation.
'A non-radioactive 96-well microtitre plate reverse transcriptase (RT) assay, based on the use of covalently bound riboadenosine homopolymer in the wells and 5-bromodeoxyuridined 5''-triphosphate (BrdUTP) as dNTP, is described. The whole assay is performed in a single well, including the quantitative detection of incorporated BrdU, which is performed immunologically using ... More
The transcription cycle of RNA polymerase II in living cells.
AuthorsKimura H, Sugaya K, Cook PR
JournalJ Cell Biol
PubMed ID12473686
'RNA polymerase II transcribes most eukaryotic genes. Its catalytic subunit was tagged with green fluorescent protein and expressed in Chinese hamster cells bearing a mutation in the same subunit; it complemented the defect and so was functional. Photobleaching revealed two kinetic fractions of polymerase in living nuclei: approximately 75% moved ... More
The use of antibody specific for bromodeoxyuridine for the immunofluorescent determination of DNA replication in single cells and chromosomes.
AuthorsGratzner HG, Leif RC, Ingram DJ, Castro A
JournalExp Cell Res
PubMed ID811484
Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors.
AuthorsNieto M, Schuurmans C, Britz O, Guillemot F
JournalNeuron
PubMed ID11239431
We have addressed the role of the proneural bHLH genes Neurogenin2 (Ngn2) and Mash1 in the selection of neuronal and glial fates by neural stem cells. We show that mice mutant for both genes present severe defects in development of the cerebral cortex, including a reduction of neurogenesis and a ... More
Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons.
AuthorsMizuguchi R, Sugimori M, Takebayashi H, Kosako H, Nagao M, Yoshida S, Nabeshima Y, Shimamura K, Nakafuku M
JournalNeuron
PubMed ID11567615
Distinct classes of neurons are generated at defined times and positions during development of the nervous system. It remains elusive how specification of neuronal identity coordinates with acquisition of pan-neuronal properties. Here we show that basic helix-loop-helix (bHLH) transcription factors Olig2 and Neurogenin2 (Ngn2) play vital roles in the coordinated ... More
Tbr1 regulates differentiation of the preplate and layer 6.
AuthorsHevner RF, Shi L, Justice N, Hsueh Y, Sheng M, Smiga S, Bulfone A, Goffinet AM, Campagnoni AT, Rubenstein JL
JournalNeuron
PubMed ID11239428
During corticogenesis, early-born neurons of the preplate and layer 6 are important for guiding subsequent neuronal migrations and axonal projections. Tbr1 is a putative transcription factor that is highly expressed in glutamatergic early-born cortical neurons. In Tbr1-deficient mice, these early-born neurons had molecular and functional defects. Cajal-Retzius cells expressed decreased ... More
Evx1 is a postmitotic determinant of v0 interneuron identity in the spinal cord.
Interneurons in the ventral spinal cord are essential for coordinated locomotion in vertebrates. During embryogenesis, the V0 and V1 classes of ventral interneurons are defined by expression of the homeodomain transcription factors Evx1/2 and En1, respectively. In this study, we show that Evx1 V0 interneurons are locally projecting intersegmental commissural ... More
Spatial distribution and specification of mammalian replication origins during G1 phase.
AuthorsLi F, Chen J, Solessio E, Gilbert DM
JournalJ Cell Biol
PubMed ID12707307
We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and ... More
Inducible dimerization of FGFR1: development of a mouse model to analyze progressive transformation of the mammary gland.
AuthorsWelm BE, Freeman KW, Chen M, Contreras A, Spencer DM, Rosen JM
JournalJ Cell Biol
PubMed ID12011115
To develop an inducible and progressive model of mammary gland tumorigenesis, transgenic mice were generated with a mouse mammary tumor virus-long terminal repeat-driven, conditional, fibroblast growth factor (FGF)-independent FGF receptor (FGFR)1 (iFGFR1) that can be induced to dimerize with the drug AP20187. Treatment of transgenic mice with AP20187 resulted in ... More
Measurement of oxidative DNA damage and repair in specific DNA sequences.
AuthorsAvrutskaya AV, Leadon SA
JournalMethods
PubMed ID11020327
We describe two methods that were developed in our laboratory to measure the production and repair of oxidative DNA damage in specific DNA sequences. Both of these methods rely on the use of monoclonal antibodies against modified nucleotides to separate DNA sequences that contain damage from those in which repair ... More
Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.
AuthorsCmarko D, Verschure PJ, Rothblum LI, Hernandez-Verdun D, Amalric F, van Driel R, Fakan S
JournalHistochem Cell Biol
PubMed ID10817672
In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing ... More
Cyclin-dependent kinases govern formation and maintenance of the nucleolus.
AuthorsSirri V, Hernandez-Verdun D, Roussel P
JournalJ Cell Biol
PubMed ID11901165
In higher eukaryotic cells, the nucleolus is a nuclear compartment assembled at the beginning of interphase, maintained during interphase, and disorganized during mitosis. Even if its structural organization appears to be undissociable from its function in ribosome biogenesis, the mechanisms that govern the formation and maintenance of the nucleolus are ... More
Simultaneous analysis of surface marker expression and cell cycle progression in human peripheral blood mononuclear cells.
AuthorsRosato MT, Jabbour AJ, Ponce RA, Kavanagh TJ, Takaro TK, Hill JP, Poot M, Rabinovitch PS, Faustman EM
JournalJ Immunol Methods
PubMed ID11516753
One method for examining cell cycle kinetics by flow cytometry uses continuous DNA labeling with bromodeoxyuridine (BrdU), a thymidine analogue. Upon incorporation into DNA, BrdU causes stoichiometric quenching of the DNA fluorochrome Hoechst 33258. After counterstaining with a secondary DNA fluorochrome (e.g., ethidium bromide), the analyst can distinguish cells in ... More
Human papillomavirus DNA replication compartments in a transient DNA replication system.
AuthorsSwindle CS, Zou N, Van Tine BA, Shaw GM, Engler JA, Chow LT
JournalJ Virol
PubMed ID9882301
Many DNA viruses replicate their genomes at nuclear foci in infected cells. Using indirect immunofluorescence in combination with fluorescence in situ hybridization, we colocalized the human papillomavirus (HPV) replicating proteins E1 and E2 and the replicating origin-containing plasmid to nuclear foci in transiently transfected cells. The host replication protein A ... More
p21-activated protein kinase gamma-PAK suppresses programmed cell death of BALB3T3 fibroblasts.
AuthorsJakobi R, Moertl E, Koeppel MA
JournalJ Biol Chem
PubMed ID11278362
In response to stress stimulants, cells activate opposing signaling pathways for cell survival and programmed cell death. p21-activated protein kinase gamma-PAK is involved in both cell survival and cell death pathways. Many stress stimulants activate gamma-PAK as a full-length enzyme and as a proteolytic fragment. Caspase-mediated proteolytic activation parallels cell ... More
Authorsvan Raaij EJ, ten Berge D, Hage W, Brouwer A, Meijlink F, Maintz JB, Verbeek FJ
JournalCytometry
PubMed ID11598942
BACKGROUND: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to detect BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographical cell proliferation analysis method is developed. METHODS: Sections stained with fluorescein-labeled ... More
N-terminal domains of the class ia phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signaling.
AuthorsHill KM, Huang Y, Yip SC, Yu J, Segall JE, Backer JM
JournalJ Biol Chem
PubMed ID11278326
Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading ... More
Advances in cytochemical methods for detection of apoptosis.
AuthorsBarrett KL, Willingham JM, Garvin AJ, Willingham MC
JournalJ Histochem Cytochem
PubMed ID11410607
In an earlier article from this laboratory, the current methods developed to detect apoptosis in cells and tissues were highlighted, along with the challenges in their interpretation. Recent discoveries concerning the underlying biochemical mechanisms of apoptotic effector pathways have made possible further assays that allow a more direct measure of ... More
Inhibition of c-Myc oncoprotein limits the growth of human melanoma cells by inducing cellular crisis.
AuthorsBiroccio A, Amodei S, Antonelli A, Benassi B, Zupi G
JournalJ Biol Chem
PubMed ID12824159
Here, we show that inhibition of c-Myc causes a proliferative arrest of M14 melanoma cells through cellular crisis, evident by the increase in size, multiple nuclei, vacuolated cytoplasm, induction of senescence-associated beta-galactosidase activity and massive apoptosis. The c-Myc-induced crisis is associated with decreased human telomerase reverse transcriptase expression, telomerase activity, ... More
Human age-related cataract and lens epithelial cell death.
AuthorsHarocopos GJ, Alvares KM, Kolker AE, Beebe DC
JournalInvest Ophthalmol Vis Sci
PubMed ID9856780
PURPOSE: To evaluate the importance of lens epithelial cell death in age-related cataract. To determine whether the large percentage of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive lens epithelial cells previously reported in human capsulotomy specimens results from apoptosis or necrosis. METHODS: Capsulotomy specimens from patients who had undergone ... More
An element in the alpha1-tubulin promoter is necessary for retinal expression during optic nerve regeneration but not after eye injury in the adult zebrafish.
AuthorsSenut MC, Gulati-Leekha A, Goldman D
JournalJ Neurosci
PubMed ID15342733
We have shown previously that a 1.696 kb upstream fragment of the goldfish alpha1-tubulin promoter was capable of driving green fluorescent protein (GFP) expression in the developing and regenerating zebrafish CNS in a pattern closely mimicking the endogenous alpha1-tubulin gene. Comparison of fish and rat alpha1-tubulin promoters identified a 64 ... More
Laser scanning cytometer (LSC) analysis of fraction of labelled mitoses (FLM).
AuthorsGorczyca W, Melamed MR, Darzynkiewicz Z
JournalCell Prolif
PubMed ID9051117
In this report we describe the successful application of a novel microscope-based multiparameter laser scanning cytometer (LSC) to measure duration of different phases of cell cycle in HL-60 human leukaemic cell lines by the fraction of labelled mitoses (FLM) method. Exponentially growing cells were harvested after various time intervals following ... More
Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection.
AuthorsCmarko D, Verschure PJ, Martin TE, Dahmus ME, Krause S, Fu XD, van Driel R, Fakan S
JournalMol Biol Cell
PubMed ID9880337
In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors ... More