Early Shape-shifting FRET Probe

BioTechniques. Feb, 2002  |  Pubmed ID: 11848401

Apoptosis of T Lymphocytes Invading Glioblastomas Multiforme: a Possible Tumor Defense Mechanism

Journal of Neurosurgery. Mar, 2002  |  Pubmed ID: 11883844

The goal of this study was to investigate whether apoptosis occurs in T lymphocytes that invade Fas ligand (FasL)-expressing glioblastomas multiforme (GBMs) and if its induction could be mediated by Fas.

In Situ DNA Ligation As a Method for Labeling Apoptotic Cells in Tissue Sections. An Overview

Methods in Molecular Biology (Clifton, N.J.). 2002  |  Pubmed ID: 12073437

Detection of Specific Double-strand DNA Breaks and Apoptosis in Situ Using T4 DNA Ligase

Methods in Molecular Biology (Clifton, N.J.). 2002  |  Pubmed ID: 12073438

In Situ Detection of Double-strand DNA Breaks with Terminal 5'OH Groups

Methods in Molecular Biology (Clifton, N.J.). 2002  |  Pubmed ID: 12073439

Caspase-3-dependent and -independent Apoptosis in Focal Brain Ischemia

Molecular Medicine (Cambridge, Mass.). Jul, 2002  |  Pubmed ID: 12393932

Although extensive caspase-3 activation has been demonstrated in experimental brain ischemia produced in neonatal rat, the role this caspase plays in the focal ischemia of adult brain is not clear, as the levels of caspase-3 in adult rat brain are extremely low. This raises the question whether caspase-3 synthesis and activation are essential for execution of the apoptotic program and DNA fragmentation in permanent brain ischemia, a condition that impairs cellular protein synthesis.

Visualization of Irreparable Ischemic Damage in Brain by Selective Labeling of Double Strand Blunt-ended DNA Breaks

Molecular Medicine (Cambridge, Mass.). Dec, 2002  |  Pubmed ID: 12606816

Double-strand DNA breaks with blunt ends represent the most serious type of DNA damage, and cannot be efficiently repaired by cells. They are generated in apoptosis or necrosis and are absent in normal or transiently damaged cells. Consequently, they can be used as a molecular marker of irreparable cellular damage. We evaluated the effects of focal brain ischemia using selective labeling of blunt-ended DNA breaks as a marker of irreversible tissue damage. A new approach permitting such analysis in situ is introduced.

Early Necrotic DNA Degradation: Presence of Blunt-ended DNA Breaks, 3' and 5' Overhangs in Apoptosis, but Only 5' Overhangs in Early Necrosis

The American Journal of Pathology. May, 2003  |  Pubmed ID: 12707041

The structure of DNA breaks in early necrosis was analyzed and compared with apoptotic DNA degradation using in vivo and cell culture models. Early necrosis (1 hour after cell death) was produced in vivo by the freezing-thawing of rat thymus and in cell culture of Jurkat cells. Apoptosis was induced in the same cell types using dexamethasone for thymus and staurosporine for Jurkat cells. Selective detection of double-strand DNA breaks with blunt ends was performed by in situ ligation. Blunt-ended breaks bearing 5' phosphates were detected in apoptotic but not in early necrotic cells. Pretreatment of apoptotic and necrotic tissue with Klenow enzyme with or without added dNTPs reduced all 3' or 5' overhangs to blunt ends. Subsequent in situ ligation with blunt-ended probes revealed no 3' overhangs in necrotic cells. However double-strand cuts with 5' overhangs were abundant in necrotic DNA. 5' Overhangs were also detected in apoptotic cells. Presence of exclusively 5' overhangs in early necrosis with absence of a variety of possible DNA ends, suggests the existence of a specific orderly mechanism of DNA degradation.

Thimerosal Induces DNA Breaks, Caspase-3 Activation, Membrane Damage, and Cell Death in Cultured Human Neurons and Fibroblasts

Toxicological Sciences : an Official Journal of the Society of Toxicology. Aug, 2003  |  Pubmed ID: 12773768

Thimerosal is an organic mercurial compound used as a preservative in biomedical preparations. Little is known about the reactions of human neuronal and skin cells to its micro- and nanomolar concentrations, which can occur after using thimerosal-containing products. A useful combination of fluorescent techniques for the assessment of thimerosal toxicity is introduced. Short-term thimerosal toxicity was investigated in cultured human cerebral cortical neurons and in normal human fibroblasts. Cells were incubated with 125-nM to 250-microM concentrations of thimerosal for 45 min to 24 h. A 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI) dye exclusion test was used to identify nonviable cells and terminal transferase-based nick-end labeling (TUNEL) to label DNA damage. Detection of active caspase-3 was performed in live cell cultures using a cell-permeable fluorescent caspase inhibitor. The morphology of fluorescently labeled nuclei was analyzed. After 6 h of incubation, the thimerosal toxicity was observed at 2 microM based on the manual detection of the fluorescent attached cells and at a 1-microM level with the more sensitive GENios Plus Multi-Detection Microplate Reader with Enhanced Fluorescence. The lower limit did not change after 24 h of incubation. Cortical neurons demonstrated higher sensitivity to thimerosal compared to fibroblasts. The first sign of toxicity was an increase in membrane permeability to DAPI after 2 h of incubation with 250 microM thimerosal. A 6-h incubation resulted in failure to exclude DAPI, generation of DNA breaks, caspase-3 activation, and development of morphological signs of apoptosis. We demonstrate that thimerosal in micromolar concentrations rapidly induce membrane and DNA damage and initiate caspase-3-dependent apoptosis in human neurons and fibroblasts. We conclude that a proposed combination of fluorescent techniques can be useful in analyzing the toxicity of thimerosal.

Semi-artificial Fluorescent Molecular Machine for DNA Damage Detection

Nano Letters. Dec, 2004  |  Pubmed ID: 17330146

The design of artificial molecular machines is complicated because the mechanics used in macromachines is not readily adaptable for nano environments. We constructed a semi-artificial molecular device, which contains a naturally occurring molecular machine-a vaccinia virus encoded protein-linked with an artificial part. The self-assembled construct makes two fluorescently labeled detector units. It is the first sensor capable of selectively detecting different types of DNA breaks, exemplifying a practical approach to the design of molecular devices.

Visualization of Individual Single-walled Carbon Nanotubes by Fluorescent Polymer Wrapping

Nano Letters. Aug, 2005  |  Pubmed ID: 16089489

Manipulating optical properties of single-walled nanotubes (SWNTs) is necessary for the development of nanoscale optical devices and probes for biomedical research. In life sciences it will make possible the direct observation of SWNTs inside living cells using optical microscopes. In the nanotechnology field it will enable the development of nanosensors with fluorescent reporting. However, the direct fluorescent labeling of SWNTs is obstructed by their strong light quenching qualities. Besides, chemical functionalization of SWNTs needed for the covalent attachment of fluorescent dyes could change favorable properties of nanotubes. Here we report that optical properties of SWNTs can be manipulated without their covalent modification by wrapping them with fluorescently labeled polymer poly(vinylpyrrolidone) (PVP-1300). Fluorescent PVP-1300 forms a monomolecular approximately 2.5 nm thick layer coiling around individual SWNTs and nanotube bundles. PVP casing is fluorescent although it is only several nanometers thick. This makes individual SWNTs observable by a fluorescent microscope. The spare polymer strands left over after wrapping around the relatively shorter nanotubes form junctions between SWNTs tying them together into new configurations, primarily Y- and psi-type junctions. The ability to use a single fluorescent polymer strand to fasten nanotubes together can be useful in assembly of nanotube-made devices. In PVP-covered SWNTs multiple fluorophores are attached to each single nanotube making them unique composite fluorophores attractive as parts of biological fluorescent probes and in the development of the new materials in photonics and nanotechnology.

Horseradish Peroxidase-driven Fluorescent Labeling of Nanotubes with Quantum Dots

BioTechniques. Mar, 2006  |  Pubmed ID: 16568818

We describe the first enzyme-driven technique for fluorescent labeling of single-walled carbon nanotubes (SWNTs). The labeling was performed via enzymatic biotinylation of nanotubes in the tyramide-horseradish peroxidase (HRP) reaction. Both direct and indirect fuorescent labeling of SWNTs was achieved using either biotinyl tyramide or fluorescently tagged tyramides. Biotinylated SWNTs later reacted with streptavidin-conjugated fluorophores. Linking semiconductor nanocrystals, quantum dots (Q-dots), to the surface of nanotubes resulted in their fluorescent visualization, whereas conventional fluorophores bound to SWNTs directly or through biotin-streptavidin linkage, were completely quenched. Enzymatic biotinylation permits fluorescent visualization of carbon nanotubes, which could be useful for a number of biomedical applications. In addition, other organic molecules such as proteins, antibodies, or DNA can be conjugated to biotinylated SWNTs using this approach.

Oscillating Probe for Dual Detection of 5'PO4 and 5'OH DNA Breaks in Tissue Sections

Methods in Molecular Biology (Clifton, N.J.). 2006  |  Pubmed ID: 16785620

Several types of DNA cuts are used as markers of apoptosis for detection of apoptotic cells in situ. We recently introduced a ligase-based in situ assay that is specific for a single type of DNA damage--a double-strand break of DNase I-type, bearing 5'PO4. Here we describe a vaccinia topoisomerase I-based approach to label another type of DNA damage in situ--a double-strand break of DNase II-type, bearing 5'OH. The assay uses a new type of probe, a molecular oscillator. The probe self-assembles in solution out of a dual-hairpin oligonucleotide and vaccinia topoisomerase I. The enzyme continuously separates and religates two fluorescently labeled hairpins, which can participate in energy transfer. We describe the successful combination of topoisomerase-and ligase-based systems into an in situ assay. The assay uses an oscillating probe for simultaneous detection of two types of DNA cuts in tissue sections.

In Situ Labeling of DNA Breaks and Apoptosis by T7 DNA Polymerase

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21057919

The native T7 DNA polymerase is a fast and highly processive enzyme that can be used for in situ detection of apoptosis and various types of DNA breaks. The technique is quick and simple, and was shown to label earlier stages of apoptosis compared to the terminal transferase technique. The in situ labeling applications of T7 DNA polymerase are presented and summarized from the DNA damage detection standpoint. The detailed protocols are provided together with the discussion of their advantages and limitations.

In Situ Ligation: a Decade and a Half of Experience

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21057920

The in situ ligation (ISL) methodology detects apoptotic cells by the presence of characteristic DNA double-strand breaks. A labeled double-stranded probe is ligated to the double-strand breaks in situ on tissue sections. Like the popular TUNEL assay, ISL detects cells in apoptosis based on the ongoing destruction of DNA by apoptotic nucleases. In comparison to TUNEL, it is more specific for apoptosis versus other causes of DNA damage, both repairable damage and necrosis. In the decade and a half since its introduction, ISL has been used in several hundred publications. Here we review the development of the method, its current status, and its uses and limitations.

In Situ Ligation Simplified: Using PCR Fragments for Detection of Double-strand DNA Breaks in Tissue Sections

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21057921

The simplified in situ ligation procedure is described. All reagents for the assay can be easily obtained in any molecular or cell biology laboratory. The technique uses ligation of double-stranded, PCR-derived DNA fragments labeled with digoxigenin or fluorophores for highly selective detection of apoptotic cells in paraffin-embedded tissue sections. Two types of DNA fragments prepared by PCR are employed. The fragment synthesized by Taq polymerase contains single-base 3' overhangs, whereas the Pfu polymerase-made fragment is blunt ended. Both fragments can be used as specific, sensitive and cost-effective DNA damage probes. After ligation to apoptotic nuclei in tissue sections, they indicate the presence of double-strand DNA breaks with single-base 3' overhangs as well as blunt ends.

5'OH DNA Breaks in Apoptosis and Their Labeling by Topoisomerase-based Approach

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21057922

Recently, the concept of apoptotic cell elimination was expanded and programed cell death is no longer viewed as an individual cellular event. The complete description of the apoptotic process now includes two phases: the self-driven cell disassembly and the externally-controlled elimination of apoptotic cell corpses by phagocytizing cells. The second, phagocytic phase is essential, highly conserved, and is even more important than the internal phase of cell disassembly. This is because it ensures the complete degradation of the dying cell's DNA, preventing the release of pathological, viral and tumor DNA, and self-immunization. In different cells and species from mammals to flies, a single conserved enzyme--DNase II is responsible for the elimination of cellular DNA in the second "mopping up" phase of apoptosis. Here, we present an assay for the selective detection of the phagocytic phase of apoptosis. The technology capitalizes on the fact that phagocytic DNase II produces identifiable signature DNA breaks, which can be labeled by vaccinia topoisomerase. The assay permits labeling of the previously underestimated phase of apoptotic execution and is a useful tool in the apoptosis detection arsenal.

Fluorescent Probes Detecting the Phagocytic Phase of Apoptosis: Enzyme-substrate Complexes of Topoisomerase and DNA

Molecules (Basel, Switzerland). 2011  |  Pubmed ID: 21642935

In apoptosis, the initial self-driven suicide phase generates cellular corpses which are digested in the phagolysosomes of professional and amateur phagocytes during the subsequent waste-management phase. This ensures the complete elimination of the genetic material which often contains pathological, viral or cancerous DNA sequences. Although the phagocytic phase is critical for the efficient execution of apoptosis, there are currently few methods specifically adapted for its detailed visualization in the fixed tissue section format. To resolve this we developed new fluorescent probes for in situ research. The probes selectively visualize active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells) which engulf and digest apoptotic cell DNA. These fluorescent probes are the covalently-bound enzyme-DNA intermediates produced in a topoisomerase reaction with specific "starting" oligonucleotides. They detect a specific marker of DNase II cleavage activity, which occurs exclusively in phagolysosomes of the cells that engulfed apoptotic nuclei. The probes provide snap-shot images of the digestion process occurring in cellular organelles responsible for the actual execution of phagocytic degradation of apoptotic cell corpses. We applied the probes for visualization of the phagocytic reaction in tissue sections of normal thymus and in several human lymphomas. We also discuss the nature, stability and properties of DNase II-type breaks as a marker of phagocytic activity. This development provides a useful fluorescent tool for studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders.