Quick Detection of DNase II-Type Breaks in Formalin-Fixed Tissue Sections.

Blunt-ended DNase II-type breaks with 5' hydroxyls are generated in phagocytic cells of any lineage during digestion of the engulfed DNA. These breaks indicate the ongoing active phagocytic reaction. They are produced by the acid deoxyribonuclease-DNase II which is the primary endonuclease responsible for DNA degradation after its engulfment.Here, we present an express approach that detects blunt-ended 5' OH DNA breaks in fixed tissue sections. The technique is simple to perform and takes only 60 min to complete. It can be useful in studies of the clearance of dying cells in oncological, inflammatory, and autoimmune disorders.

Dual Detection of Nucleolytic and Proteolytic Markers of Lysosomal Cell Death: DNase II-Type Breaks and Cathepsin D.

Lysosomes contain hydrolytic enzymes that can degrade proteins and DNA. Leakage of these reactive compounds through a compromised lysosomal membrane causes lysosomal cell death, which can have apoptotic, necrotic, or mixed morphology. Lysosomal cathepsin proteases, such as cathepsin D, and the lysosomal endonuclease, DNase II, have both been implicated in lysosome-related cell death. Here, we present a fluorescence dual-labeling technique for simultaneous visualization of these two markers of lysosomal activity linked to cell death. The approach labels the intracellular distribution of cathepsin D and the sites with DNase II-type breaks in fixed tissue sections. It determines the lysosomal or extra-lysosomal localization of the markers and can be useful in studying pathways and signals of lysosomal cell death.

Nanoblinker: Brownian motion powered bio-nanomachine for FRET detection of phagocytic phase of apoptosis.

We describe a new type of bio-nanomachine which runs on thermal noise. The machine is solely powered by the random motion of water molecules in its environment and does not ever require re-fuelling. The construct, which is made of DNA and vaccinia virus topoisomerase protein, can detect DNA damage by employing fluorescence. It uses Brownian motion as a cyclic motor to continually separate and bring together two types of fluorescent hairpins participating in FRET. This bio-molecular oscillator is a fast and specific sensor of 5'OH double-strand DNA breaks present in phagocytic phase of apoptosis. The detection takes 30 s in solution and 3 min in cell suspensions. The phagocytic phase is critical for the effective execution of apoptosis as it ensures complete degradation of the dying cells' DNA, preventing release of pathological, viral and tumor DNA and self-immunization. The construct can be used as a smart FRET probe in studies of cell death and phagocytosis.

Assessing phagocytic clearance of cell death in experimental stroke by ligatable fluorescent probes.

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

Selective transport of cationized fluorescent topoisomerase into nuclei of live cells for DNA damage studies.

The targeted delivery of fluorescently labeled, DNA-modifying proteins into cellular nuclei permits investigation of DNA damage and chromatin function in living cells. Commercially available protein delivery vectors cannot provide selective intranuclear transportation and primarily unload their cargo in the cytoplasm. Here we describe a simple approach for specific intranuclear transportation of vaccinia topoisomerase protein based on its cationization. The delivered protein can be observed and monitored by fluorescence microscopy. The technique is cost-efficient and time-saving. It can be useful in live cell studies.

In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc). This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage. Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks), fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO(4) blunt-ended break (DNase I-type breaks), if T4 DNA ligase is present in the solution. When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO(4) blunt-ended breaks, the ligase reacts with 5'PO(4) DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO(4) blunt-ended DNA breaks. This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA.

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.

Semi-artificial Fluorescent Molecular Machine for DNA Damage Detection.

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.

Caspase-3-dependent and -independent apoptosis in focal brain ischemia.

BACKGROUND: 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. MATERIALS AND METHODS: Rat middle cerebral artery was permanently occluded and histochemical detection of procaspase-3, active caspase-3 and DFF 40/CAD and apoptotic morphology analysis were performed at 6, 24, 48, and 72 hours after occlusion. RESULTS: Necrosis and two types of programmed cell death (PCD) are identified in this study of permanent focal brain ischemia. The first type of PCD is represented by active caspase-3 and DFF 40/CAD-positive cells. The second type of PCD is represented by caspase-3 and DFF40/CAD negative cells, which display morphological signs of apoptosis-like PCD: namely, nuclear chromatin condensation in lump masses and apoptotic body formation. The cells of the first type have a maximum number noted after 24 hours of ischemia. The cells of the second type are primarily seen after 48 and 72 hours of ischemia. Necrotic cells, which are also detected in the stroke, are caspase-3 negative, and have swollen nuclei, without chromatin condensation and apoptotic body formation. CONCLUSIONS: Our results indicate that in permanent brain ischemia in adult rats, PCD processes occur differently in various parts of ischemic zone. In conditions of severe energy depletion, the reactions of cellular disassembly and packaging into apoptotic bodies are accomplished without either caspase-3 expression or the activation of caspase-3-dependent deoxyribonuclease.

Visualization of irreparable ischemic damage in brain by selective labeling of double strand blunt-ended DNA breaks.

BACKGROUND: 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. MATERIALS AND METHODS: Rat brain sections taken 6, 24, 48 and 72 hr after the onset of focal brain ischemia were used. Double-strand DNA breaks were detected directly in the tissue sections via ligation of blunt-ended hairpin-shaped oligonucleotide probes. The probes were attached to the ends of the breaks by T4 DNA ligase. Conventional cresyl violet co-staining and terminal transferase based labeling (TUNEL) were employed to analyze the distribution of labeled cells. RESULTS: Double-strand blunt-ended DNA breaks rapidly accumulate in brain cells after focal brain ischemia. At 24 hr, they concentrate in the peripheral areas of stroke, which are prone to ischemia-reoxygenation. By 48-72 hr, this type of DNA damage spreads inward, covering the internal areas of the ischemic zone. CONCLUSIONS: Selective labeling of blunt-ended DNA breaks delineates the dynamics of stroke-induced irreversible DNA damage and provides highly specific detection of brain cells with irreparable DNA injury. It can be used for comparing the efficiency of various anti-ischemic drugs, particularly those that target DNA damage, as well as for monitoring stroke-induced damage.