Detection and measurement of single-strand breaks in nuclear DNA in fixed lens sections

doi:10.1016/0014-4827(72)90434-X

Experimental Cell Research

Volume 75, Issue 2, December 1972, Pages 307-313

https://doi.org/10.1016/0014-4827(72)90434-X Get rights and content

Abstract

Ethanol-fixed lens sections were incubated with calf-thymus terminal deoxynucleotidyl transferase and ³H-dATP, and the time-course of ³H-dAMP incorporation was compared among various lens cell types by quantitative autoradiography. The data show that the proportion of cell nuclei containing DNA with single-strand breaks increases during fiber cell differentiation, and that the relative number of breaks per nucleus or unit nuclear area also increase in the same sequence.

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Like DNase IIα, DNase IIβ cleaves DNA to produce 3′-phosphoryl/5′-hydroxy ends (Shiokawa and Tanuma, 1999). It has been known for many years that 3′-OH ends rather than 5′-OH ends accumulate during fiber cell denucleation (Modak and Bollum, 1972) so the realization that DNase IIβ is the major nuclease responsible for lens chromatin degradation was surprising. This apparently paradoxical finding may be explained by the presence of endogenous phosphatases, which rapidly convert 3′-PO4 ends generated by DNAse IIβ digestion into 3′-OH ends (De Maria and Bassnett, 2007).
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Whereas adult neurogenesis appears to be a universal phenomenon in the vertebrate brain, enormous differences exist in neurogenic potential between “lower” and “higher” vertebrates. Studies in the gymnotiform fish Apteronotus leptorhynchus and in zebrafish have indicated that the relative number of new cells, as well as the number of neurogenic sites, are at least one, if not two, orders of magnitude larger in teleosts than in mammals. In teleosts, these neurogenic sites include brain regions homologous to the mammalian hippocampus and olfactory bulb, both of which have consistently exhibited neurogenesis in all species examined thus far. The source of the new cells in the teleostean brain are intrinsic stem cells that give rise to both glial cells and neurons. In several brain regions, the young cells migrate, guided by radial glial fibers, to specific target areas where they integrate into existing neural networks. Approximately half of the new cells survive for the rest of the fish’s life, whereas the other half are eliminated through apoptotic cell death. A potential mechanism regulating development of the new cells is provided by somatic genomic alterations. The generation of new cells, together with elimination of damaged cells through apoptosis, also enables teleost fish rapid and efficient neuronal regeneration after brain injuries. Proteome analysis has identified a number of proteins potentially involved in the individual regenerative processes. Comparative analysis has suggested that differences between teleosts and mammals in the growth of muscles and sensory organs are key to explain the differences in adult neurogenesis that evolved during phylogenetic development of the two taxa.
Adult neurogenesis and neuronal regeneration in the teleost fish brain: Implications for the evolution of a primitive vertebrate trait
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In contrast to mammals, teleost fish exhibit an enormous potential to produce new neurons in the adult brain. Whereas in mammals this phenomenon of adult neurogenesis is restricted to the olfactory bulb and the hippocampus, in teleosts dozens of neurogenic areas have been identified, including regions homologous to the mammalian olfactory bulb and hippocampus. Furthermore, the number of new neurons relative to the total number of brain cells is orders of magnitude larger in teleosts than in mammals. In some regions of the teleostean brain, such as the optic tectum, the new cells remain near the proliferation zones in the course of their further development. In others, such as most subdivisions of the cerebellum, they migrate – typically guided by radial glial fibers – to specific target areas. Approximately 50% of the young cells undergo apoptotic cell death within the first few weeks after their generation, whereas the others appear to integrate into the existing neural networks and survive for the rest of the fish’s life. A large portion of the surviving cells differentiate into neurons. Like the potential to produce new neurons in the adult brain, teleosts are also capable of replacing neurons lost to injury rapidly and with high efficiency. This ability of neuronal regeneration is based on the elimination of damaged cells by apoptosis and on the recruitment of new neurons to the site of injury. Proteome analysis has indicated that the individual steps of this repair process are mediated by hundreds of proteins, two dozen of which have been identified thus far. Comparative analysis has suggested that adult neurogenesis is a primitive vertebrate trait, which enables teleost fish to match the number of central neurons with the growing number of (directly or indirectly connected) receptor cells and muscle fibers in the periphery. This trait became largely reduced in the branch leading to mammals, in which the number of peripheral elements remain, due to different growth mechanisms, constant during adult life. The only exception is the continuous production of primary sensory neurons in the mammalian olfactory epithelium. It is hypothesized that this proliferative activity in the olfactory epithelium is causally linked to the generation of new neurons in the olfactory bulb and the hippocampus, both of which form part of the central pathway processing olfactory information in the adult mammalian brain.
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The ocular lens is a distinct system to study cell death for the following reasons. First, during animal development, the ocular lens is crafted into its unique shape. The crafting processes include cell proliferation, cell migration, and apoptosis. Moreover, the lens epithelial cells differentiate into lens fiber cells through a process, which utilizes the same regulators as those in apoptosis at multiple signaling steps. In addition, introduction of exogenous wild-type or mutant genes or knock-out of the endogenous genes leads to apoptosis of the lens epithelial cells followed by absence of the ocular lens or formation of abnormal lens. Finally, both in vitro and in vivo studies have shown that treatment of adult lens with stress factors induces apoptosis of lens epithelial cells, which is followed by cataractogenesis. The present review summarizes the current knowledge on apoptosis in the ocular lens with emphasis on its role in lens development and pathology.
Chapter 5.6 Cellular and molecular analysis of stress-induced neurodegeneration - methodological considerations
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Citation Excerpt :
This approach detects 3′-OH ends of single-stranded DNA after the addition of labeled nucleotides to the open ends of DNA in a procedure known as in situ end-labeling (ISEL). The latter may be achieved using either E. coli polymerase (or its Klenow fragment) in a method called in situ nick-translation (ISNT), or terminal transferase in a method referred to as terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) (Modak and Bollum, 1972; Gavrieli et al., 1992; Jin et al., 1999). These methods allow the cytochemical demonstration of free DNA strand openings.
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^☆: A part of this work was conducted when one us (S. P. M.) was at the Department of Microbiology, University of Kentucky, Lexington, Ky, USA.

^☆☆: Research was supported jointly by grant 3.466.70 from the Swiss National Fund for Scientific Research and grants CA-08487 and CA-10375 from the National Cancer Institute, USA.

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Detection and measurement of single-strand breaks in nuclear DNA in fixed lens sections☆,☆☆

Abstract

J biol chem

Exptl cell res

Exptl cell res

Exptl cell res

Exptl cell res

Exptl cell res

Exptl cell res

Dev biol

Exptl cell res

Exptl cell res

Methods in cell physiol

Exptl eye res