Biomedical subjects

M A Wride

Publications and source records attributed to M A Wride.

10 recordsLinked to original sources

PubMed1999

Members of the bcl-2 and caspase families regulate nuclear degeneration during chick lens fibre differentiation.

The optical clarity of the lens is ensured by the programmed removal of nuclei and other organelles from the lens fibre cells during development. The morphology of the degenerating nuclei is similar to that observed during apoptosis and is accompanied by DNA fragmentation. Proteins encoded by the bcl-2 proto-oncogene family are important in either promoting or inhibiting apoptosis, and caspases are involved in downstream proteolytic events. Here, the expression of bcl-2 family members (bcl-2, bax, bad, and bcl-x(s/l)) and caspases-1, -2, -3, -4, and -6 was investigated through a range of stages of chick lens development using immunocytochemistry, Western blotting, and affinity labelling for caspases using biotinylated caspase inhibitors. Using differentiating lens epithelial cell cultures, it was demonstrated that the addition to cultures of synthetic peptide inhibitors of caspases -1, -2, -4, -6, and -9 brought about a 50-70% reduction in the number of degenerating nuclei per unit area of culture, as assessed by image analysis. These effects were comparable to those seen when general inhibitors of caspases were added to cultures. On the other hand, inhibitors of caspases-3 and -8 were not effective in significantly reducing the number of TUNEL-labelled nuclei. Expression of the caspase substrates poly(ADP-ribose) polymerase (PARP) and the 45-kDa subunit of DNA fragmentation factor (DFF 45) was also observed in the developing lens. Western blots of cultures to which caspase inhibitors were added revealed alterations in the PARP cleavage pattern, but not in that of DFF. These results demonstrate a role for members of the bcl-2 family and caspases in the degeneration of lens fibre cell nuclei during chick secondary lens fibre development and support the proposal that this process has many characteristics in common with apoptosis.

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PubMed1998

Nuclear degeneration in the developing lens and its regulation by TNFalpha.

DNA fragmentation in lens fibre cell nuclei undergoing programmed degeneration was identified by terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labelling (TUNEL). Lens epithelial cells in culture were induced to differentiate into lens fibre-like clumps of cells (lentoids) by insulin and it was shown that the TUNEL method was also an effective means of labelling degenerating nuclei in lentoid cells in lens epithelial cell cultures. Using immuno-fluorescence and confocal microscopy, it was shown that TNFalpha and TNF receptor (TNFR1, and TNFR2) immunoreactivity was present in sections of chick embryo lenses. TNFalpha immunoreactivity was associated with the lens epithelium and lens fibres. TNFR1 immunoreactivity was present in lens epithelial cells, cortical lens fibres, and lens fibre cell nuclei, while TNFR2 immunoreactivity had a similar distribution to that of TNFR1, but was not associated with nuclei. Similar patterns of TNFalpha, TNFR1, and TNFR2 immunoreactivity were observed in lens epithelial cell cultures. When added to lens epithelial cell cultures, TNFalpha, at concentrations of 50 to 100 ng ml-1, and agonistic antibodies to both TNFR1 and TNFR2 significantly (P<0.05) enhanced the number of degenerating (TUNEL-positive) nuclei. On the other hand, a neutralising antibody to TNFalpha significantly (P<0. 05) reduced the number of TUNEL-positive nuclei. These results demonstrate that TUNEL is an effective means of labelling degenerating lens fibre nuclei during lens fibre and lentoid differentiation, and suggest a potential role for TNFalpha-like factors and their receptors in the degeneration of lens fibre cell nuclei during lens differentiation. We further suggest that the nuclear degeneration of lens fibre cells is analogous to the nuclear events that occur during apoptosis, and that in lens cells the nuclear degeneration is uncoupled from the plasma membrane events of apoptosis that normally lead to cell death.

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PubMed1997

Roles for growth and differentiation factors in avian embryonic development.

We review the evidence for a role for transforming growth factor-beta (TGF-beta) and for tumor necrosis factor-alpha (TNF-alpha) in the development of the avian embryo. Transforming growth factor-beta is expressed in a number of locations in the early embryo with a distribution consistent with a function in epithelial-mesenchymal transformation and modulation of the composition of the extracellular matrix. During gastrulation, this factor is found in the mesoderm cell layer as well as in the endoderm underlying the primitive streak. In vivo and in vitro investigations suggest that TGF-beta may be involved in the regulation of phenotypic transformation, matrix deposition, and cell proliferation. Tumor necrosis factor-alpha and its two receptors are also located with distributions that suggest important involvement for this pleiotropic factor in early morphogenetic processes. Tumor necrosis factor-alpha is found in several cell populations from the time of gastrulation onwards, including the lens. In vitro investigations, using tissue from the gastrulating embryo as well as from the lens, suggest that this factor may be associated with the extensive cell death that occurs throughout the first 6 d of development, and with nuclear degeneration in the lens. We hypothesize that TNF-alpha, acting in a paracrine or autocrine fashion, may be involved in the signalling pathways that effect the regulation of cell death in development.

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PubMed1996

Ultrastructural identification of apoptotic nuclei using the TUNEL technique.

We describe an ultrastructural adaptation of the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) for the identification of DNA fragmentation. Thin sections of tissue embedded in hydrophilic resin were nick end labelled with biotinylated dUTP which was subsequently labelled with avidin conjugated to gold particles. The technique was validated by labelling the nuclei of L929-8 cells treated with tumour necrosis factor alpha. These cells are known to respond to treatment with the factor by undergoing apoptosis. The method was then used on tissue from the chick embryo which is known to be undergoing programmed cell death. This tissue was from the neural tube and the posterior necrotic zone of the limb bud, where cells can be identified as undergoing apoptosis based on the morphology of their nuclei. The method specifically labelled heterochromatin adjacent to the nuclear envelope as well as that associated with the nucleolus of cells from regions of the embryo where programmed cell death was expected. In addition to labelling the nuclei of cells that were clearly undergoing apoptosis, the method also identified nuclei of apparently normal cells. This method, used in conjunction with corroborating techniques, provides a means for the early detection of cells undergoing DNA fragmentation, before the onset of gross apoptotic morphology, and in cells that do not show classical apoptotic characteristics.

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PubMed1996

Cellular and molecular features of lens differentiation: a review of recent advances.

In this paper, the more recent literature pertaining to differentiation in the developing vertebrate lens is reviewed in relation to previous work. The literature reviewed reveals that the developing lens has been, and will continue to be, a useful model system for the examination of many fundamental processes occurring during embryonic development. Areas of lens development reviewed here include: the induction and early embryology of the lens; lens cell culture techniques; the role of growth factors and cytokines; the involvement of gap junctions in lens cell-cell communication; the role of cell adhesion molecules, integrins, and the extracellular matrix; the role of the cytoskeleton; the processes of programmed cell death (apoptosis) and lens fibre cell denucleation; the involvement of Pax and Homeobox genes; and crystallin gene regulation. Finally, some speculation is provided as to possible directions for further research in lens development.

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PubMed1995

Potential roles for tumour necrosis factor alpha during embryonic development.

This paper reviews the evidence indicating possible roles for tumour necrosis factor-alpha (TNF alpha) in development. It is proposed that TNF alpha may have essentially three major roles during embryonic development, which may be analogous to its roles in the immune system and during inflammation: a role in programmed cell death; a role as a cellular growth and differentiation factor; and also a role in the remodelling of extracellular matrix, and the regulation of cell adhesion molecules and integrins. The concept of the existence of a cytokine array during embryogenesis, analogous to that occurring in inflammation, is discussed, as well as potential roles for TNF alpha in the induction of ubiquitin; protective mechanisms embryonic cells may employ against TNF alpha-mediated cytotoxicity; and a consideration of the role TNF alpha may play in a "free radical theory of development".

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PubMed1995

Programmed cell death in development.

Although cell death has long been recognized to be a significant element in the process of embryonic morphogenesis, its relationships to differentiation and its mechanisms are only now becoming apparent. This new appreciation has come about not only through advances in the understanding of cell death in parallel immunological and pathological situations, but also through progress in developmental genetics which has revealed the roles played by death in the cell lineages of invertebrate embryos. In this review, we discuss programmed cell death as it is understood in developmental situations, and its relationship to apoptosis. We describe the morphological and biochemical features of apoptosis, and some methods for its detection in tissues. The occurrence of programmed cell death during invertebrate development is reviewed, as well as selected examples in vertebrate development. In particular, we discuss cell death in the early vertebrate embryo, in limb development, and in the nervous system.

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PubMed1994

Expression of TGF beta 1/beta 3 during early chick embryo development.

We have used an antibody against a TGF beta peptide fragment to localize this growth factor in the early chick embryo from laying to the ten-somite stage of development. Western blotting showed that the antibody reacted with both mammalian TGF beta 1 and chicken TGF beta 3. By immunocytochemistry we find that at the earliest developmental stage (stage X of Eyal-Giladi and Kochav) immunoreactivity to this antibody is primarily located in the cells of the area opaca and marginal zone, as well as in the most peripheral edge cells of the blastoderm. The yolk is non-reactive, except in a highly localized region subjacent to the edge cells. This pattern persists at stage XII, and at both stages individual isolated cells in the epiblast and hypoblast are also reactive. By the time of gastrulation, reactivity in the epiblast is polarized to the ventral extremity of the cells, and again some isolated cells in this layer are intensely immunoreactive. At this stage also, the endoderm cells, particularly those underlying the primitive streak, are positive, as are the mesoderm cells lateral to the streak. At somite stages, the neuroepithelium is not reactive but the ectoderm lateral to it is strongly positive. At the caudal primitive streak levels of early somite embryos, the ectoderm and endoderm are immunoreactive while the mesoderm loses the reactivity it showed at the early gastrulation stages. The neuroepithelial cells later show reactivity at their apical poles, and, as at the earlier stages, individual cells show intense labelling. These results indicate that TGF beta 1 and/or TGF beta 3 immunoreactivity is developmentally regulated from very early stages of morphogenesis in the chick, and together with data from earlier functional studies, suggest that this factor has roles in embryonic axis formation and in blastoderm expansion.

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PubMed1994

Distribution of TNF alpha-like proteins correlates with some regions of programmed cell death in the chick embryo.

Early chick embryos have previously been shown to express tumor necrosis factor-alpha-cross-reactive proteins (TNF alpha-CRPs) in a developmentally regulated manner, thus implicating these proteins in programmed cell death and in tissue remodeling. In this study, cells undergoing DNA fragmentation have been identified, using terminal deoxynucleotide transferase (TdT) mediated dUTP-biotin nick-end-labeling (TUNEL), during the embryonic development of the chick, between stages 18 and 29. DNA fragmentation is indicative of cells undergoing programmed cell death. TUNEL-positive cells were identified in several well documented areas of programmed cell death, including the limb buds, the heart, spinal motoneurons, dorsal root ganglia, and the ventral horn of the neural tube. In addition, other areas of cell death were identified including the floor plate and the mesonephros. In several locations, a close correlation was noted between the presence of TUNEL-positive cells and regions of TNF alpha-immunoreactivity. These regions included the ventral horn and marginal zone of the neural tube, spinal motoneurons, paravertebral ganglia, parts of the myotome, mesenchyme of the body wall, and the mesonephros. In addition, using the TNF alpha-sensitive L929-8 bioassay it was shown that homogenate of stage 18 chick embryos is cytotoxic to L929-8 cells and that this toxicity can be reduced using neutralizing antibodies to mouse TNF alpha. This bioassay allowed us to estimate the mean concentration of TNF alpha-like activity in embryo homogenate, which is within the range of physiological (pg/ml) levels of TNF alpha found in other systems. These results suggest that proteins with TNF alpha-like activity may have a role in programmed cell death in some tissues during early chick embryo development.

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PubMed1993

Expression of tumor necrosis factor-alpha (TNF alpha)-cross-reactive proteins during early chick embryo development.

We have investigated the expression of tumor necrosis factor-alpha (TNF alpha)-cross-reactive proteins during the early development of the chick embryo from day 1 to day 6 (H-H stages 5-29) using a polyclonal antibody and two monoclonal antibodies to recombinant mouse TNF alpha. We have confirmed the cross-reactivity of the antibodies with chicken tissue in Western blotting studies. Proteins of 50 kDa and 70 kDa, showing anti-TNF alpha cross-reactivity, have been identified during early chick development. In addition, both monoclonal antibodies recognize a 120 kDa protein. These molecules probably represent cytosolic or transmembrane TNF-alpha-like proteins, similar to those previously identified on the surface of cytotoxic T-lymphocytes. We show by ultrastructural cytochemistry that immunoreactivity can be detected at the surfaces of some cells, suggesting that at least some of the antigen is membrane-associated. The proteins are shown to have a widespread tissue distribution during this period of development. Immunoreactivity is first detected in the gastrulating embryo, in the mesoderm and the endoderm. By day 2, expression is confined to the ectoderm and the endoderm, while at day 3 expression appears in the myotome, the notochord, and in nervous tissue. At day 4 the distribution of reactivity is more extensive and includes the notochord, the sclerotome, and the myotome, while the cranial and spinal nerves also become intensely immunoreactive. Also at this stage, neural tube reactivity becomes localized to the marginal neuroepithelial zone, and the lens fibers become positive. This distribution of staining then persists until 6 days of development. We hypothesize that the expression of TNF alpha-cross-reactive proteins in early development could be indicative of a role for them in programmed cell death (apoptosis) during differentiation of the notochord, the lens, and the nervous system, and in tissue remodeling.

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