Stephen E. Bloom

A technique is described for obtaining well-spread metaphases from solid tissues of fishes without the use of methodologies that rely on tissue grinders, centrifuges, digestive enzymes, or tissue culture. This procedure involves the formation of a cell suspension from acetic alcohol fixed tissues using 50% acetic acid. The suspension is applied to a warm (50 °C) slide using a micropipette.Solid tissue preparations may be stained by any of the conventional dyes or treated to reveal Q-bands, C-bands, and nucleolar organizers. Large numbers of slides offish chromosomes can be made easily and rapidly using this procedure.

A decrease in chondrocyte numbers is one characteristic of osteoarthritic cartilage. This decrease may be the result of apoptosis or other forms of cell death induced by mechanical damage. Furthermore, cell death may contribute to the structural and metabolic changes found in osteoarthritic cartilage. Therefore, we investigated cell viability and the mode of cell death in cartilage subjected to an increasing severity of impact loads expected to cause compositional damage and osteoarthritic-like metabolic alterations. Canine cartilage explants were subjected to cyclic indentation impacts of 5 megapascals at 0.3 Hz for 0, 2, 20, and 120 min and then kept in culture for 2, 4, 48, and 144 h. Cell death was assessed by the TUNEL assay and by uptake of propidium iodide. Viable cells were detected by the ability to metabolize fluorescein diacetate. Nuclear morphology and ultrastructure of the cell were examined using Hoechst 33342 fluorescent staining and transmission electron microscopy (TEM). As controls for necrosis and apoptosis, cartilage was, respectively, frozen and thawed or incubated with mitomycin-C, an apoptosis inducer. In cartilage that had been loaded for 2 h, 32% of the chondrocytes in the loaded core took up propidium iodide within 2 h after loading. Most of these were in the middle to superficial zones and reflected leaky cell membranes usually characteristic of necrosis. Less than 1% of these chondrocytes were positive in the TUNEL assay after 4 h. After additional culture for 2 days, however, the proportion of chondrocytes which were positive in the TUNEL assay reached 73%. A dose dependent response to duration of loading was detected with the TUNEL assay at this time. The TUNEL assay was not specific for apoptosis since 92% of chondrocytes in freeze/thawed cartilage were TUNEL positive. However, some cells with apoptotic bodies and chromatin condensation characteristic of apoptosis were found in the transition zone between necrotic and normal chondrocytes, but not in the superficial and upper zones, in impact damaged cartilage. We concluded that in this study, necrosis occurred first, followed by apoptosis.

Cytolethal distending toxin (CDT) is a heterotrimeric AB-type genotoxin produced by several clinically important Gram-negative mucocutaneous bacterial pathogens. Irrespective of the bacterial species of origin, CDT causes characteristic and irreversible cell cycle arrest and apoptosis in a broad range of cultured mammalian cell lineages. The active subunit CdtB has structural homology with the phosphodiesterase family of enzymes including mammalian DNase I, and alone is necessary and sufficient to account for cellular toxicity. Indeed, mammalian cells treated with CDT initiate a DNA damage response similar to that elicited by ionizing radiation-induced DNA double strand breaks resulting in cell cycle arrest and apoptosis. The mechanism of CDT-induced apoptosis remains incompletely understood, but appears to involve both p53-dependent and -independent pathways. While epithelial, endothelial and fibroblast cell lines respond to CDT by undergoing arrest of cell cycle progression resulting in nuclear and cytoplasmic distension that precedes apoptotic cell death, cells of haematopoietic origin display rapid apoptosis following a brief period of cell cycle arrest. In this review, the ecology of pathogens producing CDT, the molecular biology of bacterial CDT and the molecular mechanisms of CDT-induced cytotoxicity are critically appraised. Understanding the contribution of a broadly conserved bacterial genotoxin that blocks progression of the mammalian cell cycle, ultimately causing cell death, should assist with elucidating disease mechanisms for these important pathogens.