The radiosensitivity of cultured human and mouse keratinocytes

Clonogenic survival assays after gamma-radiation in vitro were performed on freshly isolated and subcultured keratinocytes from mouse skin, mouse tongue and human skin. Survival curves were constructed by fitting the data to a multi-target model of cell survival. When subcultured, keratinocytes from all sites produced survival curves which showed a reduced shoulder region and an increased D0 when compared with their freshly isolated counterparts. Freshly isolated human skin keratinocytes were more radiosensitive than mouse keratinocytes from either skin or tongue.     

A reproducible technique combining tritiated thymidine autoradiography with immunodetection of bromodeoxyuridine for double labelling studies of cell proliferation in paraffin sections of tissues

Abstract. A method is described to combine tritiated thymidine autoradiography with immunoperoxidase detection of bromodeoxyuridine on the same paraffin sections. It overcomes the varied technical artefacts we encountered when first attempting to combine these techniques and results in preparations with extremely low peroxidase and autoradiographic backgrounds. In particular, we find it is important to avoid the use of detergents during immunostaining, otherwise grain counts are reduced and autoradiograph exposures need to be greatly increased, and to avoid excessive peroxidase staining which makes it difficult to visualize silver grains in the overlying emulsion. The advantages of a method to remove emulsion films using acid-alcohol, allowing the same sections to be dipped twice with a long and a short autoradiographic exposure, are presented. The routine combination of high quality tritiated thymidine autoradiography with clean immunoperoxidase staining of bromodeoxyuridine-positive nuclei provides a new and powerful cell kinetic, double-labelling method to augment existing techniques e.g. by labelling the same cells undergoing DNA synthesis in successive cell cycles.     

Influence of pH,exchangeable aluminium and 0.02M CaCl2-extractable aluminium on the growth and nitrogen-fixing activity of white clover (Trifolium repens) in some New Zealand soils

The effects of soil acidity on the growth and N₂-fixing activity of white clover in seven acid topsoils and subsoils of New Zealand were investigated using a glasshouse experiment.The application of phosphate (Ca(H₂PO₄)₂) to the soils resulted in very large increases in white clover growth on all soils. The application of phosphate, as well as increasing P supply, also decreased 0.02M CaCl₂-extractable Al levels, but had little effect on exchangeable Al levels.Where adequate phosphate was applied, increasing rates of lime (CaCO₃) resulted in increased plant growth on most soils. N₂[C₂H₂]-fixing activity was increased by the first level of lime for one soil, but generally remained approximately constant or declined slightly at higher rates of lime. Up to the point of maximum yield, white clover top weight was more highly correlated with 0.02M CaCl₂-extractable soil Al than with exchangeable Al or pH. At pH values greater than 5.5, plant yield declined on some soils, apparently because of Zn deficiency. The data suggest that white clover is unlikely to be affected by Al toxicity at 0.02M CaCl₂-extractable Al levels of less than about 3.3 g g^–1. However, there were differences between soils in apparent plant tolerance to 0.02M CaCl₂-extractable Al, which appeared to be caused by differing C levels in the 0.02M CaCl₂ extracts.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.