Non-histone chromosomal proteins from virus-transformed and untransformed 3T3 mouse fibroblasts.

A peak in the non-histone chromosomal protein polyacrylamide gel electrophoresis profiles has been detected which is higher in log phase 3T3 and 3T3/SV40 cells than in density-inhibited 3T3 cells. Radioactive incorporation is substantially higher into this peak in log phase 3T3 than in 3T3/SV40 and density-inhibited 3T3 cells. Reversion of 3T3/SV40 cells with dibutyryl cyclic AMP and theophylline produces increased radioactive incorporation into the peak. Electrophoresis of non-histone chromosomal proteins extracted at different stages of the cell cycle in density inhibited 3T3 cells following serum stimulation shows a cyclic variation in the amount of this peak with maximum accumulation in late G1. In contrast the height of an equivalent peak in synchronously growing 3T3/SV40 cells remains constant throughout the cell cycle. It is postulated that the protein(s) of this peak may have a regulatory role in cell growth.

The synthesis of polyadenylated RNA and other RNA species in synchronised CHK cells.

Chinese hamster kidney cells have been synchronised by the double thymidine block technique. The rate of incorporation of [5-3H]uridine into the polyadenylated RNA fraction has been analysed throughout two complete cell cycles and compared with the rate of incorporation into other RNA species and the UTP pool. In contrast with other reports, we have found that synthesis of 18 S rRNA continues steadily throughout the cell cycle, as does synthesis of 5 S RNA and tRNA. In the case of the polyadenylated RNA, however, our data indicate a major peak of synthesis during S phase and a smaller peak coinciding with G1.

The half-life of polyadenylated polysomal RNA from normal and transformed cells in monolayer culture.

The small genotypic differences between normal and transformed cells are insufficient to account directly for all their wide phenotypic differences, which probably in some cases at least involve alterations in control of gene expression. To ascertain whether such alterations involved changes in mRNA stability, RNA half-lives were estimated in five monolayer cell lines, including two pairs of normal cells and their transformed counterparts. The results for the polyadenylated fractions in all cases fit with those expected from a model in which the whole fraction has a single half-life, of less than one generation time. From both the transformed/untransformed cell pairs, there is evidence that a relationship exists between cell generation time and the half-life of the polyadenylated polysomal RNA fraction, which persists even through the process of transformation. Considerable alteration in the pattern of RNA stability is therefore unlikely to be obligatory in in vitro transformation.

Dopamine release in the amygdaloid complex of the rat, studied by brain microdialysis.

The dopaminergic projection from the ventral tegmental area to the amygdaloid complex may be modulatory on the processes of associative learning in the latter region. We measured dopamine in four different amygdaloid subfields in the rat, using brain microdialysis. Extracellular levels of dopamine in two sites in the lateral nucleus were not consistently measurable, even after treatment with amphetamine. However, basal dopamine levels were measurable in more medial locations (basolateral and central nuclei), with higher concentrations in the caudal than in the rostral probe placement, and were increased around 3-fold by systemic amphetamine. Similarly, dopamine levels in caudal-medial amygdala were increased by local potassium stimulation and by mild footshock in a calcium-dependent manner, indicating a neurotransmitter origin.

Modification of the cytotoxicity of aflatoxin B1 in rat liver cells by steroids.

The addition of steroids with aflatoxin B1 (AFB1) to rat liver cells in culture has been shown to increase the toxin's inhibitory action on growth and protein synthesis. In contrast the inhibition of RNA synthesis by AFB1 was unaffected. The steroid potentiates the direct action of AFB1 at initiation of translation.

The effect of aflatoxin B1 on translation in cells in culture.

The effect of aflatoxin B1 (AFB1) upon translation has been studied in two cell lines in culture, which differ markedly in their sensitivity to the toxin. It was found that AFB1 predominantly inhibits initiation of translation in both cell lines.

Inhibitory effects of methyl glyoxal on DNA, RNA and protein synthesis in cultured guinea pig keratocytes.

The inhibitory action of methyl glyoxal upon protein, RNA and DNA synthesis in guinea pig keratocytes has been investigated. Methyl glyoxal directly inhibits each of these processes. Inhibition of protein synthesis results from an action upon initiation of translation. Methyl glyoxal induces polysome degradation which is prevented by pretreatment with cycloheximide. Further analysis of translation indicates that elongation and termination are not affected. Inhibition of ribosomal RNA synthesis results from inhibition of maturation. Methyl glyoxal was found to inhibit the appearance of 28S rRNA in the cytoplasm from pulse-labelled precursors. The significance of these results is discussed in relation to cell injury by ketals.

The mechanism of action of 4-hydroxynonenal in cell injury.

The effect of the C9 ketoaldehyde, 4-hydroxynonenal (HNE), a cytotoxic product of lipid peroxidation, on DNA, RNA and protein synthesis has been investigated in cells in culture. Macromolecular synthesis is powerfully inhibited by this agent which readily enters the lipid-rich membranes and is considerably more toxic than the polar ketoaldehyde, methyl glyoxal (MG). The entry of HNE into membranes lowers their glutathione GSH content. This is associated with an increased lipid peroxidation measured in vitro which is blocked by added GSH or alpha-tocopherol. It is proposed that this latter sequence of events is the underlying cause of the cytopathic effect of HNE in cells in culture.

The relationship between the turnover of UTP and RNA synthesis in cultured cells treated with aflatoxin B1.

Methods have been adapted to measure the specific activity of UTP in cells in monolayer culture. In HeLa cells labelled with [3H]uridine and treated with aflatoxin B1 there was reduced radioactivity in crude acid extracts, but the toxin did not affect the radioactive incorporation into UTP. Using cells in which the UTP was pre-labelled, the subsequent addition of aflatoxin B1 inhibited UTP incorporation into RNA. Accordingly aflatoxin B1 did not inhibit the uptake of uridine or the latter's conversion to UTP but inhibited the incorporation of UTP into RNA.