We have examined the methylation profiles of the asparagine synthetase (ASY) promoter in a number of human leukemic cell lines in relation to their asparagine (ASN) requirements in vitro. Cells in which the promoter is highly methylated are auxotrophs and express ASY at very low levels. Electromobility shift assays (EMSA) of nuclear extracts with oligomers from the promoting region show, in addition to recognized transcription factor binding, a novel methyl binding protein specific for a 12 base consensus sequence, which includes a single methylated CpG. This sequence overlaps that of the amino-acid response unit of the ASY promoter, which is activated byATF4 and C/EBP. Competition by the methyl binding protein could account for the observed failure of the methylated promoter to bind these transcription factors and consequently, although other mechanisms can also be operative, for the specific repression of the gene. The ASY methyl binding protein (ASMB) is present in leukemic lymphoid and myeloid cells irrespective of their methylation status, and in normal lymphocytes after phytohemagglutinin stimulation. It has been purified by affinity chromatography and has a molecular size of 40 kDa in 10% SDS-polyacrylamide gels.

We report the validation of a reversed-phase gradient HPLC method allowing simultaneous quantification of retinol, retinyl esters, tocopherols and selected carotenoids in lung, liver and plasma of mouse, rat and guinea pig (gp) using a diode array detector. A significant species difference was observed regarding the distribution of retinol and retinyl esters. The levels of total retinol in lung, liver and plasma were in the following order: mouse >> rat > gp; rat >mouse > gp; and gp >> rat > mouse, respectively. Furthermore, comparison studies revealed similarities between the vitamin A profiles of human and gp lung samples.

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