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Methylazoxymethanol (MAM) and nitrogen mustard (HN2) are two established genotoxicants that reproducibly disrupt neuronal development when administered during the fetal or neonatal period of CNS development (Cattabeni and Di Luca 1997; Ferguson 1996; Graef et al.
Because the majority of neurodevelopmental disorders in children occur during the migration of immature neurons, gene expression profiling was used to identify the specific molecular networks targeted by MAM or the related alkylating agent HN2 in cultures of young postmitotic cerebellar neurons.
We fed cell cultures weekly by adding fresh culture media to the wells and maintained the cells for 7 days (neurons) or 3-4 weeks (astrocytes) before treatment with 10-1,000 [micro]M MAM or 0.1-20 [micro]M mechlorethamine hydrochloride (HN2).
Mouse neuronal and astrocyte cell cultures treated with control media or media supplemented with various concentrations of MAM or HN2 were examined for cell viability using the fluorochrome acetoxymethyl ester, as previously described (Kisby et al.
N7-mG and GMOH levels were determined in samples and from a standard curve (r = 0.99) of CT-DNA alkylated with MAM or HN2, respectively.
Primary cerebellar neuronal cultures treated for 24 hr with MAM or HN2 were examined for DNA fragmentation using TUNEL with the NeuroTacs staining kit according to the manufacturer's instructions (Trevigen, Gaithersburg, MD).