Mapping of a functional recombination motif that defines isotype specificity for mu-->gamma3 switch recombination implicates NF-kappaB p50 as the isotype-specific switching factor.

Ig class switch recombination (CSR) requires expression of activation-induced deaminase (AID) and production of germline transcripts to target S regions for recombination. However, the mechanism of CSR remains unclear. Here we show that an extrachromosomal S plasmid assay is AID dependent and that a single consensus repeat is both necessary and sufficient for isotype-specific CSR. Transfected switch substrates specific for mu-->gamma3 and mu-->gamma1 are stimulated to switch with lipopolysaccharide (LPS) alone or LPS and interleukin-4, respectively. An Sgamma3/Sgamma1 substrate containing only three Sgamma3-associated nucleotides reconstituted LPS responsiveness and permitted mapping of a functional recombination motif specific for mu-->gamma3 CSR. This functional recombination motif colocalized with a binding site for NF-kappaB p50, and p50 binding to this site was previously established. We show a p50 requirement for plasmid-based mu-->gamma3 CSR using p50-deficient B cells. Switch junctions from p50-deficient B cells showed decreased lengths of microhomology between Smu and Sgamma3 relative to wild-type cells, indicating a function for p50 in the mechanics of CSR. We note a striking parallel between the affects of p50 and Msh2 deficiency on Smu/Sgamma3 junctions. The data suggest that p50 may be the isotype-specific factor in mu-->gamma3 CSR and epistatic with Msh2.

Benzodiazepines block alpha2-containing inhibitory glycine receptors in embryonic mouse hippocampal neurons.

Inhibitory glycine receptors (GlyRs) in the mammalian cortex probably contribute to brain development and to maintaining tonic inhibition. Given their presence throughout the cortex, their modulation likely has important physiological consequences. Although benzodiazepines potentiate gamma-aminobutyric acidA receptors (GABAARs), they may also modulate GlyRs because binding studies initially suggested that they act at GlyRs. Furthermore, their diminished ability to potentiate neonatal GABAARs suggests that they may exert their beneficial clinical effects at another site in the developing brain. Therefore we examined the effect of benzodiazepines on whole cell currents mediated by GlyRs in cultured embryonic mouse hippocampal neurons. First, we determined the GlyR subunit composition in this preparation. Glycine, beta-alanine, and taurine activate strychnine-sensitive chloride currents in a dose-dependent manner. Maximal concentrations of the three agonists produce equal, nonadditive responses as expected of full agonists. The pharmacological properties of the GlyR currents including their pattern of modulation by picrotoxinin, picrotin, and tropisetron indicate that GlyRs consist of alpha2beta heteromers and alpha2 homomers. Reverse transcriptase polymerase chain reaction (RTPCR) studies confirmed the presence of alpha2 and beta subunits. Second, we found that micromolar concentrations of some benzodiazepines, including chlordiazepoxide and nitrazepam, inhibit GlyR currents. Nitrazepam inhibition of GlyRs is noncompetitive, is not voltage dependent, and does not reflect enhanced desensitization. Thus benzodiazepines allosterically inhibit alpha2-containing GlyRs in embryonic mouse hippocampal neurons via a "low"-affinity site.