The opioid antagonist naltrexone inhibits activity and alters expression of alpha7 and alpha4beta2 nicotinic receptors in hippocampal neurons: implications for smoking cessation programs.

This study was designed to investigate whether naltrexone, an opioid antagonist that has been evaluated clinically as a co-adjuvant in smoking cessation programs, affects function and expression of neuronal nicotinic receptors (nAChRs). Whole-cell current recordings from rat hippocampal neurons in culture and in slices demonstrated that alpha7 nAChRs can be inhibited non-competitively by naltrexone (IC(50) approximately 25 microM). The voltage dependence of the effect suggested that naltrexone acts as an open-channel blocker of alpha7 nAChRs. Naltrexone also inhibited activation of alpha4beta2 nAChRs in hippocampal neurons; however its IC(50) was higher ( approximately 141 microM). At a concentration as high as 300 microM (which is sufficient to block by 100% and 70% the activity of alpha7 and alpha4beta2 nAChRs, respectively), naltrexone had no effect on kainate and AMPA receptors, blocked by no more than 20% the activity of NMDA and glycine receptors, and reduced by 35% the activity of GABA(A) receptors. A 3-day exposure of cultured hippocampal neurons to naltrexone (30 microM) or nicotine (10 microM, a concentration that fully desensitized alpha7 nAChRs) resulted in a 2-fold increase in the average amplitude of alpha7 nAChR-subserved currents. Naltrexone did not augment the maximal up-regulation of alpha7 nAChRs induced by nicotine, indicating that both drugs act via a common mechanism. In addition to increasing alpha7 nAChRs-mediated responses per neuron, nicotine increased the number of neurons expressing functional non-alpha7 nAChRs (probably alpha4beta2 nAChRs); this effect was blocked by naltrexone (0.3 and 30 microM). Therefore, naltrexone may affect dependence on cigarette smoking by differentially altering function and expression of alpha7 and alpha4beta2 nAChRs in the central nervous system.

Interdependence of the position and orientation of SoxS binding sites in the transcriptional activation of the class I subset of Escherichia coli superoxide-inducible promoters.

SoxS is the direct transcriptional activator of the member genes of the Escherichia coli superoxide regulon. At class I SoxS-dependent promoters, e.g. zwf and fpr, whose SoxS binding sites ('soxbox') lie upstream of the -35 region of the promoter, activation requires the C-terminal domain of the RNA polymerase alpha-subunit, while at class II SoxS-dependent promoters, e.g. fumC and micF, whose binding sites overlap the -35 region, activation is independent of the alpha-CTD. To determine whether SoxS activation of its class I promoters shows the same helical phase-dependent spacing requirement as class I promoters activated by catabolite gene activator protein, we increased the 7 bp distance between the 20 bp zwf soxbox and the zwf -35 promoter hexamer by 5 bp and 11 bp, and we decreased the 15 bp distance between the 20 bp fpr soxbox and the fpr -35 promoter hexamer by the same amounts. In both cases, displacement of the binding site by a half or full turn of the DNA helix prevented transcriptional activation. With constructs containing the binding site of one gene fused to the promoter of the other, we demonstrated that the positional requirements are a function of the specific binding site, not the promoter. Supposing that opposite orientation of the SoxS binding site at the two promoters might account for the positional requirements, we placed the zwf and fpr soxboxes in the reverse orientation at the various positions upstream of the promoters and determined the effect of orientation on transcription activation. We found that reversing the orientation of the zwf binding site converts its positional requirement to that of the fpr binding site in its normal orientation, and vice versa. Analysis by molecular information theory of DNA sequences known to bind SoxS in vitro is consistent with the opposite orientation of the zwf and fpr soxboxes.

Characterization of the recombinant MutY homolog, an adenine DNA glycosylase, from yeast Schizosaccharomyces pombe.

The mutY homolog (SpMYH) gene from a cDNA library of Schizosaccharomyces pombe encodes a protein of 461 amino acids that displays 28 and 31% identity to Escherichia coli MutY and human MutY homolog (MYH), respectively. Expressed SpMYH is able to complement an E. coli mutY mutant to reduce the mutation rate. Similar to E. coli MutY protein, purified recombinant SpMYH expressed in E. coli has adenine DNA glycosylase and apurinic/apyrimidinic lyase activities on A/G- and A/7,8-dihydro-8-oxoguanine (8-oxoG)-containing DNA. However, both enzymes have different salt requirements and slightly different substrate specificities. SpMYH has greater glycosylase activity on 2-aminopurine/G and A/2-aminopurine but weaker activity on A/C than E. coli MutY. Both enzymes also have different substrate binding affinity and catalytic parameters. Although SpMYH has great affinity to A/8-oxoG-containing DNA as MutY, the binding affinity to A/G-containing DNA is substantially lower for SpMYH than MutY. SpMYH has similar reactivity to both A/G- and A/8-oxoG-containing DNA; however, MutY cleaves A/G-containing DNA about 3-fold more efficiently than it does A/8-oxoG-containing DNA. Thus, SpMYH is the functional eukaryotic MutY homolog responsible for reduction of 8-oxoG mutational effect.

Ambidextrous transcriptional activation by SoxS: requirement for the C-terminal domain of the RNA polymerase alpha subunit in a subset of Escherichia coli superoxide-inducible genes.

Purified MalE-SoxS fusion protein specifically stimulated in vitro transcription of the Escherichia coli zwf, fpr, fumC, micF, nfo, and sodA genes, indicating that activation of the superoxide regulon requires only SoxS. As in vivo, a 21 bp sequence adjacent to the zwf promoter was able to activate transcription of an heterologous promoter in vitro. Activation of zwf and fpr transcription required the C-terminal domain (CTD) of the RNA polymerase alpha subunit, while stimulation of fumC, micF, nfo, and sodA transcription was independent of CTD truncation. Thus, like the catabolite gene activator protein (CAP), SoxS is an 'ambidextrous' activator, activation only requiring the alpha CTD in a subset of regulated promoters. Indeed, the -35 hexamers of the zwf and fpr promoters lie downstream of the respective MalE-SoxS binding sites, while the binding sites of fumC, micF, nfo, and sodA overlap their -35 promoter hexamers.

Genetic definition of the Escherichia coli zwf "soxbox," the DNA binding site for SoxS-mediated induction of glucose 6-phosphate dehydrogenase in response to superoxide.

In Escherichia coli K-12, transcription of zwf, the gene for glucose 6-phosphate dehydrogenase, is subject to growth rate-dependent regulation and is activated by SoxS in response to superoxide stress. To define genetically the site of SoxS activation, we undertook a detailed deletion analysis of the zwf promoter region. Using specifically targeted 5' and 3' deletions of zwf sequences, we localized the SoxS activation site to a 21-bp region upstream of the zwf promoter. This minimal "soxbox" was able to confer paraquat inducibility when placed upstream of a normally unresponsive gnd-lacZ protein fusion. In addition, we used these findings as the basis for resecting unnecessary sequences from the region upstream of the promoters of two other SoxS-regulated genes, sodA and nfo. Like the zwf soxbox, the regions required for SoxS activation of sodA and nfo appear to lie just upstream or overlap the -35 hexamers of the corresponding promoters. Importantly, the sequence boundaries established here by deletion analysis agree with the primary SoxS recognition sites of zwf, sodA, and nfo that we previously identified in vitro by gel mobility shift and DNase I protection assays with a purified MalE-SoxS fusion protein.

Purification of a MalE-SoxS fusion protein and identification of the control sites of Escherichia coli superoxide-inducible genes.

In Escherichia coli, the soxRS genes effect the cell's defence against superoxide by activating the transcription of more than 14 genes, including zwf, sodA, nfo, micF and fumC. Previous work from other laboratories has indicated that SoxR is the sensor of oxidative stress and induces synthesis of SoxS, which in turn activates transcription of the regulon's target genes. Although SoxS appears to be a DNA-binding protein, its ability to bind to the promoter regions of target genes has not been demonstrated. To facilitate purification and characterization of SoxS, we constructed a fusion of soxS to malE, which encodes maltose-binding protein, and demonstrated that the in vivo expression of the MalE-SoxS fusion protein can provide SoxS function to a soxRS deletion mutant. We purified the fusion protein by affinity chromatography on an amylose column. The purified fusion protein stimulated in vitro expression of zwf in a coupled transcription-translation system and formed specific complexes with DNA fragments carrying target gene promoters. Moreover, MalE-SoxS protected from DNase I attack 22-27 bp segments immediately adjacent to or overlapping the -35 hexamers of the zwf, sodA, nfo, micF, and fumC promoters. The protected regions revealed a consensus 'soxbox' sequence.

Molecular characterization of mutations affecting expression level and growth rate-dependent regulation of the Escherichia coli zwf gene.

We characterized three cis dominant mutations which elevate glucose 6-phosphate dehydrogenase level. Growth rate-dependent regulation and oxidative stress control of enzyme level were altered by the mutations. DNA sequencing and transcript mapping showed that the "up" mutations created new promoters whose hyperactive expression overrides the normal regulation of the native promoter.