Hydrogen peroxide induces a rapid production of nitric oxide in mung bean (Phaseolus aureus).

Nitric oxide (NO) has recently been identified as an important signaling molecule in plant immune response. The present study aims to investigate the signaling pathway that leads to NO production. Using the NO specific fluorescent dye DAF-2DA, we observed rapid production of NO in mung bean leaves after the addition of 10 mM hydrogen peroxide (H(2)O(2)). NO was probably produced by a NOS-like enzyme in plants, as the NO production was inhibited by l-NAME, a NOS inhibitor. The NOS-like activity in the total leaf protein preparation of mung bean (Phaseolus aureus) was elevated 8.3-fold after 10 mM H(2)O(2) treatment, as demonstrated using the chemiluminescence NOS assay. The NOS-like activity was BH(4) dependent: omitting BH(4) in the reaction mixture of NOS assay reduced the NOS activity by 76%. We also found that the H(2)O(2) induced NO production was mediated via calcium ion flux, as it was blocked in the presence of a calcium ion channel blocker, verapamil. Results from the present study identified H(2)O(2) as an upstream signal that leads to NO production in plants. H(2)O(2) and NO, besides acting as two independent signaling molecules in plant immune response, may interrelate to form an oxidative cell death (OCD) cycle.

The human HMGB1 promoter is modulated by a silencer and an enhancer-containing intron.

The highly conserved, ubiquitous high mobility group protein HMGB1 (formerly named as HMG1) is an architectural transcription factor encoded by a single functional gene in human. HMGB1 is expressed in almost all cell or tissue types studied. In general, it is expressed at a basal level in most cells but at a slightly elevated level of 2--3-fold in actively proliferating tissues or estrogen stimulated breast cancer cells. To understand the regulatory mechanism controlling expression of the human HMGB1 gene, we cloned and analyzed the upstream region as well as the first intron of this gene. We found that transcription of the human HMGB1 gene in the breast cancer MCF-7 cells starts at one major site 57 nucleotides upstream from the first exon-intron boundary. Expression of the human HMGB1 gene is under the control of a very strong TATA-less promoter, which has an activity more than 18-fold that of the SV40 promoter. Immediately upstream, a silencer element is present. This silencer can repress the activity of the HMGB1 promoter down to just one-sixth. The first intron of the human HMGB1 gene contains enhancer elements, which can increase the human HMGB1 promoter activity by 2--3-fold. We postulate that the human HMGB1 gene is capable of being expressed at a very high level. The basal level of expression observed in most cells is probably a result of the strong promoter being held in check by the silencer. The 2--3-fold increase in HMGB1 expression observed in proliferating cells or breast cancer cells stimulated by estrogen may probably result from the action of the enhancer elements in intron 1.

The chicken genome contains no HMG1 retropseudogenes but a functional HMG1 gene with long introns.

We have cloned the genomic sequence coding for the high mobility group 1 (HMG1) protein in chickens. Multiple sequence alignment shows that the chicken HMG1 gene is highly homologous to the human and the mouse HMG1 genes. The gene structure of chicken HMG1 is similar to that of the mouse and the human HMG1 genes, with the same exon-intron boundaries. However, in contrast to other avian genes that have shorter introns, the chicken HMG1 gene has introns that are twice as long as their mammalian homologues. In addition to the functional, intron-containing HMG1 gene, all mammalian genomes contain more than 50 copies of HMG1 retropseudogenes each, while in the chicken genome there are no HMG1 retropseudogenes. This finding suggests that the HMG1 retropseudogenes arose in mammals after their divergence away from the birds.