Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A- and sigma B-dependent stress-responsive promoters.

The opuE gene from Bacillus subtilis encodes a transport system (OpuE) for osmoprotective proline uptake and is expressed from two osmoregulated promoters: opuE P-1 recognized by the vegetative sigma factor A (sigma A and opuE P-2 dependent on the stress-induced transcription factor sigma B (sigma B). The contributions of these two promoters to osmoregulation of opuE were analysed. Genetic studies using chromosomal opuE-treA operon fusions revealed that opuE transcription is rapidly induced after an osmotic upshock. The strength of opuE expression is proportionally linked to the osmolarity of the growth medium. Deletion analysis of the opuE regulatory region identified a 330 bp DNA segment carrying all sequences required in cis for full and osmoregulated transcription. The proper rotational orientation of the upstream region present within this fragment was essential for the function of both opuE promoters. Mutant opuE-treA fusions with defects in either the sigma A-or the sigma B-dependent promoters revealed different contributions of these sequences to the overall osmoregulation of opuE. opuE P-2 (sigma B) activity increased transiently after an osmotic upshock and did not significantly contribute to the level of opuE expression in cells subjected to long-term osmotic stress. In contrast, transcription initiating from opuE P-1 (sigma A) rose in proportion to the external osmolarity and was maintained at high levels. Moreover, both promoters exhibited a different response to the osmoprotectant glycine betaine in the medium. Our results suggest that at least two different signal transduction pathways operate in B. subtilis to communicate osmotic changes in the environment to the transcription apparatus of the cell.

Urinary 6-sulphatoxymelatonin excretion reflects pineal melatonin secretion in the Djungarian hamster (Phodopus sungorus).

To monitor pineal function in the Djungarian hamster (Phodopus sungorus), we measured the urinary excretion of the melatonin metabolite 6-sulphatoxymelatonin (aMT6s) at 3-hr intervals by radioimmunoassay. Hamsters maintained in either long photoperiod (LP, LD 16:8) or short photoperiod (SP, LD 8:16) showed marked daily rhythms in aMT6s excretion, with elevated levels during the dark phase. In both photoperiods, we found large interindividual differences, mainly in the amplitude of the signal. However, the amplitude as well as the duration of nocturnal aMT6s excretion was higher in SP than in LP. Light exposure at night (180 mW/m2, 30 min) caused a decrease in aMT6s excretion, indicating that the pineal gland is the major source of urinary aMT6s. Moreover, there was a significant correlation between nocturnal pineal/plasma melatonin contents and 24-hr aMT6s excretion. We conclude that, measurements of aMT6s provide a valid and quantitative index of pineal melatonin synthesis in this hamster species. As an advantage in determining pineal melatonin contents, this approach will allow noninvasive long-term studies of individual animals under varying environmental conditions.

Short isoform of POU factor Brn-3b can form a heterodimer with Brn-3a that is inactive for octamer motif binding.

The POU proteins Brn-3a and Brn-3b belong to a family of DNA binding transcription factors that share stretches of extensive homology. Both Brn-3a and Brn-3b are expressed as shorter and longer isoforms. The long form of Brn-3a is able to oncogenically transform primary fibroblasts. By contrast, the short form of Brn-3b (Brn-3b(s)) cannot transform fibroblasts but is able to specifically inhibit the transforming activity of Brn-3a(1). Moreover, Brn-3a(1) can act as a transcriptional transactivator, while Brn-3b(s) is not only unable to do so but in addition specifically inhibits the tranactivating activity of Brn-3a(1). Here, we show that the opposite and antagonistic activities of Brn-3a(1) and Brn-3b(s) proteins are due to their different DNA binding properties; Brn-3a(1) but not Brn-3b(s) can form stable complexes with several octamer-related target DNA sequences. The presence of Brn-3b(s) completely inhibits the binding of Brn-3a(1) to DNA by preventing the formation of Brn-3a(1)-DNA complexes as well as by disrupting preformed complexes. Experiments with GST fusion proteins and in vitro binding studies suggest that the inhibition of Brn-3a(1) activity by Brn-3b(s) occurs via direct interaction of the two transcription factors in solution. Therefore, we hypothesize that Brn-3b(s) can act as a direct antagonist of Brn-3a(1) by inhibiting its DNA binding through the formation of an inactive hetero-oligomeric complex.

Multiple regulatory steps are involved in the control of lipoprotein lipase activity in brown adipose tissue.

Lipoprotein lipase (LPL) supplies brown adipose tissue with fatty acids for nonshivering thermogenesis. In brown adipose tissue of the Djungarian hamster we studied i) the molecular mechanisms involved in cold-induced stimulation of LPL activity, ii) the adrenergic control of LPL expression, and iii) compared LPL expression in brown and white adipose tissue. i) After 8 h cold exposure we detected a 2-fold increase in LPL activity and protein level in brown adipose tissue, whereas LPL mRNA level remained unchanged. A cold-induced increase (1.5-fold) in LPL activity was observed in brown adipose tissue of hamsters treated with actinomycin D prior to 4 h cold exposure, whereas cycloheximide treatment completely abolished LPL stimulation. Thus, these data suggest that during the initial phase (< 24 h) of cold exposure the stimulation of LPL activity in brown adipose tissue is most likely due to increased translation. In contrast, during prolonged cold exposure, we detected a maximal 7-fold increase in LPL activity and a 2- to 3-fold increase in LPL mRNA level in brown adipose tissue indicating LPL regulation at the pretranslational level. Furthermore, comparison of LPL protein and activity in brown adipose tissue during prolonged (> 24 h) cold exposure provides some evidence that the active fraction of the enzyme pool in brown adipose tissue is increased in response to cold. ii) Surgical denervation and noradrenaline treatment revealed a complex role of the sympathetic innervation in the control of LPL expression in brown adipose tissue. Denervation decreased LPL mRNA level, but increased LPL activity. Noradrenaline treatment stimulated LPL activity to a similar extent as cold exposure. However, cold-induced stimulation of LPL activity was not impaired by denervation. iii) Cold exposure significantly elevated LPL mRNA content of inguinal white adipose tissue, although LPL activity was not affected. Posttranscriptional mechanisms appear to be involved in the tissue specific control of LPL expression.