Shift Work in Rats Results in Increased Inflammatory Response after Lipopolysaccharide Administration: A Role for Food Consumption.

The suprachiasmatic nucleus (SCN) drives circadian rhythms in behavioral and physiological variables, including the inflammatory response. Shift work is known to disturb circadian rhythms and is associated with increased susceptibility to develop disease. In rodents, circadian disruption due to shifted light schedules (jet lag) induced increased innate immune responses. To gain more insight into the influence of circadian disruption on the immune response, we characterized the inflammatory response in a model of rodent shift work and demonstrated that circadian disruption affected the inflammatory response to lipopolysaccharide (LPS) both in vivo and in vitro. Since food consumption is a main disturbing element in the shift work schedule, we also evaluated the inflammatory response to LPS in a group of rats that had no access to food during their working hours. Our results demonstrated that the shift work schedule decreased basal TNF-α levels in the liver but not in the circulation. Despite this, we observed that shift work induced increased cytokine response after LPS stimulation in comparison to control rats. Also, Kupffer cells (liver macrophages) isolated from shift work rats produced more TNF-α in response to in vitro LPS stimulation, suggesting important effects of circadian desynchronization on the functionality of this cell type. Importantly, the effects of shift work on the inflammatory response to LPS were prevented when food was not available during the working schedule. Together, these results show that dissociating behavior and food intake from the synchronizing drive of the SCN severely disturbs the immune response.

The HvDOF19 transcription factor mediates the abscisic acid-dependent repression of hydrolase genes in germinating barley aleurone.

Upon germination of seed cereals, mobilization of the reserves stored in the endosperm is regulated by the phytohormones gibberellins (GA) and abscisic acid (ABA). In barley, the cis regulatory elements and the trans-acting factors mediating the ABA response of hydrolase genes remain elusive. Two new barley genes, HvDof17 and HvDof19, encoding transcription factors (TFs) of the DNA binding with one finger (DOF) class have been characterized and their role upon germination investigated. HvDOF19 binds in a specific manner to the pyrimidine box within the GARC of a thiol-protease gene (Al21), and mediates the ABA repression of this gene in the barley aleurone. Silencing of HvDof19 in transient expression assays diminishes the inhibitory effect of ABA upon expression of the Al21 gene promoter. Transcripts from HvDof17 and HvDof19 accumulate early in germinating aleurones with a peak at 16 h after seed imbibition (hai), whereas the mRNAs of the GA-induced activator GAMYB remain little expressed. At 48 hai, mRNA content of both genes is comparatively insignificant compared with that of GAMYB, which reaches a maximum. Both TFs repress, in transient expression assays, the GA- and the GAMYB-mediated activation of this thiol-protease gene (Al21). In addition, HvDOF17 and HvDOF19 interact with GAMYB in plant cell nuclei, and HvDOF17, but not HvDOF19, interferes with the DNA binding of GAMYB to its target site in the promoter of the Al21 gene. A regulatory model of hydrolase gene expression upon germination is proposed.

The barley cystatin gene (Icy) is regulated by DOF transcription factors in aleurone cells upon germination.

A genomic clone (Icy gene) encoding a barley cystatin has been characterized. The gene contains one intron interrupting its ORF that encodes a protein of 107 amino acid residues. A DNA fragment of -1058 bp upstream of the ATG translation initiation codon has been sequenced and several promoter deletions fused to the beta-glucuronidase (GUS; uidA gene) reporter gene obtained. Transient expression assays in different barley tissues have indicated that a -631 bp promoter fragment was sufficient for full activity. In bombarded barley aleurone layers the GUS-driven expression by this promoter is repressed by GA(3) incubation, as is the accumulation of the Icy transcripts detected. A spatial and temporal location of cystatin transcripts by in situ hybridization techniques indicates that this gene is ubiquitously expressed and its transcripts are particularly abundant in leaves and roots, and in seeds, both during development and upon germination. Two DOF transcription factors, SAD and BPBF, previously described as involved in gene expression regulation in seeds, interact specifically in vitro with oligonucleotides containing DOF binding-sites derived from the Icy gene promoter. Transient expression experiments in co-bombarded aleurone layers demonstrate that BPBF strongly represses transcription of the native Icy promoter, even when co-transfected with SAD that behaves as an activator in this in vivo system.