Conformation of the c-Fos/c-Jun complex in vivo: a combined FRET, FCCS, and MD-modeling study.

The activator protein-1 transcription factor is a heterodimer containing one of each of the Fos and Jun subfamilies of basic-region leucine-zipper proteins. We have previously shown by fluorescence cross-correlation spectroscopy (FCCS) that the fluorescent fusion proteins Fos-EGFP and Jun-mRFP1, cotransfected in HeLa cells, formed stable complexes in situ. Here we studied the relative position of the C-terminal domains via fluorescence resonance energy transfer (FRET) measured by flow cytometry and confocal microscopy. To get a more detailed insight into the conformation of the C-terminal domains of the complex we constructed C-terminal labeled full-length and truncated forms of Fos. We developed a novel iterative evaluation method to determine accurate FRET efficiencies regardless of relative protein expression levels, using a spectral- or intensity-based approach. The full-length C-terminal-labeled Jun and Fos proteins displayed a FRET-measured average distance of 8 +/- 1 nm. Deletion of the last 164 amino acids at the C-terminus of Fos resulted in a distance of 6.1 +/- 1 nm between the labels. FCCS shows that Jun-mRFP1 and the truncated Fos-EGFP also interact stably in the nucleus, although they bind to nuclear components with lower affinity. Thus, the C-terminal end of Fos may play a role in the stabilization of the interaction between activator protein-1 and DNA. Molecular dynamics simulations predict a dye-to-dye distance of 6.7 +/- 0.1 nm for the dimer between Jun-mRFP1 and the truncated Fos-EGFP, in good agreement with our FRET data. A wide variety of models could be developed for the full-length dimer, with possible dye-to-dye distances varying largely between 6 and 20 nm. However, from our FRET results we can conclude that more than half of the occurring dye-to-dye distances are between 6 and 10 nm.

Involvement of the caudal raphe nuclei in the feeding behavior of rats

Involvement of the caudal raphe nuclei (raphe pallidus, RPa; raphe magnus, RMg, and raphe obscurus, ROb) in feeding behavior of adult rats was studied by measuring c-Fos protein expression, in animals submitted to the "meal-feeding" model of food restriction in which the rats were fed ad libitum only from 7:00 to 9:00 h, for 15 days. The experimental groups submitted to chronic fasting, named 'search for food' (SF), 'ingestion of food' (IF) and 'satiety of food' (SaF) were scheduled after a previous study in which the body weight and the general and feeding behaviors were evaluated by daily monitoring. Acute, 48-h fasting (AF) was used as control. In the chronic group, the animals presented a 16 percent reduction in body weight in the first week, followed by a continuous, slow rise in weight over the subsequent days. Entrainment of the sleep-wake cycle to the schedule of food presentation was also observed. The RPa was the most Fos immunopositive nucleus in the chronic fasting group, followed by the RMg. The ANOVA and Tukey test (P<0.05) confirmed these results. The IF group was significantly different from the other three groups, as also was the number of labeled cells in the RPa in SF and IF groups. Nevertheless, no significant difference was observed between RMg and RPa, or RMg and ROb in the SaF and AF. However, it is interesting to observe that the groups in which the animals were more active, searching for or ingesting food, presented a larger number of labeled cells. These results suggest a different involvement of the caudal raphe nuclei in the somatic and autonomic events of feeding behavior, corroborating the functions reported for them earlier

Ultrastructure of Fos-labeled neurons relating to nociceptive primary afferent and substance P terminals in rat spinal superficial laminae.

The presence of Fos-labeled neurons at ultrastructural level was confirmed in the spinal superficial laminae following an injection of formalin into rat hindpaw in the present study. The Fos-like immunoreactive products were found exclusively in regions associated with the euchromatin in the nuclei of Fos-labeled neurons. By the methods used-of anterograde transport of horseradish peroxidase conjugated to wheat-germ agglutinin and immunocytochemistry-it was observed that some Fos-labeled neuronal bodies received synaptic contacts from, or were apposed directly to, small diameter primary afferent terminals in the spinal superficial laminae. By means of double-labeled immunocytochemistry, a direct apposition was often observed and a synaptic relationship was occasionally found between Fos-labeled neuronal bodies and substance P-like immunoreactive terminals.

[Molecular factors of cerebral hypoxia-ischemia]. / Factores moleculares en la hipoxia-isquemia cerebral.

In recent years much has been learned about the cellular and molecular events underlying cerebral hypoxia-ischemia (HI). We review, from a molecular standpoint, the main pathogenetic theories in hypoxic-ischemic cerebral injury, including excitotoxicity, free radical damage, and the role of growth factors, proto-oncogenes and heat shock proteins. The various forms of cell death in the developing and adult brain (necrosis, apoptosis and delayed neuronal death) are reviewed, with an emphasis on gene regulation of naturally-occurring and HI-associated cell death. We report the expression of the immediate early gene c-fos and c-jun mRNAs and of HSP72 mRNA and protein in several models of cerebral HI. Gel agarose electrophoresis of extracted DNA and in situ end-labeling of fragmented DNA revealed that cell death in these models was associated with endonuclease(s) activation. We also pre-treated some animals with dexamethasone, a neuroprotective drug in a model of perinatal HI. High-dose dexamethasone prevented c-fos induction in cerebral regions sensitive to HI. This effect may be due to a functional antagonism, at the transcriptional level, between Fos and the glucocorticoid receptor.