Feeding and circadian clocks.

The mammalian genome encodes at least a dozen of genes directly involved in the regulation of the feedback loops constituting the circadian clock. The circadian system is built up on a multitude of oscillators organized according to a hierarchical model in which neurons of the suprachiasmatic nuclei of the hypothalamus may drive the central circadian clock and all the other somatic cells may possess the molecular components allowing tissues and organs to constitute peripheral clocks. Suprachiasmatic neurons are driving the central circadian clock which is reset by lighting cues captured and integrated by the melanopsin cells of the retina and define the daily rhythms of locomotor activity and associated physiological regulatory pathways like feeding and metabolism. This central clock entrains peripheral clocks which can be synchronized by non-photic environmental cues and uncoupled from the central one depending on the nature and the strength of the circadian signal. The human circadian clock and its functioning in central or peripheral tissues are currently being explored to increase the therapeutic efficacy of timed administration of drugs or radiation, and to offer better advice on lighting and meal timing useful for frequent travelers suffering from jet lag and for night workers' comfort. However, the molecular mechanism driving and coordinating the central and peripheral clocks through a wide range of synchronizers (lighting, feeding, physical or social activities) remains a mystery.

Clock genes of Mammalian cells: practical implications in tissue culture.

The clock genes family is expressed by all the somatic cells driving central and peripheral circadian rhythms through transcription/translation feedback loops. The circadian clock provides a local time for a cell and a way to integrate the normal environmental changes to smoothly adapt the cellular machinery to new conditions. The central circadian rhythm is retained in primary cultures by neurons of the suprachiasmatic nuclei. The peripheral circadian rhythms of the other somatic cells are progressively dampened down up to loss unless neuronal signals of the central clock are provided for re-entrainment. Under typical culture conditions (obscurity, 37 +/- 1 degrees C, 5-7% CO(2)), freshly explanted peripheral cells harbor chaotic expression of clock genes for 12-14 h and loose, coordinated oscillating patterns of clock components. Cells of normal or cancerous phenotypes established in culture harbor low levels of clock genes idling up to the re-occurrence of new synchronizer signals. Synchronizers are physicochemical cues (like thermic oscillations, short-term exposure to high concentrations of serum or single medium exchange) able to re-induce molecular oscillations of clock genes. The environmental synchronizers are integrated by response elements located in the promoter region of period genes that drive the central oscillator complex (CLOCK:BMAL1 and NPAS2:BMAL1 heterodimers). Only a few cell lines from different species and lineages have been tested for the existence or the functioning of a circadian clockwork. The best characterized cell lines are the immortalized SCN2.2 neurons of rat suprachiasmatic nuclei for the central clock and the Rat-1 fibroblasts or the NIH/3T3 cells for peripheral clocks. Isolation methods of fragile cell phenotypes may benefit from research on the biological clocks to design improved tissue culture media and new bioassays to diagnose pernicious consequences for health of circadian rhythm alterations.

Three-dimensional binding of epidermal growth factor peptides in colonic tissues produced from rotating bioreactor.

Epidermal growth factor peptide binding was analyzed on primary cultures of colonic cells and along crypts by fluorescent laser-scanning confocal microscopy, using a three-dimensional image analysis software (Quant3D, Linux/Unix). Structural, proliferative units from primary cultures grown in rotating bioreactor for 41 d were arranged according to a tubular symmetry or on a parallelepiped sheet. Mean width, height, and depth of 23 tissue-like masses (+/- standard error) were 125 microm (+/-16), 152 microm (+/-23), and 29 microm (+/-3), respectively. Mean density of nuclei in tissue-like masses, expressed as the number of nuclei per cubic millimeter (+/- standard error of the mean), was 1.8 x 10(5) (+/-0.7 x 10(5)) nuclei per cubic millimeter, which corresponded to a density that was five to six times lower than that estimated for the colonic crypt isolated by chelation. Spots of high epidermal growth factor (EGF) peptide binding that corresponded to microlesions in crypt monolayers or to active colonization of microcarriers by epithelial and stromal cells in tissue-like masses were observed. The relative intensities of EGF peptide binding that were obtained below cell position 8 on crypts were very homogeneous and were representative of the profile obtained with crypts isolated from adult rats adapted to a normal diet and used to develop primary cultures of colonocytes in our laboratory. A microscopic multidimensional analytic system to record the expression profiles of biomarkers along intestinal tissues should enhance the use of primary cultures of colonocytes for in vitro testing of new food products.