Evaluation of light-emitting diodes (LED) effect on skin biology (in vitro study).

BACKGROUND: Interest in anti-aging approaches has grown significantly in recent years. The most popular are the non invasive methods to decrease the signs of aging. One such method is LED-based therapy. METHODS: This study investigated the potential of two different wavelengths, 590 nm and 630 nm, combined or not, in the photobiomodulation of proteins involved in the slowdown of the skin aging. RESULTS: These in vitro results on cell viability, cell shape, and mitochondrial function support and build on previous studies suggested that LED treatment is safe. Regarding its biological functions, our data indicated that the combination of two different wavelengths acted in synergy to enhance the impact of each irradiation alone. Combined, the LED wavelengths could improve in vitro the cell shape, the cell proliferation, and the level of major proteins involved in the healing process. CONCLUSION: These benefits may lead to reinforcement of the skin organization and structure. This hypothesis will be checked in future clinical studies.

Maintenance of the ubiquitin-proteasome system activity correlates with visible skin benefits.

Researches on longevity and anti-ageing molecules have clearly evidenced the potential to increase lifespan of the cells. These recent scientific data raise interests and questions on the capacity of the cells to live longer and maintain their fundamental mechanisms of protection, reparation or degradation of abnormal proteins to maintain their capital of healthy and functional cellular activity. In this concern, this study was focused on the ubiquitin-proteasome system as an essential cellular tool to maintain the pool of functionally active proteins allowing renewal of proteins and degradation of damaged proteins. As the proteasome keeps the 'cells health capital', it should be particularly interesting to associate the maintenance of the proteasome activity with increasing longevity. Indeed, although oxidative stress damage increases with ageing leading to collagen and cellular membrane alterations, it also leads to a reduction in the proteasome activity which is critical for the cells. The aim of this study was to better understand the cellular role of the proteasome and to provide new data showing the skin beneficial effects in activating the overall system of ubiquitination and proteasomal degradation. For this purpose, in vitro, ex vivo and in vivo experiments were performed to evaluate the effects of maintaining the ubiquitin-proteasome activity in basal and stress conditions on young versus aged cells. Experiments have included evaluation of a newly developed dimerized tripeptide targeting specifically the ubiquitin-proteasome pathway. Our results have demonstrated that maintenance of this essential mechanism that participates in abnormal protein elimination and protein renewal allows maintaining cellular integrity that correlates with visible skin benefits.

The rat dermorphin-like immunoreactivity is supported by an aminopeptidase resistant peptide.

Site-directed antibodies against synthetic related dermorphin peptides were previously produced and characterized. One of them, which specifically recognizes the crucial 'opioid message' (the N-terminal part of the dermorphin molecule (i.e. Tyr-D-Ala-Phe-Gly) was selected in order to detect and locate endogenous dermorphin-like molecules in rat, mouse and guinea pig tissues. Dermorphin-like peptides were found to be present in tissues known to contain peptides such as neurons in the central nervous system, nerve fibers in the gut and B and T immune cells. With all the tissues assayed, the HPLC profile obtained on the immunoreactive material showed the same main peak eluted at a retention time of 32 +/- 1 min. The results of biochemical experiments in which enzymatic treatments were performed on the dermorphin-like immunoreactivity indicate the immunoreactivity is a peptide resistant to aminopeptidase hydrolysis. This finding suggests the presence of a residue conferring resistance to proteolytic processes of this kind, which is likely to be a D-amino acid residue.

Hemorphin peptides are released from hemoglobin by cathepsin D. radioimmunoassay against the C-part of V-V-hemorphin-7: an alternative assay for the cathepsin D activity.

In order to investigate the putative physiological role of the in vivo release of hemorphins from hemoglobin in tissues, an immunological approach was developed. Specific and sensitive antiserum were raised against the C-part of the V-V-hemorphin-7. The antisera recognized to the same extent the related hemorphins V-V-hemorphin-7 and L-V-V-hemorphin-7. The validity of our immunological approach was analyzed by studying the in vitro release of hemorphin from hemoglobin by cathepsin D and compared to the pepsin hydrolysis. These two enzymes led to the release of these same products suggesting that cathepsin D acted as an accurate pepsin-like enzyme. Moreover, considering the poor sensitivity of the available methods of detection for the in vitro Cathepsin D activity, our specific and sensitive V-V-hemorphin-7 radioimmunoassay seems to be a useful alternative assay for this enzymatic activity.

Production and characterization of site-directed antibodies against dermorphin and dermorphin-related peptides.

To detect and purify endogenous dermorphin-like molecules in mammalian tissues, an immunological approach was developed. Site-directed antibodies against synthetic dermorphin and related dermorphin peptides were produced. The immunogenic forms of dermorphin were selected to obtain antibodies recognizing different epitopes overlapping the whole dermorphin molecule. One of them specifically recognized the crucial "opioid message" (the N-terminal part of the molecule), which is required for a ligand to exert its full opioid activity. The validity of our immunological approach was analyzed by studying the dermorphin-related peptide distribution in Phyllomedusa sauvagei skin. The finding that tetrapeptide Y-A-G-F-OH was present in Phyllomedusa sauvagei extracts suggested that either the Tyr3-Pro6 peptidic bond may be relatively unstable or endogenous proteolytic enzymes present in Phyllomedusa skin may inactivate this peptidic bond.

Anti-idiotypic antibodies: a useful alternative for studying the biochemical expression of mu/delta opioid binding sites in mammalian brain.

A polyclonal antiserum was produced against opioid binding sites using an anti-idiotypic approach whereby antibodies directed against the opioid agonist DSLET were used as immunogen. The anti-idiotypic antiserum recognized specific brain proteins with molecular masses of 76 +/- 4, 73 +/- 4 and 59 +/- 3 kDa, respectively. The immunolabeling of these proteins was mainly inhibited by mu, delta opioid agonists and a general antagonist, naloxone. The inhibition of immunoprecipitation by opioid agonists and antagonist and the developmental expression of these immunoreactive proteins found to occur during brain ontogeny strongly suggest that these three proteins were mu, delta but not kappa opioid binding sites. The anti-idiotypic antiserum both inhibits 3H-DADLE binding and mimics the inhibitory agonist effects on the stimulated cAMP level of NG 108-15 cells which expressed delta opiate receptors. Numerous mammalian brain opioid binding sites were labeled, due to the fact that the binding site was the epitope recognized by the anti-idiotypic antibodies. From the numerous studies performed with a view to characterizing the specificity of the anti-idiotypic antibodies, it was strongly suggested that the anti-idiotypic antibodies specifically recognize mu/delta opioid binding sites and they can therefore be powerful tools for studying the biochemical expression of these opioid binding sites in mammalian brains.

Distribution of enkephalin immunoreactivity in sympathetic prevertebral ganglia and digestive tract of guinea-pigs and rats.

The aim of the present study was to determine the distribution of methionine-enkephalin (ME) and leucine-enkephalin (LE) immunoreactivity in the sympathetic prevertebral ganglia (coeliac plexus and inferior mesenteric ganglion) and in the myenteric plexus-muscular layer complex of the digestive tract in guinea-pigs and rats. This study was performed using the same immunological approaches including radioimmunoassays and HPLC characterization as those used previously on cats in order to be able to make inter-region and inter-species comparisons. In rat and guinea-pig prevertebral ganglia, the distributions of the enkephalin immunoreactivities were comparable and were characterized by a low ME/LE concentration ratio, of less than 1. In the digestive tract of rats, the enkephalin immunoreactivities were homogeneously distributed, whereas in guinea-pigs, they were found to be very low in the lower oesophageal sphincter and high in the duodenum. In both species, the ME/LE concentration ratio was around 2. The ME/LE concentration ratio determined in the present study in peripheral nervous structures was much lower than that determined previously in the rat brain. Radioimmunoassay and biochemical data might indicate that different mechanisms are responsible for the processing and/or degradation of enkephalins in the central and peripheral nervous systems. The present study provides further evidences that there are tissue- and species-dependent differences in the distribution of enkephalin immunoreactivities. These differences should be taken into consideration when dealing with the effects and the role of enkephalins in the nervous control of intestinal motility in mammals.

Ultrastructural study of delta-opioid receptors in the dorsal horn of the rat spinal cord using monoclonal anti-idiotypic antibodies.

The ultrastructural localization of delta-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti-D-Ala2-D-Leu5-enkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, delta or mu, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3-4 h before addition of the anti-idiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10(-9) M while dermorphin or dermorphin Lys7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between the two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for delta-opioids in the spinal cord, providing that these receptors are functional. delta-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.

Purification and characterization of enkephalin-related peptides released by in vitro peptic digestion of bovine plasma proteins.

In vitro pepsin treatment of plasma proteins generates biologically active peptides such as enkephalin-related peptides. These peptides were characterized using chromatographic techniques along with a radioimmunoassay procedure involving the use of Leu-enkephalin and Met-enkephalin antisera. Serum albumin is the only existing source of Met-enkephalin-immunoreactive peptides. One of these peptides consists of nine residues with the sequence NH2-Glu-Lys-Leu-Gly-Glu-Tyr-Gly-Phe-Gln; a second immunoreactive peptide might be the hexapeptide NH2-Gly-Glu-Tyr-Gly-Phe-Gln, which has been already identified in a rat serum albumin hydrolysate. Our results indicate that immunoglobulins constitute the main source of Leu-enkephalin-immunoreactive peptides. Immunoreactive NH2-Tyr-Phe-Leu was isolated from pepsin-treated bovine immunoglobulins. Binding experiments and cyclic nucleotide measurements suggested that this peptide was an enkephalin-related peptide. Similar experiments could be carried out to identify the proteins that contain enkephalin-like peptide sequences with the view to investigating the various biological processes occurring in enzymatically treated proteins.

Chromatographic analysis of the enkephalin immunoreactivity in the nucleus locus coeruleus of the cat after local colchicine administration.

Cell bodies immunoreactive for methionine- and leucine-enkephalin are found in the area of the locus coeruleus (dorsolateral pons) of the cat after injection of colchicine in the ascending projections of the nucleus. Using radioimmunoassay procedures, it is shown that colchicine induces a significant increase in methionine- and leucine-enkephalin-immunoreactive material in this area of the brain. High pressure liquid chromatography analysis demonstrated that the immunoreactive materials were authentic methionine- and leucine-enkephalin. The methionine- and leucine-enkephalin patterns were identical in the colchicine injected and non-injected sides of the dorsolateral pons. It is suggested that, in this area of the brain, colchicine (i) does not significantly modify the processing of proenkephalin to form the pentapeptides methionine- and leucine-enkephalin, and (ii) does not induce the appearance of new substances reactive to the enkephalin antisera employed.