Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age‐related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi‐mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro‐inflammatory cytokines and extracellular matrix–degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi‐related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non‐senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.     

Reciprocal relationships between general (Propofol) anesthesia and circadian time in rats.

Several common postdischarge symptoms, such as sleep disorders, headache, drowsiness or general malaise, evoke disturbances of circadian rhythms due to jet lag (ie crossing time zones) or shift work rotation. Considering that general anesthesia is associated with numerous effects on the central nervous system, we hypothesized that it may also act on the circadian timing system. We first determined the effects of the circadian timing on general anesthesia. We observed that identical doses of propofol showed marked circadian fluctuations in duration of effects, with a peak at the middle of the resting period (ie 7 h after lights on). Then, we examined the effects of general anesthesia on circadian timing, by analysing stable free-running circadian rhythms (ie in constant environmental conditions), an experimental approach used widely in circadian biology. Free-running rats were housed in constant darkness and temperature to assess possible phase-shifting effects of propofol anesthesia according to the time of the day. When administered around (+/-2 h) the daily rest/activity transition point, a 30-min propofol anesthesia induced a 1-h phase advance in the free-running rest-activity rhythm, while anesthesia had no significant resetting effect at other times of the day. Anesthesia-induced hypothermia was not correlated with the phase-shifting effects of propofol anesthesia. From our results, anesthesia itself can reset circadian timing, and acts as a synchronizing cue for the circadian clock.     

Production of a human monoclonal anti-epithelial cell surface antibody derived from a patient with pemphigus vulgaris.

The production of monoclonal autoantibodies derived from individuals with autoimmune diseases constitutes a powerful tool to analyse an autoimmune process at both the antigen and antibody levels. We established a human anti-epithelial cell surface monoclonal antibody by applying hybridoma technology using peripheral blood lymphocytes from a patient with pemphigus vulgaris using a heteromyeloma as the fusion partner. The F12 monoclonal antibody displays four major characteristics: (1) it belongs to the IgM, kappa class; (2) it binds to the cell surface of stratified squamous and simple epithelia; (3) it recognizes an antigenic determinant associated with the desmosomal complex as demonstrated by indirect immunoelectron microscopy; (4) by immunoblotting analysis, it reacts with a 185 kDa polypeptide which was also recognized by a few pemphigus vulgaris sera. Although the F12 monoclonal antibody does not have the immunochemical properties of classical pemphigus vulgaris autoantibodies, several arguments suggest its relevance to the pemphigus vulgaris autoimmune response and, therefore, the heterogeneity of the antigen/antibody systems involved in this autoimmune disorder.     

Role of the EGF-like domains in mammalian tolloid (mTLD) secretion and procollagen C-proteinase activity

Introduction Bone morphogenetic protein (BMP)‐1 and its larger splice variant mammalian tolloid (mTLD) belong to the tolloid group of astacin‐like metalloproteinases that are fundamental to tissue patterning and extracellular matrix assembly. BMP‐1 and mTLD exhibit similar substrate specificity in vitro; however, BMP‐1 is a much better procollagen C‐proteinase than mTLD. mTLD consists of a prodomain (which is cleaved by a furin‐like enzyme) ( Leighton & Kadler 2003 ), a zinc metalloproteinase domain and a C‐terminal part comprising five CUB domains thought to be important for protein–protein interactions ( Hartigan et al. 2003 ), and two EGF‐like domains, which in other proteins are involved in calcium ion binding. BMP‐1 lacks the most C‐terminal two CUB domains and one EGF‐like domain. mTLD activity is known to be calcium ion dependent, as demonstrated for the chick homologue ( Hojima et al. 1985 ). In our current work, we are studying the role of the EGF‐like domains in the secretion and procollagen C‐proteinase activity of mTLD, and the contribution that these domains made to calcium ion dependency. Materials and methods We designed proteins lacking EGF1, EGF2 or both. NotI sites were introduced by PCR at the borders of the EGF domain of a cDNA clone encoding a V5‐His mTLD. Restriction enzyme digestion was used to delete individual domains. The mutant constructs in pCEP4 were stably transfected into 293‐EBNA cells. Expression was analysed by Western blot. The wild‐type and the mutant enzymes were purified on a nickel ion column, and their activity was determined by cleavage of type‐I procollagen in the presence or absence of 5 mm CaCl₂. Results We showed that (1) the mTLD proteins lacking EGF1, EGF2 or EGF1 + EGF2 were poorly secreted into the culture medium compared to mTLD and (2) the EGF deletion mutants remained calcium ion dependent, but some differences were seen. Most notably, the ΔEGF2 and ΔEGF1 + ΔEGF2 mutants were found to be better C‐proteinases than the wild‐type enzyme in the presence of calcium ions. Conclusion From these preliminary data, we concluded that (1) the EGF domains are necessary for efficient secretion (2) both EGF1 and EGF2 domains contribute to the calcium ion dependency of mTLD and (3) the EGF2 domain might be a Ca²⁺‐activated hinge that ‘swings’ the CUB‐4 and CUB‐5 domains away from the active site. The ?EGF2 mTLD might be expected to have an open conformation, thereby making it a better C‐proteinase than the wild‐type enzyme, and (?4) Ca²⁺ ions are bound by other domains in mTLD and not only by the EGF‐like domains.     

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