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.     

Anti-idiotypic antibodies against a monoclonal antibody specific for the trichothecene mycotoxin T-2

A BALB/c murine monoclonal antibody against the trichothecene mycotoxin T-2 was generated. The antibody, designated HD11, specifically bound T-2 mycotoxin. The binding of HD11 to T-2 conjugated to bovine serum albumin was inhibited by free T-2 toxin but not by the water-soluble heterocyclic guanidines saxitoxin and tetrodotoxin. The T-2 detection limit in an enzyme-linked immunosorbent assay with HD11 was in the nanogram range. The in vitro cytotoxicity of T-2, as measured by the inhibition of radiolabeled leucine uptake of the human epidermoid carcinoma Hep-2 and KB cell lines, was completely reversed by the addition of HD11. Rabbit anti-idiotypic antibodies specific for HD11 were generated and characterized.     

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.     

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.     

Why are children living in poverty getting fatter?

In recent decades, there has been a dramatic increase in unhealthy weight for both children and adults. The Canadian standard of living has changed in favour of more easily prepared, calorie-dense foods and sedentary practices. Many family characteristics have also changed over the past 50 years. More Canadian families are living in disadvantaged situations, forecasting a host of unhealthy behaviours and attitudes in adults. The poor are not only getting poorer, they are also becoming heavier. Children from disadvantaged families seem to be leading the trend in increasing prevalence of unhealthy weight. Because they live in neighbourhoods that are perceived as unsafe, these children are likely spending more time indoors. This is associated with watching more television, which not only displaces other forms of educational and active entertainment but also places them at risk of learning inaccurate information about proper eating. Social science research helps identify factors contributing most to the rise in excess weight within this population, thus providing essential clues for effective approaches to its eradication.     

Interstitial telomeric DNA sequences of Chinese hamster cells are hypersensitive to nitric oxide damage, and DNA-PKcs has a specific local role in its repair

The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was used to analyze DNA single-strand breaks (SSBs) and alkali-labile sites induced by exposure to the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3-morpholinosydnomine hydrochloride (SIN-1) in the whole genome and in long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cells. The relative density of DNA damage generated in the ITRS by X-rays was similar to that induced in the genome overall, whereas it was 1.7 times higher when the alkylating agent MNNG was assayed. Nevertheless, after SNP or SIN-1 treatment, ITRSs proved to be 2.8 and 2.7 times relatively more damaged, respectively, than the whole genome. When the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was not active, as in XR-C1 mutant cells, the repair kinetics in the whole genome did not differ from that in the parental cell line with X-ray or SNP exposure. However, whereas the SSBs and alkali-labile sites induced in the ITRS by X-rays exhibited rejoining kinetics similar to that of the parental cell line, the damage induced by SNP was more slowly rejoined. This implies a role for DNA-PKcs in the repair of DNA damage induced by NO, especially in ITRSs. The results demonstrated intragenomic heterogeneity of NO-induced DNA damage and repair; there was a higher density of DNA damage in the ITRS blocks, possibly because of their guanine richness. This suggests that a parallel process may occur in the terminal telomeres, which has implications for premature aging and neoplastic development by chronic NO exposure in vivo.     

Pubertal neuromaturation, stress sensitivity, and psychopathology

Normal adolescent development is often accompanied by transient emotional and behavioral problems. For most individuals with postpubertal-onset adjustment problems, there is a resolution by early adulthood and relative stability through the adult life span. But for a minority, adjustment problems escalate during adolescence and portend the development of serious mental illness in adulthood. In this article, we explore adolescent behavioral changes and neurodevelopmental processes that might contribute to stress sensitivity and vulnerability for the emergence of the mental disorders. Of particular interest is the role that hormonal changes might play in the expression of genetic vulnerabilities for psychopathology. Drawing on recent findings from clinical research and behavioral neuroscience, we describe the ways in which postpubertal hormones might alter brain function and, thereby, behavior. It is concluded that there are both activational and organization effects of hormones on the adolescent brain, and these contribute to developmental discontinuities in behavioral adjustment. Implications for adult psychopathology and preventive intervention are discussed.