Metal chelatorNNN’ N’-tetrakis-(2-pyridylmethyl)ethylene diamine inhibits the induction of heat shock protein 70 synthesis by heat in cultured keratinocytes

Heat shock protein (HSP) synthesis results from various types of injury, including heat shock (HS) and some oxidants. The intracellular signals leading to HSP synthesis are not yet fully elucidated. We have studied the influence ofNNN’N’-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a metal chelator known to induce cellular zinc and copper deprivation, on resistance to heat and on hsp70 synthesis in HaCaT keratinocytes. TPEN was shown to sensitize HaCaT cells to heat shock. The effect of TPEN was neutralized by equimolar Zn²⁺. By the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and Western blotting characterization of hsp70, it was shown that cultured HaCaT cells constitutively express the inducible form of hsp70. The application of TPEN alone slightly increases the level of hsp70 but inhibits its induction by HS. This inhibitory effect is related to metal deprivation, because it is eliminated when Cu²⁺ or Zn²⁺ ions are supplied together with TPEN. These results suggest that these metals are involved in the expression by keratinocytes of a stress protein which has a protective action against environmental stress.     

Development of a Simple Antioxidant Screening Assay Using Human Skin Fibroblasts

The purpose of this study was to develop a simple antioxidant screening assay for quantifying the protective effects of antioxidant enzymes, inhibitors and scavengers against extracellularly generated oxygen species on human skin fibroblast cytotoxicity. Different in vitro oxidative stresses have been studied: xanthine oxidase-hypoxanthine, flavin mononucleotide-NADH, and hydrogen peroxide. Cytotoxicity and protection were evaluated by two procedures: evaluation of the living cells using a colorimetric method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT), and ability of the viable cells to adherate and proliferate. Hypoxanthine-xanthine oxidase and H^b0₂ induced a dose dependent cytotoxicity only when we considered the delayed toxicity. The influence of the cell density was also investigated. The delayed toxicity was higher when cell density increased. One hundred percent protection against free radical cytotoxicity induced by the three systems were obtained with catalase (500 U/ml). When the oxidative stress used was H₂0₂ 90-96% protection was obtained with deferoxamine an iron chelating agent that prevents iron catalysed radical reactions. Using the colorimetric method no significant protection was obtained when SOD was added before and during the stresses. Using the fibroblasts ability to proliferate SOD (10-150 μ/ml) reduced xanthine oxidase (20 U/1)-hypoxanthine (0.10-0.30mM) or H₂0₂ (1-6mM) cytotoxicity by 15-20%. SOD did not act as antioxidant when the applied stress was mediated by flavin. In this study we showed a paradoxical effect and the cytotoxicity of flavin-NADH system increased when we added SOD to the cell medium. This simple and reliable antioxidant screening assay required no costly or radioactive equipment.     

Calcium binding protein changes of sarcoplasmic reticulum from rat denervated skeletal muscle

Two Ca²⁺ sequestering proteins were studied in fast-twitch (EDL) and slow-twitch (soleus) muscle sarcoplasmic reticulum (SR) as a function of denervation time. Ca²⁺-ATPase activity measured in SR fractions of normal soleus represented 5% of that measure in SR fractions of normal EDL. Denervation caused a severe decrease in activity only in fast-twich muscle. Ca²⁺-ATPase and calsequestrin contents were affected differently by denervation. In EDL SR, Ca²⁺-ATPase content decreased progressively, whereas in soleus SR, no variation was observed. Calsequestrin showed a slight increase in both muscles as a function of denervation time correlated with increased⁴⁵Ca-binding.These results indicate first that Ca²⁺-ATPase activity in EDL was under neural control, and that because of low Ca²⁺-ATPase activity and content in slow-twitch muscle no variation could be detected, and secondly that greater calsequestrin content might represent a relative increasing of heavy vesicles or decreasing of light vesicles as a function of denervation time in the whole SR fraction isolated in both types of muscles.     

The Free Fatty Acid Receptor G Protein-coupled Receptor 40 (GPR40) Protects from Bone Loss through Inhibition of Osteoclast Differentiation

The mechanisms linking fat intake to bone loss remain unclear. By demonstrating the expression of the free fatty acid receptor G-coupled protein receptor 40 (GPR40) in bone cells, we hypothesized that this receptor may play a role in mediating the effects of fatty acids on bone remodeling. Using micro-CT analysis, we showed that GPR40^−/− mice exhibit osteoporotic features suggesting a positive role of GPR40 on bone density. In primary cultures of bone marrow, we showed that GW9508, a GRP40 agonist, abolished bone-resorbing cell differentiation. This alteration of the receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation occurred via the inhibition of the nuclear factor κB (NF-κB) signaling pathway as demonstrated by decrease in gene reporter activity, inhibitor of κB kinase (IKKα/β) activation, inhibitor of κB (IkBα) phosphorylation, and nuclear factor of activated T cells 1 (NFATc1) expression. The GPR40-dependent effect of GW9508 was confirmed using shRNA interference in osteoclast precursors and GPR40^−/− primary cell cultures. In addition, in vivo administration of GW9508 counteracted ovariectomy-induced bone loss in wild-type but not GPR40^−/− mice, enlightening the obligatory role of the GPR40 receptor. Then, in a context of growing prevalence of metabolic and age-related bone disorders, our results demonstrate for the first time in translational approaches that GPR40 is a relevant target for the design of new nutritional and therapeutic strategies to counter bone complications.     

Development of serotonergic neurons of dorsal raphe nuclei in mice with knockout of monoamine oxidase a and 5-HT1A and 5-HT1B autoreceptor

The morphological changes in the development of serotonergic neurons of the dorsal raphe nuclei in the medulla oblongata was studied by immunocytochemistry in mice with knockout of 1A and 1B serotonin autoreceptors as well as monoamine oxidase A. Serotonin autoreceptors regulate electric activity of serotonergic neurons as well as the synthesis and release of the neurotransmitter, while monoamine oxidase A catalyzes its degradation. These genetic modifications proved to have no effect on the number of serotonergic neurons in the medulla oblongata but induced morphofunctional changes. Decreased cell size and increased intracellular serotonin level were observed in the case of monoamine oxidase A deficiency, while excessive cell size and decreased intracellular serotonin level were observed in the case of autoreceptor deficiency. The data obtained confirm the hypothesis of autoregulation of serotonergic neurons in development.     

Crosstalk between TGF-β and hedgehog signaling in cancer

Hedgehog (HH) and TGF-β signals control various aspects of embryonic development and cancer progression. While their canonical signal transduction cascades have been well characterized, there is increasing evidence that these pathways are able to exert overlapping activities that challenge efficient therapeutic targeting. We herein review the current knowledge on HH signaling and summarize the recent findings on the crosstalks between the HH and TGF-β pathways in cancer.     

Low-quality females prefer low-quality males when choosing a mate

Mate choice studies routinely assume female preferences for indicators of high quality in males but rarely consider developmental causes of within-population variation in mating preferences. By contrast, recent mate choice models assume that costs and benefits of searching or competing for high-quality males depend on females'' phenotypic quality. A prediction following from these models is that manipulation of female quality should alter her choosiness or even the direction of her mating preferences. We here provide (to our knowledge) the first example where an experimental manipulation of female quality induced a mating preference for low-quality males. Zebra finches (Taeniopygia guttata) reared in small or large experimental broods became high- or low-quality adults, respectively. Only high-quality females preferred high-quality males'' mate-advertising songs, while all low-quality females preferred low-quality males'' song. Subsequent breeding trials confirmed this pattern: latency until egg laying was shortest in quality-matched pairs, indicating that quality-matched birds were accepted faster as partners. Females produced larger eggs when mated with high-quality males, regardless of their own quality, indicating consensus regarding male quality despite the expression of different choices. Our results demonstrate the importance of considering the development of mating preferences to understand their within-population variation and environmentally induced change.