CDKL5 expression is modulated during neuronal development and its subcellular distribution is tightly regulated by the C-terminal tail

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment.     

NAADP+ is an agonist of the human P2Y11 purinergic receptor

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is an intracellular second messenger releasing Ca2+ from intracellular stores in different cell types. In addition, it is also active in triggering [Ca2+](i) increase when applied extracellularly and various underlying mechanisms have been proposed. Here, we used hP2Y(11)-transfected 1321N1 astrocytoma cells to unequivocally establish whether extracellular NAADP+ is an agonist of the P2Y(11) receptor, as previously reported for beta-NAD+ [I. Moreschi, S. Bruzzone, R.A. Nicholas, et al., Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes, J. Biol. Chem. 281 (2006) 31419-31429]. Extracellular NAADP+ triggered a concentration-dependent two-step elevation of [Ca2+](i) in 1321N1-hP2Y(11) cells, but not in wild-type 1321N1 cells, secondary to the intracellular production of IP(3), cAMP and cyclic ADP-ribose (cADPR). Specifically, the transient [Ca2+](i) rise proved to be related to IP(3) overproduction and to consequent Ca2+ mobilization, while the sustained [Ca2+](i) elevation was caused by the cAMP/ADP-ribosyl cyclase (ADPRC)/cADPR signalling cascade and by influx of extracellular Ca2+. In human granulocytes, endogenous P2Y(11) proved to be responsible for the NAADP+-induced cell activation (as demonstrated by the use of NF157, a selective and potent inhibitor of P2Y(11)), unveiling a role of NAADP+ as a pro-inflammatory cytokine. In conclusion, we provide unequivocal evidence for the activation of a member of the P2Y receptor subfamily by NAADP+.     

Catalase T and Cu,Zn-superoxide dismutase in the acetic acid-induced programmed cell death in Saccharomyces cerevisiae

To investigate the role of catalase and superoxide dismutase (SOD) in the acetic acid (AA) induced yeast programmed cell death (AA-PCD), we compared Saccharomyces cerevisiae cells (C-Y) and cells individually over-expressing catalase T (CTT1-Y) and Cu,Zn-SOD (SOD1-Y) with respect to cell survival, hydrogen peroxide (H2O2) levels and enzyme activity as measured up to 200 min after AA treatment. AA-PCD does not occur in CTT1-Y, where H2O2 levels were lower than in C-Y and the over-expressed catalase activity decreased with time. In SOD1-Y, AA-PCD was exacerbated; high H2O2 levels were found, SOD activity increased early, remaining constant en route to AA-PCD, but catalase activity was strongly reduced.     

CD14 major role during lipopolysaccharide-induced inflammation in chick embryo cardiomyocytes

CD14 is a surface differentiation antigen that functions as a receptor for bacterial lipopolysaccharide. The cellular signaling events that lead to lipopolysaccharide-induced production of inflammatory mediators are the primary cause of myocardial dysfunction observed in sepsis. Here, we evaluated the role of CD14 in chick embryo cardiomyocytes stimulated with lipopolysaccharide. CD14 expression was detected by confocal laser microscopy observation and by immunoblotting analysis. Moreover, we provided evidence for CD14-dependent functional responses of lipopolysaccharide-stimulated cardiomyocytes in terms of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production. Attenuated TNF-alpha and NO secretion, following anti-CD14 treatment of cardiomyocytes, suggested a role for this receptor in lipopolysaccharide-mediated cell responses. We also evidenced that labeled lipopolysaccharide was internalized and localized next to the Golgi complex, at the level of lysosomes, and in the perinuclear zone. The intracytoplasmatic transport seems to depend on the contractile apparatus, because cell pretreatment with cytochalasin D prevented lipopolysaccharide internalization and reduced both TNF-alpha and NO release. Lipopolysaccharide internalization was dependent on CD14 receptor, since anti-CD14 pre-treatment prevented endotoxin uptake by cardiomyocytes. Results demonstrated: (1) CD14 is expressed on the surface membrane of cardiomyocytes; (2) CD14 is involved in cytoskeletal dependent lipopolysaccharide internalization at specific cytoplasmatic locations; (3) CD14 plays a role in lipopolysaccharide-mediated responses by cardiomyocytes after lipopolysaccharide internalization.     

FAK-independent alphavbeta3 integrin-EGFR complexes rescue from anoikis matrix-defective fibroblasts

Extracellular matrix (ECM) binding to integrin receptors regulates cell cycle progression and survival. In adherent cells, ECM disassembly induces anoikis, the apoptotic pathway switched on by loss of adhesion. ECM-deficient Ehlers-Danlos syndrome (EDS) fibroblasts, to adhere to rare fibronectin (FN) fibrils, and to proliferate, only organize, as FN receptor, the alphavbeta3 integrin. We report that in EDS cells the alphavbeta3 integrin is bound to talin and vinculin, but not to tensin, and that actin cytoskeleton is disorganized. Furthermore, in EDS cells Bcl-2 is down-regulated and caspases are active. We provide evidence that the antibody-mediated alphavbeta3 integrin or the FN inhibition induces anoikis in EDS cells. The alphavbeta3 integrin transduces survival signals to pp60src-mediated tyrosine phosphorylated paxillin, instead than to FAK, and interacts with EGF receptor (EGFR). This complex, when activated by EGF and FN, signals for the rescue of EDS cells from anoikis. Therefore, EDS cells, through the alphavbeta3 integrin-EGFR complexes, engage a paxillin- but not FAK-mediated pathway of cell survival.     

Impaired carotenogenesis can affect organization and functionality of etioplast membranes

The effects of impaired carotenogenesis on plastid membrane organization, functionality and stability were studied in etiolated barley plants grown at 20 and 30°C. The plants were treated with norflurazon or amitrole, two herbicides affecting phytoene desaturation and lycopene cyclization, respectively. At 20°C, the amitrole-treated etioplasts, which accumulated lycopene in their inner membranes, exhibited disorganized prolamellar bodies, containing a prevalent form of non-phototransformable protochlorophyllide (Pchlide). They also showed a certain difficulty in reducing the phototransformable pigment to chlorophyllide when exposed to light, and were unable to reform the active ternary complex [protochlorophyllide–oxidoreductase (POR)–Pchlide–NADPH] when placed back in darkness. No ultrastructural alterations were found in norflurazon-treated etioplasts, with carotenogenesis inhibited at the phytoene desaturation step. In these latter organelles, Pchlide, whose forms were comparable with those of the control etioplasts, was photoreduced quickly after illumination and the ternary complex was reformed during a subsequent dark period. Thus, the impaired carotenogenesis leading to the accumulation of lycopene showed greater interference with the etioplast membrane arrangement and functionality than did the earlier interruption of the biosynthetic pathway at the phytoene level. This might be due to the different interactions of the distinct carotenoid precursors with other membrane components. However, in etioplasts of norflurazon-treated plants, a rise in growth temperature caused a partial demolition of prolamellar bodies, showing a lowered thermostability of the carotenoid-deficient membranes. This latter effect strengthens the concept that a correct and complete carotenogenesis pathway, leading to the synthesis of polar carotenoids (i.e. xanthophylls), is required for the maintenance of stable plastid membranes.     

Synthesis of radioactive and photoactivable ganglioside derivatives for the study of ganglioside-protein interactions

The procedures for the preparation of radioactive and photoactivable ganglioside derivatives have been continuously developed from 1989, when for the first time the synthesis of photoactivable tritium labeled GM1 ganglioside was presented. We described previously the synthesis of photoactivable derivatives of GM3 and GM1 gangliosides, tritium-labeled at acetyl group of sugar units, and of photoactivable GM1 and GD1b gangliosides, tritium-labeled at position 6 of the external galactose. These procedures are reviewed in detail in the present paper. The use of these ganglioside derivatives to study the ganglioside-protein interactions and to identify proteins that specifically interact with gangliosides (including GPI-anchored proteins of the outer membrane leaflet, proteins anchored to the cytoplasmic side of the plasma membrane through a fatty acyl chain, transmembrane proteins, and soluble cytoplasmic proteins) is discussed. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.