Expression and functional activity of ryanodine receptors (RyRs) during skeletal muscle development

Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment.     

Guanosine inhibits CD40 receptor expression and function induced by cytokines and beta amyloid in mouse microglia cells

Growing evidence implicates CD40, a member of the TNFR superfamily, as contributing to the pathogenesis of many neurodegenerative diseases. Thus, strategies to suppress its expression may be of benefit in those disorders. To this aim, we investigated the effect of guanosine, a purine nucleoside that exerts neurotrophic and neuroprotective effects. CD40 expression and function are increased by exposure of mouse microglia cultures or the N9 microglia cell line to IFN-gamma (10 ng/ml) plus TNF-alpha (50 ng/ml) or beta amyloid (Abeta) peptide (Abeta(1-42); 500 nM). Culture pretreatment with guanosine (10-300 microM), starting 1 h before cytokine or Abeta addition, dose-dependently inhibited the CD40-induced expression as well as functional CD40 signaling by suppressing IL-6 production promoted by IFN-gamma/TNF-alpha challenge in the presence of CD40 cross-linking. Moreover, guanosine abrogated IFN-gamma-induced phosphorylation on Ser(727) and translocation of STAT-1alpha to the nucleus as well as TNF-alpha-/Abeta-induced IkappaBalpha and NF-kappaB p65/RelA subunit phosphorylation, thus inhibiting NF-kappaB-induced nuclear translocation. Guanosine effects were mediated by an increased phosphorylation of Akt, a PI3K downstream effector, as well as of ERK1/2 and p38 in the MAPK system, because culture pretreatment with selective ERK1/2, p38 MAPK, and PI3K antagonists (U0126, SB203580, or LY294002, respectively) counteracted guanosine inhibition on IFN-gamma/TNF-alpha-induced CD40 expression and function as well as on STAT-1alpha or NF-kappaB nuclear translocation. These findings suggest a role for guanosine as a potential drug in the experimental therapy of neuroinflammatory/neurodegenerative diseases, particularly Alzheimer's disease.     

Dielectric Properties of the Plasma Membrane of Cultured Murine Fibroblasts Treated with a Nonterpenoid Extract of Azadirachta indica Seeds

Neem oil is a natural product obtained from the seeds of the tree Azadirachta indica. In this report, we investigate the alterations of the biophysical properties of the plasma membrane caused by treatment with the nonterpenoid fraction of neem oil that we defined as methanolic extract (MEX). The dose-response effect was evaluated and a MEX-dependent cytoxicity evidenced. The effect of MEX on the plasma membrane was studied by a well-established dielectric spectroscopy technique: electrorotation, which allows single-cell analysis. Our results show a structural/functional alteration of the plasma membrane with an evident increase of specific capacitance and conductance. The biological implications of this effect are discussed.     

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.     

Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8(+) T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and alpha-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.     

ZAP-70 kinase regulates HIV cell-to-cell spread and virological synapse formation

HIV efficiently spreads in lymphocytes, likely through virological synapses (VSs). These cell-cell junctions share some characteristics with immunological synapses, but cellular proteins required for their constitution remain poorly characterized. We have examined here the role of ZAP-70, a key kinase regulating T-cell activation and immunological synapse formation, in HIV replication. In lymphocytes deficient for ZAP-70, or expressing a kinase-dead mutant of the protein, HIV replication was strikingly delayed. We have characterized further this replication defect. ZAP-70 was dispensable for the early steps of viral cycle, from entry to expression of viral proteins. However, in the absence of ZAP-70, intracellular Gag localization was impaired. ZAP-70 was required in infected donor cells for efficient cell-to-cell HIV transmission to recipients and for formation of VSs. These results bring novel insights into the links that exist between T-cell activation and HIV spread, and suggest that HIV usurps components of the immunological synapse machinery to ensure its own spread through cell-to-cell contacts.     

The first agmatine/cadaverine aminopropyl transferase: biochemical and structural characterization of an enzyme involved in polyamine biosynthesis in the hyperthermophilic archaeon Pyrococcus furiosus

We report here the characterization of the first agmatine/cadaverine aminopropyl transferase (ACAPT), the enzyme responsible for polyamine biosynthesis from an archaeon. The gene PF0127 encoding ACAPT in the hyperthermophile Pyrococcus furiosus was cloned and expressed in Escherichia coli, and the recombinant protein was purified to homogeneity. P. furiosus ACAPT is a homodimer of 65 kDa. The broad substrate specificity of the enzyme toward the amine acceptors is unique, as agmatine, 1,3-diaminopropane, putrescine, cadaverine, and sym-nor-spermidine all serve as substrates. While maximal catalytic activity was observed with cadaverine, agmatine was the preferred substrate on the basis of the k(cat)/K(m) value. P. furiosus ACAPT is thermoactive and thermostable with an apparent melting temperature of 108 degrees C that increases to 112 degrees C in the presence of cadaverine. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. The crystal structure of the enzyme determined to 1.8-A resolution confirmed its dimeric nature and provided insight into the proteolytic analyses as well as into mechanisms of thermal stability. Analysis of the polyamine content of P. furiosus showed that spermidine, cadaverine, and sym-nor-spermidine are the major components, with small amounts of sym-nor-spermine and N-(3-aminopropyl)cadaverine (APC). This is the first report in Archaea of an unusual polyamine APC that is proposed to play a role in stress adaptation.     

New 1,8-naphthyridine and quinoline derivatives as CB2 selective agonists

A series of new 1,8-naphthyridine and quinoline derivatives were synthesized and evaluated for their cannabinoid receptor affinity. In particular, compounds 2, 5, 11, and 13 showed a high CB(2) affinity and CB(2) versus CB(1) selectivity, in agreement with molecular modeling studies. Furthermore, compound 2 also exhibited in vivo antinociceptive effects.