Protection against progressive leishmaniasis by IFN-beta

Type I IFNs (IFN-alphabeta) exert potent antiviral and immunoregulatory activities during viral infections, but their role in bacterial or protozoan infections is poorly understood. In this study, we demonstrate that the application of low, but not of high doses of IFN-beta protects 60 or 100% of BALB/c mice from progressive cutaneous and fatal visceral disease after infection with a high (10(6)) or low (10(4)) number of Leishmania major parasites, respectively. IFN-beta treatment of BALB/c mice restored the NK cell cytotoxic activity, increased the lymphocyte proliferation, and augmented the production of IFN-gamma and IL-12 in the draining lymph node. Low, but not high doses of IFN-beta caused enhanced tyrosine phosphorylation of STAT1 and STAT4, suppressed the levels of suppressor of cytokine signaling-1, and up-regulated the expression of inducible NO synthase in vivo. The IFN-beta-induced increase of IFN-gamma production was dependent on STAT4. Protection by IFN-beta strictly required the presence of inducible NO synthase. In the absence of STAT4 or IL-12, IFN-beta led to an amelioration of the cutaneous and visceral disease, but was unable to prevent its progression. These results identify IFN-beta as a novel cytokine with a strong, dose-dependent protective effect against progressive cutaneous leishmaniasis that results from IL-12- and STAT4-dependent as well as -independent events.     

Pacemaking by HCN channels requires interaction with phosphoinositides

Hyperpolarization-activated, cyclic-nucleotide-gated (HCN) channels mediate the depolarizing cation current (termed I(h) or I(f)) that initiates spontaneous rhythmic activity in heart and brain. This function critically depends on the reliable opening of HCN channels in the subthreshold voltage-range. Here we show that activation of HCN channels at physiologically relevant voltages requires interaction with phosphoinositides such as phosphatidylinositol-4,5-bisphosphate (PIP(2)). PIP(2) acts as a ligand that allosterically opens HCN channels by shifting voltage-dependent channel activation approximately 20 mV toward depolarized potentials. Allosteric gating by PIP(2) occurs in all HCN subtypes and is independent of the action of cyclic nucleotides. In CNS neurons and cardiomyocytes, enzymatic degradation of phospholipids results in reduced channel activation and slowing of the spontaneous firing rate. These results demonstrate that gating by phospholipids is essential for the pacemaking activity of HCN channels in cardiac and neuronal rhythmogenesis.     

Soluble adenylyl cyclase mediates nerve growth factor-induced activation of Rap1

Nerve growth factor (NGF) and the ubiquitous second messenger cyclic AMP (cAMP) are both implicated in neuronal differentiation. Multiple studies indicate that NGF signals to at least a subset of its targets via cAMP, but the link between NGF and cAMP has remained elusive. Here, we have described the use of small molecule inhibitors to differentiate between the two known sources of cAMP in mammalian cells, bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC) and G protein-regulated transmembrane adenylyl cyclases. These inhibitors, along with sAC-specific small interfering RNA, reveal that sAC is uniquely responsible for the NGF-elicited rise in cAMP and is essential for the NGF-induced activation of the small G protein Rap1 in PC12 cells. In contrast and as expected, transmembrane adenylyl cyclase-generated cAMP is responsible for Rap1 activation by the G protein-coupled receptor ligand PACAP (pituitary adenylyl cyclase-activating peptide). These results identify sAC as a mediator of NGF signaling and reveal the existence of distinct pathways leading to cAMP-dependent signal transduction.     

Alterations in the cytoplasmic domain of CLCN2 result in altered gating kinetics.

Mutations in the human ClC-2 Cl(-) channel have been described to influence its function dramatically. To test for naturally occurring gene variants in a human population and their functionality, all 24 CLCN2 exons from a Central African population were sequenced. Six single amino acid exchanges in the intracellular N-terminus (P48R, R68H), in the pore domain (G199A), or in the intracellular C-terminus (R646Q, R725W, R747H) were identified at low frequency. Heterologous expression of these polymorphisms in Xenopus laevis oocytes demonstrated their functional significance as determined by two-electrode voltage-clamp. The polymorphisms R68H, R725W, and R747H exhibited faster voltage-stimulated gating as compared to the wild type channel, resulting in higher steady state currents of R725W. Probably due to decreased surface expression P48R, R68H, and R646Q mutants generated lower currents than the wild type channels. The inward currents of the mutated channels R725W, R747H, and G199A failed to increase during hypotonic swelling, a defect paralleled by impaired swelling-accelerated voltage-gating in one mutant (G199A). In conclusion, the Africans' gene pool comprises CLCN2 gene variants in the N-terminus, the C-terminus or the pore domain that affect surface expression and voltage- or cell-swelling-stimulated channel gating.