Nitric oxide inhibits execution of apoptosis at two distinct ATP-dependent steps upstream and downstream of mitochondrial cytochrome c release

The endogenous mediator nitric oxide (NO) blocked apoptosis of Jurkat cells elicited by staurosporine, anti-CD95 or chemotherapeutics, and switched death to necrosis. The switch in the mode of cell death was dependent on the ATP loss elicited by NO. This affected two distinct steps of the apoptotic cascade. First, the release of cytochrome c from mitochondria was delayed by NO. Second, processing of procaspases-3/7 to the active proteases was prevented even after cytochrome c had been released. Thus, NO interferes with execution steps of apoptosis both upstream and downstream of cytochrome c release.     

Extension of the Messapia x dicoccoides linkage map of Triticum turgidum (L.) Thell

A set of recombinant inbred lines (RIL) derived from a cross between the cultivar Messapia of durum wheat (Triticum turgidum var. durum) and the accession MG4343 of T. turgidum var. dicoccoides was analysed to increase the number of assigned markers and the resolution of the previously constructed genetic linkage map. An updated map of the durum wheat genome consisting of 458 loci was constructed. These loci include 261 Restriction Fragment Length Polymorphisms (RFLPs), 91 microsatellites (Simple Sequence Repeats, SSRs), 87 Amplified Fragment Length Polymorphisms (AFLPs), two ribosomal genes, and nine biochemical (seven seed storage proteins and two isozymes) and eight morphological markers. The loci were mapped on all 14 chromosomes of the A and B genomes, and covered a total distance of 3038.4 cM with an average distance of 6.7 cM between adjacent markers. The molecular markers were evenly distributed between the A and the B genomes (240 and 218 markers, respectively). An additional forty loci (8.8%) could not be assigned to a specific linkage group. A fraction (16.4%) of the markers significantly deviated from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on the 1B, 2A, 2B, 3A, 4A, 7A and 7B chromosomes. The genetic lengths of the chromosomes range from 148.8 cM (chromosome 6B) to 318.0 cM (chromosome 2B) and approximately concur with their physical lengths. Chromosome 2B has the largest number of markers (47), while the chromosomes with the fewest markers are 3A and 6B (23). There are two gaps larger than 40 cM on chromosomes 2A and 3B. The durum wheat map was compared with the published maps of bread and durum wheats; the order of most common RFLP and SSR markers on the 14 chromosomes of the A and B genomes were nearly identical. A core-map can be extracted from the high-density Messapia x dicoccoides map and a subset of uniformly distributed markers can be used to detect and map quantitative trait loci.     

Solution chemistry and cytotoxic properties of novel organogold(III) compounds

The solution behaviour of some novel organogold(III) compounds was investigated, and their cytotoxic properties evaluated against a few human tumour cell lines (A2780/S, A2780/R, MCF7, HT29 and A549). Specifically, the following compounds were considered: [Au(bipy(dmb)-H)(2,6-xylidine-H)][PF(6)] (AuXyl) and [Au(bipy(dmb)-H)(p-toluidine-H)][PF(6)] (AuTol) (in which bipy(dmb)=6-(1,1-dimethylbenzyl)-2,2'-bipyridine), [Au(py(dmb)-H)(AcO)(2)] (AuPyAcO) (in which py(dmb)=2-(1,1-dimethylbenzyl)-pyridine) and [Au(pz(Ph)-H)Cl(3)]K (AuPzCl) (in which pz(Ph)=1-phenylpyrazole). The solution chemistry of these compounds, under physiological-like conditions, was investigated through UV-vis absorption and (1)H NMR spectroscopies. Significant cytotoxic effects in vitro were observed in selected cases.     

Cigarette smoke extract induces oxidative stress and apoptosis in human lung fibroblasts

Cigarette smoke is a mixture of chemicals having direct and/or indirect toxic effects on different lung cells. We investigated the effect of cigarette smoke on human lung fibroblasts (HFL-1) oxidation and apoptosis. Cells were exposed to various concentrations (1, 5, and 10%) of cigarette smoke extract (CSE) for 3 h, and oxidative stress and apoptosis were assessed by fluorescence-activated cell sorting and confocal laser fluorescence microscopy. Both oxidative stress and apoptosis exhibited a dose-response relationship with CSE concentrations. Lung fibroblasts also showed marked DNA fragmentation at the Comet assay after exposure to 10% CSE. Coincubation of HLF-1 cells with N-acetylcysteine (1 mM) during CSE exposure significantly reduced oxidative stress, apoptosis, and DNA fragmentation, whereas preincubation (3 h) with the glutathione-depleting agent buthionine sulfoximine (125 microM) produced a significant increase of oxidative stress. Cigarette smoke is a potent source of oxidative stress, DNA damage, and apoptosis for HFL-1 cells, and we speculate that this could contribute to the development of pulmonary emphysema in the lungs of smokers.     

Sodium ions and GTP decrease the potency of [Nphe1]N/OFQ(1-13)NH2 in blocking nociceptin/orphanin FQ receptors coupled to cyclic AMP in N1E-115 neuroblastoma cells and rat olfactory bulb

The pseudopeptide [Nphe(1)]N/OFQ(1-13)NH(2) (Nphe) has been shown to act as a pure, selective and competitive antagonist of nociceptin/orphanin FQ (N/OFQ) receptors in different tissues. However, Nphe displayed a highly variable potency, with pA(2) values ranging from 5.96 to 8.45. In the present study, we show that sodium ions and GTP markedly affect the potency of Nphe in blocking N/OFQ receptors coupled to cyclic AMP inhibition in different cellular systems. In intact N1E-115 neuroblastoma cells, the pA(2) value of Nphe increased from 7.13 to 8.02 when the extracellular sodium concentration was reduced from 138 to 2.5 mM. When N/OFQ inhibition of adenylyl cyclase activity was assayed in cell membranes, 100 mM NaCl decreased the pK(i) value of Nphe from 8.38 to 7.32, but increased that of the nonpeptide N/OFQ receptor antagonist CompB from 8.61 to 8.92. Similar effects of sodium ions on the potencies of Nphe and CompB were observed when the compounds were used to antagonize the N/OFQ inhibition of adenylyl cyclase activity in membranes of the external plexiform layer of the rat olfactory bulb. In the same assay, the increase of GTP concentration from 0.1 to 200 micro M decreased Nphe potency by 8-fold. These data demonstrate that sodium ions and GTP affect the potency of Nphe in a manner similar to that of agonists but not of pure antagonists and suggest that these factors may contribute to the reported variability of Nphe affinity constant.     

A route for prion neuroinvasion

The astonishing recognition that self-propagating changes in protein structure may be at the basis of spongiform encephalopathies is changing our views of transmissible diseases. Infectious agents consisting of abnormally folded proteins (i.e., the prion) can propagate in host organisms using previously unrecognized routes and invade the brain after a dangerous liaison with the immune system. The identification of the nodal points of neuroinvasion and new techniques to amplify minute amounts of misfolded proteins may open the possibility for post exposure prophylaxis.     

Disialoganglioside GD3 is released by microglia and induces oligodendrocyte apoptosis

Increased brain ganglioside levels are a hallmark of various neuroinflammatory pathologies. Here, we provide evidence that murine microglia can secrete disialoganglioside GD3 upon exposure to inflammatory stimuli. Comparison of different neural cell types revealed a particular and specific sensitivity of oligodendrocytes towards exogenous GD3. Oligodendrocyte death triggered by GD3 was preceded by degeneration of cellular processes, and associated with typical features of apoptosis, such as chromatin condensation, exposure of phosphatidylserine, release of cytochrome c from mitochondria, and loss of mitochondrial membrane potential, followed by the loss of plasma membrane integrity and detachment of disintegrated oligodendrocytes. Overexpression of bcl-2 partially protected oligodendrocytes from death. In contrast, treatment with the pan-caspase inhibitor zVAD-fmk did not prevent phosphatidylserine exposure, chromatin margination at the nuclear periphery, and death, although caspase-3 was blocked. Thus, GD3 produced by microglia under neuroinflammatory conditions may function as a novel mediator triggering mitochondria-mediated, but caspase-independent, apoptosis-like death of oligodendrocytes.