Role of xanthine oxidase activation and reduced glutathione depletion in rhinovirus induction of inflammation in respiratory epithelial cells

Rhinoviruses are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. We previously reported rapid rhinovirus induction of intracellular superoxide anion, resulting in NF-kappaB activation and pro-inflammatory molecule production. The mechanisms of rhinovirus superoxide induction are poorly understood. Here we found that the proteolytic activation of the xanthine dehydrogenase/xanthine oxidase (XD/XO) system was required because pretreatment with serine protease inhibitors abolished rhinovirus-induced superoxide generation in primary bronchial and A549 respiratory epithelial cells. These findings were confirmed by Western blotting analysis and by silencing experiments. Rhinovirus infection induced intracellular depletion of reduced glutathione (GSH) that was abolished by pretreatment with either XO inhibitor oxypurinol or serine protease inhibitors. Increasing intracellular GSH with exogenous H2S or GSH prevented both rhinovirus-mediated intracellular GSH depletion and rhinovirus-induced superoxide production. We propose that rhinovirus infection proteolytically activates XO initiating a pro-inflammatory vicious circle driven by virus-induced depletion of intracellular reducing power. Inhibition of these pathways has therapeutic potential.     

TGF-beta1 genotypes in cirrhosis: relationship with the occurrence of liver cancer

This study aimed to verify whether specific single nucleotide polymorphisms (SNPs) of the transforming growth factor-beta1 (TGF-beta1) may predispose to end-stage liver disease and/or hepatocellular carcinoma (HCC). One hundred eighty-eight consecutive patients transplanted for liver cirrhosis (HBV N=21, HCV N=68, alcoholic N=55 and others N=23) and a control group of 140 healthy blood donors were investigated. Four SNPs were studied by restriction fragment length assays: -800G>A, -509C>T, Leu10Pro and Arg25Pro. Patients were found to possess the -509T/ * (TT 53/188, CT 85/188, CC 50/188 vs TT 22/140, CT 61/140, CC 57/140; p<0.002) and Arg25Pro C/ * genotypes (CC 1/188, CG 31/188, GG 156/188 vs CC 0/140, CG 13/140, GG 127/140; p<0.05) more frequently than controls. Patients with cirrhosis complicated by HCC possessed more frequently the Leu10Pro T/ * genotype than patients without HCC (TT 20/54, CT 26/54, CC 8/54 vs TT 31/134, CT 69/134, CC 34/134; p<0.05). The analysis of molecular variance detected significant genotypic differentiations between controls and cirrhotics but not between cirrhotics with or without HCC. In conclusion, TGF-beta1 SNPs probably facilitate the development of liver cirrhosis, while they seem to have a limited role in predicting the occurrence of HCC.     

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.     

A DIGE approach for the assessment of rat soleus muscle changes during unloading: effect of acetyl-L-carnitine supplementation

After hind limb suspension, a remodeling of postural muscle phenotype is observed. This remodeling results in a shift of muscle profile from slow-oxidative to fast-glycolytic. These metabolic changes and fiber type shift increase muscle fatigability. Acetyl-L-carnitine (ALCAR) influences the skeletal muscle phenotype of soleus muscle suggesting a positive role of dietary supplementation of ALCAR during unloading. In the present study, we applied a 2-D DIGE, mass spectrometry and biochemical assays, to assess qualitative and quantitative differences in the proteome of rat slow-twitch soleus muscle subjected to disuse. Meanwhile, the effects of ALCAR administration on muscle proteomic profile in both unloading and normal-loading conditions were evaluated. The results indicate a modulation of troponin I and tropomyosin complex to regulate fiber type transition. Associated, or induced, metabolic changes with an increment of glycolytic enzymes and a decreased capacity of fat oxidation are observed. These metabolic changes appear to be counteracted by ALCAR treatment, which restores the mitochondrial mass and decreases the glycolytic enzyme expression, suggesting a normalization of the metabolic shift observed in unloaded animals. This normalization is accompanied by a maintenance of body weight and seems to prevent a switch of fiber type.     

HPV16 E7-Dependent Transformation Activates NHE1 through a PKA-RhoA-Iinduced Inhibition of p38alpha

Background

Neoplastic transformation originates from a large number of different genetic alterations. Despite this genetic variability, a common phenotype to transformed cells is cellular alkalinization. We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 (HPV16), that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na/⁺H⁺ exchanger isoform 1 (NHE1) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice.

Methodology

Here, we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1.

Principal Findings

We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell. Further, using pharmacological agents and transient transfections with dominant interfering, constitutively active, phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1, we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein, RhoA, and its subsequent inhibition.

Conclusions

All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation.     

Defence syndromes in lodgepole – whitebark pine ecosystems relate to degree of historical exposure to mountain pine beetles

Warming climate is allowing tree‐killing bark beetles to expand their ranges and access naïve and semi‐naïve conifers. Conifers respond to attack using complex mixtures of chemical defences that can impede beetle success, but beetles exploit some compounds for host location and communication. Outcomes of changing relationships will depend on concentrations and compositions of multiple host compounds, which are largely unknown. We analysed constitutive and induced chemistries of Dendroctonus ponderosae's primary historical host, Pinus contorta, and Pinus albicaulis, a high‐elevation species whose encounters with this beetle are transitioning from intermittent to continuous. We quantified multiple classes of terpenes, phenolics, carbohydrates and minerals. Pinus contorta had higher constitutive allocation to, and generally stronger inducibility of, compounds that resist these beetle–fungal complexes. Pinus albicaulis contained higher proportions of specific monoterpenes that enhance pheromone communication, and lower induction of pheromone inhibitors. Induced P. contorta increased insecticidal and fungicidal compounds simultaneously, whereas P. albicaulis responses against these agents were inverse. Induced terpene accumulation was accompanied by decreased non‐structural carbohydrates, primarily sugars, in P. contorta, but not P. albicaulis, which contained primarily starches. These results show some host species with continuous exposure to bark beetles have more thoroughly integrated defence syndromes than less‐continuously exposed host species.     

Acridine Orange/exosomes increase the delivery and the effectiveness of Acridine Orange in human melanoma cells: A new prototype for theranostics of tumors

Specifically targeted drug delivery systems with low immunogenicity and toxicity are deemed to increase efficacy of cancer chemotherapy. Acridine Orange (AO) is an acidophilic dye with a strong tumoricidal action following excitation with a light source at 466?nm. However, to date the clinical use of AO is limited by the potential side effects elicited by systemic administration. The endogenous nanocarrier exosomes have been recently introduced as a natural delivery system for therapeutic molecules. In this article, we show the outcome of the administration to human melanoma cells of AO charged Exosomes (Exo-AO), in both monolayer and spheroid models. The results showed an extended drug delivery time of Exo-AO to melanoma cells as compared to the free AO, improving the cytotoxicity of AO. This study shows that Exo-AO have a great potential for a real exploitation as a new theranostic approach against tumors based on AO delivered through the exosomes.     

Microphytobenthos activity and fluxes at the sediment-water interface: interactions and spatial variability

In this study oxygen and nutrient fluxes and denitrification rates across the sediment-water interface were measured via intact core incubations with a twofold aim: show whether microphytobenthos activity affects these processes and analyse the dispersion of replicate measurements. Eighteen intact sediment cores (i.d. 8 cm) were randomly sampled from a shallow microtidal brackish pond at Tjarno, on the west coast of Sweden, and were incubated in light and in darkness simulating in situ conditions. During incubation O₂, inorganic N and SiO₂ fluxes and denitrification rates (isotope pairing) were measured. Assuming mean values of 18 cores as best estimate of true average (BEA), the accuracy of O₂, NH₄ ⁺, NO₃ ^- and SiO₂ fluxes calculated for an increasing number of subsamples was tested. At the investigated site, microalgae strongly influenced benthic O₂, inorganic N and SiO₂ fluxes and coupled (D_n) and uncoupled (D_w) denitrification through their photosynthetic activity. In the shift between dark and light conditions NH₄ ⁺ and SiO₂ effluxes (60 and 110 µmol m^-2h^-1) and D_n (5 µmol m^-2 h^-1) dropped to zero, NO₃ ^- uptake (70 µmol m^-2 h^-1) showed a 30% increase, while D_w (20 µmol m^-2 h^-1) showed an 80% decrease. For O₂ and NO₃ ^- dark fluxes, 4 core replicates were sufficient to obtain averages within 5-10% of the best estimated mean, while 10-20% accuracy was obtained with 4-12 replicates for SiO₂ and >10 replicates for NH₄ ⁺ dark fluxes. Mean accuracy was considerably lower for all light incubations, probably due to the patchy distribution of the benthic microalgal community.     

Local adaptation and ecological genetics of host-plant specialization in a leaf beetle

The tendency of insect species to evolve specialization to one or a few plant species is probably a major reason for the remarkable diversity of herbivorous insects. The suggested explanations for this general trend toward specialization include a range of evolutionary mechanisms, whose relative importance is debated. Here we address two potentially important mechanisms: (i) how variation in the geographic distribution of host use may lead to the evolution of local adaptation and specialization; (ii) how selection for specialization may lead to the evolution of trade‐offs in performance between different hosts. We performed a quantitative genetic experiment of larval performance in three different populations of the alpine leaf beetle Oreina elongata reared on two of its main host plants. Due to differences in host availability, each population represents a distinctly different selective regime in terms of host use including selection for specialization on one or the other host as well as selection for utilizing both hosts during the larval stage. The results suggest that selection for specialization has lead to some degree of local adaptations in host use: both single‐host population had higher larval growth rate on their respective native host plant genus, while there was no difference between plant treatments in the two‐host population. However, differences between host plant treatments within populations were generally small and the degree of local adaptation in performance traits seems to be relatively limited. Genetic correlations in performance traits between the hosts ranged from zero in the two‐host population to significantly positive in the single‐host populations. This suggests that selection for specialization in single host populations typically also increased performance on the alternative host that is not naturally encountered. Moreover, the lack of a positive genetic correlation in the two host‐population give support for the hypothesis that performance trade‐offs between two host plants may typically evolve when a population have adapted to both these plants. We conclude that although there is selection for specialization in larval performance traits it seems as if the genetic architecture of these traits have limited the divergence between populations in relative performance on the two hosts.     

Inhibition of Mitochondrial ATP Generation by Nitric Oxide Switches Apoptosis to Necrosis

Under pathological conditions, the mode of cell death, apoptosis or necrosis, is relevant for the subsequent fate of the tissue. Cell demise may be shaped by endogenous mediators such as nitric oxide (NO) which interfere with subroutines of the death program. Here we show that apoptosis of Jurkat cells elicited by either staurosporine (STS) or anti-CD95 antibodies in glucose-free medium is converted to necrosis by NO donors. In the presence of NO, release of mitochondrial cytochrome c was delayed and activation of execution caspases was prevented. Stimulated cells died nonetheless. The switch in the mode of cell death was due to NO-dependent failure of mitochondrial energy production. Restoration of intracellular ATP by glucose supplementation recovered the cells' ability to activate caspases and undergo apoptosis. In this system, the apoptosis/necrosis conversion promoted by NO was not mediated by cyclic guanosine monophosphate-dependent mechanisms, poly-(ADP-ribose)-polymerase (PARP) activation, or inhibition of caspases due to S-nitrosylation and glutathione depletion. In contrast, depleting intracellular ATP with rotenone, an inhibitor of mitochondrial complex I mimicked the effect of NO. The findings presented here suggest that NO can decide the shape of cell death by lowering intracellular ATP below the level required to allow the coordinated execution of apoptosis.