Helper-dependent adenoviral vector-mediated delivery of woodchuck-specific genes for alpha interferon (IFN-alpha) and IFN-gamma: IFN-alpha but not IFN-gamma reduces woodchuck hepatitis virus replication in chronic infection in vivo

Alpha interferon (IFN-alpha) and IFN-gamma are able to suppress hepadnavirus replication. The intrahepatic expression of high levels of IFN may enhance the antiviral activity. We investigated the effects of woodchuck-specific IFN-alpha (wIFN-alpha) and IFN-gamma(wIFN-gamma) on woodchuck hepatitis virus (WHV) replication in vivo by helper-dependent adenoviral (HD-Ad) vector-mediated gene transfer. The expression of biologically active IFNs was demonstrated in vitro after transduction of woodchuck cells with HD-Ad vectors encoding wIFN-alpha (HD-AdwIFN-alpha) or wIFN-gamma (HD-AdwIFN-gamma). The transduction efficacy of the HD-Ad vector in woodchuck liver in vivo was tested with a vector expressing green fluorescence protein (GFP). Immunohistochemical staining of liver samples on day 5 after injection showed expression of GFP in a high percentage of liver cells surrounding the central vein. The transduction of livers of WHV carriers in vivo with HD-AdwIFN-alpha or HD-AdwIFN-gamma induced levels of biologically active IFN, which could be measured in the sera of these animals. Expression of wIFN-alpha in the liver reduced intrahepatic WHV replication and WHV DNA in sera of about 1 log step in two of two woodchucks. Transduction with HD-AdwIFN-gamma, however, reduced WHV replicative intermediates only slightly in two of three animals, which was not accompanied with significant changes in the WHV DNA in sera. We demonstrated for the first time the successful HD-Ad vector-mediated transfer of genes for IFN-alpha and IFN-gamma in vivo and timely limited reduction of WHV replication by wIFN-alpha, but not by wIFN-gamma.     

Architecture of the Qo site of the cytochrome bc1 complex probed by superoxide production

Although several X-ray structures have been determined for the mitochondrial cytochrome (cyt) bc(1) complex, none yet shows the position of the substrate, ubiquinol, in the quinol oxidase (Q(o)) site. In this study, the interaction of molecular oxygen with the reactive intermediate Q(o) semiquinone is used to probe the Q(o) site. It has been known for some time that partial turnover of the cyt bc(1) complex in the presence of antimycin A, a Q(i) site inhibitor, results in accumulation of a semiquinone at the Q(o) site, which can reduce O(2) to superoxide (O(2)(*)(-)). It was more recently shown that myxothiazol, which binds close to the cyt b(L) heme in the proximal Q(o) niche, also induces O(2)(*)(-) production. In this work, it is shown that, in addition to myxothiazol, a number of other proximal Q(o) inhibitors [including (E)-beta-methoxyacrylate-stilbene, mucidin, and famoxadone] also induce O(2)(*)(-) production in the isolated yeast cyt bc(1) complex, at approximately 50% of the V(max) observed in the presence of antimycin A. It is proposed that proximal Q(o) site inhibitors induce O(2)(*)(-) production because they allow formation, but not oxidation, of the semiquinone at the distal niche of the Q(o) site pocket. The apparent K(m) for ubiquinol at the Q(o) site in the presence of proximal Q(o) site inhibitors suggests that the "distal niche" of the Q(o) pocket can act as a fully independent quinol binding and oxidation site. Together with the X-ray structures, these results suggest substrate ubiquinol binds in a fashion similar to that of stigmatellin with H-bonds between H161 of the Rieske iron-sulfur protein and E272 of the cyt b protein. When modeled in this way, mucidin and ubiquinol can bind simultaneously to the Q(o) site with virtually no steric hindrance, whereas progressively bulkier inhibitors exhibit increasing overlap. The fact that partial turnover of the Q(o) site is possible even with bound proximal Q(o) site inhibitors is consistent with the participation of two separate functional Q(o) binding niches, occupied simultaneously or sequentially.     

Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.     

The magnetofection method: using magnetic force to enhance gene delivery

In order to enhance and target gene delivery we have previously established a novel method, termed magnetofection, which uses magnetic force acting on gene vectors that are associated with magnetic particles. Here we review the benefits, the mechanism and the potential of the method with regard to overcoming physical limitations to gene delivery. Magnetic particle chemistry and physics are discussed, followed by a detailed presentation of vector formulation and optimization work. While magnetofection does not necessarily improve the overall performance of any given standard gene transfer method in vitro, its major potential lies in the extraordinarily rapid and efficient transfection at low vector doses and the possibility of remotely controlled vector targeting in vivo.     

Removal of Pex3p is an important initial stage in selective peroxisome degradation in Hansenula polymorpha

Selective degradation of peroxisomes (macropexophagy) in Hansenula polymorpha involves the sequestration of individual organelles to be degraded by membranes prior to the fusion of this compartment with the vacuole and subsequent degradation of the whole organelle by vacuolar hydrolases. Here we show that Pex3p, a peroxisomal membrane protein essential for peroxisome biogenesis, escapes this autophagic process. Upon induction of macropexophagy, Pex3p is removed from the organelle tagged for degradation prior to its sequestration. Our data indicate that Pex3p degradation is essential to allow the initiation of the organellar degradation process. Also, in a specific peroxisome degradation-deficient (pdd) mutant in which sequestration still occurs but the vacuolar fusion event is disturbed, the turnover of Pex3p is still observed. Taken together, our data suggest that degradation of Pex3p is part of the initial degradation machinery of individual peroxisomes.     

Biochemical characterization of a membrane-bound manganese-containing superoxide dismutase from the cyanobacterium Anabaena PCC 7120

The filamentous cyanobacterium Anabaena PCC 7120 (now renamed Nostoc PCC 7120) possesses two genes for superoxide dismutase (SOD). One is an iron-containing (FeSOD) whereas the other is a manganese-containing superoxide dismutase (MnSOD). Localization experiments and analysis of the sequence showed that the FeSOD is cytosolic, whereas the MnSOD is a membrane-bound homodimeric protein containing one transmembrane helix, a spacer region, and a soluble catalytic domain. It is localized in both cytoplasmic and thylakoid membranes at the same extent with the catalytic domains positioned either in the periplasm or the thylakoid lumen. A phylogenetic analysis revealed that generally the highly homologous MnSODs of filamentous cyanobacteria are unique in being membrane-bound. Two recombinant variants of Anabaena MnSOD lacking either the hydrophobic region (MnSOD(Delta 28)) or the hydrophobic and the linker region (MnSOD(Delta 60)) are shown to exhibit the characteristic manganese peak at 480 nm, an almost 100% occupancy of manganese per subunit, a specific activity using the ferricytochrome assay of (660 +/- 90) unit mg-1 protein and a dissociation constant for the inhibitor azide of (0.84 +/- 0.05) mm. Using stopped-flow spectroscopy it is shown that the decay of superoxide in the presence of various (MnSOD(Delta 28)) or (MnSOD(Delta 60)) concentrations is first-order in enzyme concentration allowing the calculation of catalytic rate constants which increase with decreasing pH: 8 x 10(6) m-1 s-1 (pH 10) and 6 x 10(7) m-1 s-1 (pH 7). The physiological relevance of these findings is discussed with respect to the bioenergetic peculiarities of cyanobacteria.     

Cutting edge: a critical role for IL-10 in induction of nasal tolerance in experimental autoimmune myocarditis

Appropriate treatment of autoimmune myocarditis following virus infection remains a major clinical problem. Induction of nasal tolerance may provide a new approach to treatment. However, the exact mechanism of nasal tolerance is unknown. To assess the mechanism of nasal tolerance, we examined the role of IL-10 in the induction and suppression of autoimmune myocarditis. First we showed that blocking IL-10 concurrent with nasal administration of Ag abolished the disease-suppressing effect of nasal tolerization. It also led to increased cardiac myosin-specific IL-1 and TNF-alpha production. Then we demonstrated that blocking IL-10 during the effector phase increased not only the incidence and severity of disease but also Ag-specific IL-2, IL-4, and TNF-alpha production as well as cardiac myosin-specific IgG1 and IgG2b production, whereas blocking IL-10 during the induction phase had no effect. This study implicates IL-10 in the induction of nasal tolerance and in limiting inflammation later during the disease process.     

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding beta-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.     

PPAR activators inhibit endothelial cell migration by targeting Akt

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose metabolism and exert several vascular effects that may provide a dual benefit of these receptors on metabolic disorders and atherosclerotic vascular disease. Endothelial cell migration is a key event in the pathogenesis of atherosclerosis. We therefore investigated the effects of lipid-lowering PPARalpha-activators (fenofibrate, WY14643) and antidiabetic PPARgamma-activators (troglitazone, ciglitazone) on this endothelial cell function. Both PPARalpha- and PPARgamma-activators significantly inhibited VEGF-induced migration of human umbilical vein endothelial cells (EC) in a concentration-dependent manner. Chemotactic signaling in EC is known to require activation of two signaling pathways: the phosphatidylinositol-3-kinase (PI3K)-->Akt- and the ERK1/2 mitogen-activated protein kinase (ERK MAPK) pathway. Using the pharmacological PI3K-inhibitor wortmannin and the ERK MAPK-pathway inhibitor PD98059, we observed a complete inhibition of VEGF-induced EC migration. VEGF-induced Akt phosphorylation was significantly inhibited by both PPARalpha- and gamma-activators. In contrast, VEGF-stimulated ERK MAPK-activation was not affected by any of the PPAR-activators, indicating that they inhibit migration either downstream of ERK MAPK or independent from this pathway. These results provide first evidence for the antimigratory effects of PPAR-activators in EC. By inhibiting EC migration PPAR-activators may protect the vasculature from pathological alterations associated with metabolic disorders.