Role of endothelial nitric oxide synthase in endothelial activation: insights from eNOS knockout endothelial cells

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N-monomethyl-L-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation. vascular biology; atherosclerosis; mouse models     

Effects of exposure uncertainties in the TSCE model and application to the Colorado miners data

The simulations in this paper show that exposure measurement error affects the parameter estimates of the biologically motivated two-stage clonal expansion (TSCE) model. For both Berkson and classical error models, we show that likelihood-based techniques of correction work reliably. For classical errors, the distribution of true exposures needs to be known or estimated in addition to the distribution of recorded exposures conditional on true exposures. Usually the exposure uncertainty biases the model parameters toward the null and underestimates the precision. But when several parameters are allowed to be dependent on exposure, e.g. initiation and promotion, then their relative importance is also influenced, and more complicated effects of exposure uncertainty can occur. The application part of this paper shows for two different types of Berkson errors that a recent analysis of the data for the Colorado plateau miners with the TSCE model is not changed substantially when correcting for such errors. Specifically, the conjectured promoting action of radon remains as the dominant radiation effect for explaining these data. The estimated promoting action of radon increases by a factor of up to 1.2 for the largest assumed exposure uncertainties.     

Characterization of the Arabidopsis TU8 Glucosinolate Mutation,an Allele of TERMINAL FLOWER2

Glucosinolates are a group of defense-related secondary metabolites found in Arabidopsis and other cruciferous plants. Levels of leaf glucosinolates are regulated during plant development and increase in response to mechanical damage or insect feeding. The Arabidopsis TU8 mutant has a developmentally altered leaf glucosinolate profile: aliphatic glucosinolate levels drop off more rapidly, consistent with the early senescence of the mutant, and the levels of two indole glucosinolates are uniformly low. In TU8 seeds, four long-chain aliphatic glucosinolates have significantly increased levels, whereas the indolyl-3-methyl glucosinolate level is significantly reduced relative to wild type. Genetic mapping and DNA sequencing identified the TU8 mutation as tfl2-6, a new allele of TERMINAL FLOWER2 (TFL2), the only Arabidopsis homolog of animal HETEROCHROMATIN PROTEIN1 (HP1). TU8 (tfl2-6) has other previously identified tfl2 phenotypes, including an early transition to flowering, altered meristem structure, and stunted leaves. Analysis of two additional alleles, tfl2-1 and tfl2-2, showed glucosinolate profiles similar to those of line TU8 (tfl2-6).     

Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis

The multiple-locus variable-number tandem-repeats analysis (MLVA) method is currently being used as the primary typing tool for Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates in our laboratory. Our published initial MLVA was performed using a single fluorescent dye and the different patterns were assigned from gel images. Here we present a new and significantly improved assay using multiple dye colors, enhanced PCR multiplexing and the introduction of two new loci for better adaptation to capillary electrophoresis with increased speed. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from gel images. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. A total of 106 human, bird, animal and food isolates of S. Typhimurium, including 16 with definite type (DT) 104, were used for the development of the improved MLVA. The method is based on capillary separation of multiplexed PCR products from five VNTR loci in the S. Typhimurium genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned allele numbers, which were used for strain comparison.     

Changes in dissolved lignin-derived phenols, neutral sugars, uronic acids, and amino sugars with depth in forested Haplic Arenosols and Rendzic Leptosols

A major part of the dissolved organic matter produced in the organic layers of forest ecosystems and leached into the mineral soil is retained by the upper subsoil horizons. The retention is selective and thus dissolved organic matter in the subsoils has different composition than dissolved organic matter leached from the forest floor. Here we report on changes in the composition of dissolved organic matter with soil depth based on C-to-N ratios, XAD-8 fractionation, wet-chemical analyses (lignin-derived CuO oxidation products, hydrolysable sugars and amino sugars) and liquid-state ¹³C nuclear magnetic resonance (NMR) spectroscopy. Dissolved organic matter was sampled directly beneath the forest floor using tension-free lysimeters and at 90cm depth by suction cups in Haplic Arenosols under Scots pine (Pinus sylvestris L.) and Rendzic Leptosols under European beech (Fagus sylvatica L.) forest. At both sites, the concentrations of dissolved organic carbon (DOC) decreased but not as strongly as reported for deeply weathered soils. The decrease in DOC was accompanied by strong changes in the composition of dissolved organic matter. The proportion of the XAD-8-adsorbable (hydrophobic) fraction, carboxyl and aromatic C, and the concentrations of lignin-derived phenols decreased whereas the concentrations of sugars, amino sugars, and nitrogen remained either constant or increased. A general feature of the compositional changes within the tested compound classes was that the ratios of neutral to acidic compounds increased with depth. These results indicate that during the transport of dissolved organic matter through the soils, oxidatively degraded lignin-derived compounds were preferentially retained while potentially labile material high in nitrogen and carbohydrates tended to remain dissolved. Despite the studied soils' small capacity to sorb organic matter, the preferential retention of potentially refractory and acidic compounds suggests sorption by the mineral soil matrix rather than biodegradation to govern the retention of dissolved organic matter even in soils with a low sorption capacity.     

Long-term behaviour of 15N in an alpine grassland ecosystem

Nitrogen dynamics in semi-natural environments is crucial for the development and ecological stability of these systems. The present paper shows the results of the reinvestigation of a ¹⁵N-tracer experiment, which was established in the Grossglockner massif in Austria at 2300ma.s.l. in 1974/1975. We show that large quantities of nitrogen introduced by a single pulse labelling (amounting to approximately 1.7% of the nitrogen in the system) into an alpine grassland remain in the soil–plant system, with only 55% being lost during 27–28 years. In the first 10cm of the four investigated soil profiles 40% of ¹⁵N was recovered, being mainly bound in organic forms. A simple site specific model was established on the basis of the results considering a biological, residual and labile N-pool, the latter being the source for N-losses. By the model a long mean residence time close to 100 years was derived for the remaining ¹⁵N.     

Effect of hypercholesterolemia and of oxidative stress on the nitric oxide-cGMP pathway

Soluble guanylyl cyclase (sGC) is a key enzyme of the NO-cGMP pathway which is believed to mediate vasoprotective actions. In cardiovascular diseases such as hypercholesterolemia and atherosclerosis, these important functions of the vascular endothelium are strongly impaired. One of the major reasons for this so-called endothelial dysfunction is the increased vascular generation of reactive oxygen species such as superoxide and peroxynitrite. We aimed to investigate whether superoxide and peroxynitrite impacts on the expression and function of sGC and if such a mechanism occurs in a hypercholestemia-induced atherosclerosis. Our experiments with isolated rat aortic rings showed that extracellular superoxide has no effect on expression and function of sGC, while subjection of these rings to continuously generated extracellular peroxynitrite reduced sGC activity. Furthermore, intracellular superoxide as generated by LY85385 almost completely inhibited sGC-activity and increased its expression. In the cholesterol-fed White New Zealand rabbit, we found a 3.5-fold upregulation of sGC, while basal and NO-stimulated sGC-activities were only slightly enhanced and the vasodilator potency of SNAP was decreased by 10-fold. A great portion of the overexpressed dysfunctional sGC is located in intimal lesions. Finally, platelet sGC-activity and the anti-aggregatory effect of SNAP were not changed. These data suggest that endothelial dysfunction in hypercholesterolemia is associated with an oxidative stress-dependent and reversible overexpression of a dysfunctional vascular sGC, while inhibition of platelet sGC-activity is most likely not involved in hypercholesterolemia-induced platelet hyperreactivity.     

Missorting of LaCrosse virus nucleocapsid protein by the interferon-induced MxA GTPase involves smooth ER membranes

The interferon-induced human MxA protein belongs to the class of dynamin-like, large guanosine-5'-triphosphatases that are involved in intracellular vesicle trafficking and organelle homeostasis. MxA shares many properties with the other members of this protein superfamily, including the propensity to self-assemble and to associate with lipid membranes. However, MxA is unique in that it has antiviral activity and inhibits the replication of several RNA viruses. Here, we determined the role of membranes for the antiviral function of MxA using LaCrosse-bunyavirus (LACV). We show that MxA does not affect trafficking and sorting of viral glycoproteins but binds and mislocates the viral nucleocapsid (N) protein into membrane-associated, large perinuclear complexes. We further demonstrate that MxA localizes to a subcompartment of the smooth endoplasmic reticulum where the viral N protein accumulates. In infected MxA-expressing cells, oligomeric MxA/N complexes are formed in close association with COP-I-positive vesicular-tubular membranes. Our results suggest that this membrane compartment is the preferred place where MxA and N interact, leading to efficient sequestration and missorting of an essential viral component.