The minor receptor group of human rhinovirus (HRV) includes HRV23 and HRV25, but the presence of a lysine in the VP1 HI loop is not sufficient for receptor binding

Like all 10 minor receptor group human rhinoviruses (HRVs), HRV23 and HRV25, previously classified as major group viruses, are neutralized by maltose binding protein (MBP)-V33333 (a soluble recombinant concatemer of five copies of repeat 3 of the very-low-density lipoprotein receptor fused to MBP), bind to low-density lipoprotein receptor in virus overlay blots, and replicate in intercellular adhesion molecule 1 (ICAM-1)-negative COS-7 cells. From phylogenetic analysis of capsid protein VP1-coding sequences, they are also known to cluster together with other minor group strains. Therefore, they belong to the minor group; there are now 12 minor group and 87 major group HRV serotypes. Sequence comparison of the VP1 capsid proteins of all HRVs revealed that the lysine in the HI loop, strictly conserved in the 12 minor group HRVs, is also present in 9 major group serotypes that are neutralized by soluble ICAM-1. Despite the presence of this lysine, they are not neutralized by MBP-V33333 and fail to replicate in COS-7 cells and in HeLa cells in the presence of an ICAM-1-blocking antibody. These nine serotypes are therefore "true" major group viruses.     

Gene expression profiling of serum- and interleukin-1 beta-stimulated primary human adult articular chondrocytes--a molecular analysis based on chondrocytes isolated from one donor

In order to understand the cellular disease mechanisms of osteoarthritic cartilage degeneration it is of primary importance to understand both the anabolic and the catabolic processes going on in parallel in the diseased tissue. In this study, we have applied cDNA-array technology (Clontech) to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one donor cultured under anabolic (serum) and catabolic (IL-1beta) conditions. Significant differences between the different in vitro cultures tested were detected. Overall, serum and IL-1beta significantly altered gene expression levels of 102 and 79 genes, respectively. IL-1beta stimulated the matrix metalloproteinases-1, -3, and -13 as well as members of its intracellular signaling cascade, whereas serum increased the expression of many cartilage matrix genes. Comparative gene expression analysis with previously published in vivo data (normal and osteoarthritic cartilage) showed significant differences of all in vitro stimulations compared to the changes detected in osteoarthritic cartilage in vivo. This investigation allowed us to characterize gene expression profiles of two classical anabolic and catabolic stimuli of human adult articular chondrocytes in vitro. No in vitro model appeared to be adequate to study overall gene expression alterations in osteoarthritic cartilage. Serum stimulated in vitro cultures largely reflected the results that were only consistent with the anabolic activation seen in osteoarthritic chondrocytes. In contrast, IL-1beta did not appear to be a good model for mimicking catabolic gene alterations in degenerating chondrocytes.     

Effect of auranofin on the mitochondrial generation of hydrogen peroxide. Role of thioredoxin reductase

The mitochondrial production of hydrogen peroxide, in the presence of different respiratory substrates (succinate, glutamate, malate and isocitrate), is stimulated by submicromolar concentrations of auranofin, a highly specific inhibitor of thioredoxin reductase. This effect is particularly evident in the presence of antimycin. Auranofin was also able to unmask the production of hydrogen peroxide occurring in the presence of rotenone. However, at variance with whole mitochondria, auranofin does not stimulate hydrogen peroxide production in submitochondrial particles indicating that it does not alter the formation of hydrogen peroxide by the respiratory chain but prevents its removal. As the mitochondrial metabolism of hydrogen peroxide proceeds through the peroxidases linked to glutathione or thioredoxin, the relative efficiency of the two systems and the effects of auranofin were tested. In conclusion, the inhibition of thioredoxin reductase determines an increase of the basal flow of hydrogen peroxide leading to a more oxidized condition that alters the mitochondrial functions.     

Oxygen therapy at low flow causes oxidative stress in chronic obstructive pulmonary disease: Prevention by N-acetyl cysteine

Exposure to high oxygen concentration produces toxicity by free radical release. We aimed to study: whether stable chronic obstructive pulmonary disease (COPD) patients present an unbalance in the blood redox status; the effect of oxygen administration on blood redox balance; the efficacy of N-acetyl-cysteine (NAC) treatment against the oxidative stress-induced by oxygen administration and whether it is dose-related. To this, 45 stable state III COPD patients were recruited and reduced glutathione (GSH) and oxidised glutathione (GSSG) in erythrocytes and thiol proteins (P-SH) and carbonyl proteins (PC) in both erythrocytes and plasma were evaluated. All COPD patients underwent 2 l/m oxygen for 18 h and NAC at 1200 or 1800 mg/day or placebo for 48 h starting with oxygen administration. Blood samples were collected at basal conditions, after 8 and 18 h of oxygen administration and 24 h after oxygen withdrawal. Results: COPD patients present an unstable redox equilibrium mainly due to plasma sulphydryl protein depletion. Oxygen administration oxidize erythrocyte GSH, decrease P-SH and increase PC levels in both plasma and erythrocytes. NAC administration counteract the oxidative stress and at the highest dose completely prevent protein oxidation. In conclusion, stable state III COPD patients present an unstable redox balance; long term low flow oxygen administration induces systemic oxidative stress, which is prevented by NAC treatment.     

Root-derived cytokinins as long-distance signals for NO3--induced stimulation of leaf growth

Leaf growth of many plant species shows rapid changes in response to alterations of the form and the level of N supply. In hydroponically-grown tomato (Lycopersicon esculentum L.), leaf growth was rapidly stimulated by NO(3)(-) application to NH(4)(+) precultured plants, while NH(4)(+) supply or complete N deprivation to NO(3)(-) precultured plants resulted in a rapid inhibition of leaf growth. Just 10 microM NO(3)(-) supply was sufficient to stimulate leaf growth to the same extent as 2 mM. Furthermore, continuous NO(3)(-) supply induced an oscillation of leaf growth rate with a 48 h interval. Since changes in NO(3)(-) levels in the xylem exudate and leaves did not correlate with NO(3)(-)-induced alterations of leaf growth rate, additional signals such as phytohormones may be involved. Levels of a known inhibitor of leaf growth, abscisic acid (ABA), did not consistently correspond to leaf growth rates in wild-type plants. Moreover, leaf growth of the ABA-deficient tomato mutant flacca was inhibited by NH(4)(+) without an increase in ABA concentration and was stimulated by NO(3)(-) despite its excessive ethylene production. These findings suggest that neither ABA nor ethylene are directly involved in the effects of N form on leaf growth. However, under all experimental conditions, stimulation of leaf growth by NO(3)(-) was consistently associated with increased concentration of the physiologically active forms of cytokinins, zeatin and zeatin riboside, in the xylem exudate. This indicates a major role for cytokinins as long-distance signals mediating the shoot response to NO(3)(-) perception in roots.     

Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen

The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N(2)O (2NO + 2H(+) + 2e(-) --> N(2)O + H(2)O), which is an obligatory step in the sequential reduction of nitrate to dinitrogen known as denitrification. NOR has four redox-active cofactors, namely, two low-spin hemes c and b, one high-spin heme b(3), and a non-heme iron Fe(B), and belongs to same superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electron acceptor; this catalytic activity was investigated in this study. We show that the product in the steady-state reduction of oxygen is water. A single turnover of the fully reduced NOR with oxygen was initiated using the flow-flash technique, and the progress of the reaction monitored by time-resolved optical absorption spectroscopy. Two major phases with time constants of 40 micros and 25 ms (pH 7.5, 1 mM O(2)) were observed. The rate constant for the faster process was dependent on the O(2) concentration and is assigned to O(2) binding to heme b(3) at a bimolecular rate constant of 2 x 10(7) M(-)(1) s(-)(1). The second phase (tau = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protons from the bulk solution. The rate constant for this phase shows a pH dependence consistent with rate limitation by proton transfer from an internal group with a pK(a) = 6.6. This group is presumably an amino acid residue that is crucial for proton transfer to the catalytic site also during NO reduction.     

Tracking of proinflammatory collagen-specific T cells in early and late collagen-induced arthritis in humanized mice

Rheumatoid arthritis is a chronic inflammatory disease associated with certain HLA-DR4 subtypes. The target autoantigen(s) is unknown, but type II collagen (CII) is a candidate, with a single immunodominant DR4-restricted 261-273 T cell epitope (CII(261-273)). In the present study, we have prepared HLA-DR4:CII(261-273) tetramers and analyzed peripheral blood, lymph node, and synovial fluid cells from DR4-transgenic mice with early and late collagen-induced arthritis to draw a fuller picture of the role of CII-reactive Th cells in disease development. Their frequencies increased approximately 20-fold in blood 1-2 wk postimmunization, and even more in acutely arthritic joints. Our data strongly suggest that CII-specific Th cells are necessary, but not sufficient for collagen-induced arthritis. The CII-specific Th cells displayed an activated proinflammatory Th1 phenotype, and their expansion correlated with onset and severity of arthritis and also with anti-CII Ab levels. Surprisingly, shortly after the first clinical signs of arthritis, activated HLA-DR4:CII tetramer(+) cells became undetectable in the synovial fluid and rare in the blood, but persisted in lymph nodes. Consequently, future human studies should focus on patients with early arthritis, and on their synovial cells, to re-evaluate the occurrence and pathogenic importance of CII-specific or other Th cells in rheumatoid arthritis.     

Dendritic cells exposed to herpes simplex virus in vivo do not produce IFN-alpha after rechallenge with virus in vitro and exhibit decreased T cell alloreactivity

Plasmacytoid dendritic cells (DC) are known to produce large amounts of IFN-alpha when stimulated with virus in vivo and in vitro. Immunohistological staining of spleens from mice taken at different times after HSV infection revealed an early infiltration of plasmacytoid DC whereas both the myeloid DC and lymphoid-related DC had different kinetics. Upon rechallenge with virus in vitro, total splenic DCs from viral-infected mice were unable to produce IFN-alpha when compared with DC from mice that received an initial in vivo injection with PBS. Furthermore, DC from mice that were infected with increasing doses of HSV expressed high levels of accessory and activation molecules compared with control mice. However, when cultured in vitro together with allogeneic T cells, DC from mice that had been exposed to the highest viral titers in vivo induced the lowest levels of T cell proliferation. DC exposed to PBS in vivo promoted a Th1 response upon coculture with CD4(+) T cells whereas T cells cultured with DC exposed to increasing viral titers in vivo resulted in a gradually decreased Th1 response. The data suggest HSV induces DC maturation and at higher titers, exhaustion, diminishing T cell proliferation, and IFN-gamma secretion.     

Direct and ecological costs of resistance and tolerance in the stinging nettle

Plant resistance and tolerance to herbivores, parasites, pathogens, and abiotic factors may involve two types of costs. First, resistance and tolerance may be costly in terms of plant fitness. Second, resistance and tolerance to multiple enemies may involve ecological trade-offs. Our study species, the stinging nettle ( Urtica dioica L.) has significant variation among seed families in resistance and tolerance as well as costs of resistance and tolerance to the holoparasitic plant Cuscuta europaea L. Here we report on variation among seed families (i.e. genetic) in tolerance to nutrient limitation and in resistance to both mammalian herbivores (i.e. number of stinging trichomes) and an invertebrate herbivore (i.e. inverse of the performance of a generalist snail, Arianta arbustorum). Our results indicate direct fitness costs of snail resistance in terms of host reproduction whereas we did not detect fitness costs of mammalian resistance or tolerance to nutrient limitation. We further tested for ecological trade-offs among tolerance or resistance to the parasitic plant, herbivore resistance, and tolerance to nutrient limitation in the stinging nettle. Tolerance of nettles to nutrient limitation and resistance to mammalian herbivores tended to correlate negatively. However, there were no significant correlations among resistance and tolerance to the different natural enemies (i.e. parasitic plants, snails, and mammals). The results of this greenhouse study thus suggest that resistance and tolerance of nettles to diverse enemies are free to evolve independently of each other but not completely without direct costs in terms of plant fitness.