Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults

Human rotavirus-specific CD4(+) and CD8(+) T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8(+) and CD4(+) T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4(+) and CD8(+) T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4(+) and CD8(+) T cells and the frequencies of IL-13-secreting CD4(+) T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4(+) cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.     

Mitochondrial reactive oxygen species control the transcription factor CHOP-10/GADD153 and adipocyte differentiation: a mechanism for hypoxia-dependent effect

Recent reports emphasize the importance of mitochondria in white adipose tissue biology. In addition to their crucial role in energy homeostasis, mitochondria are the main site of reactive oxygen species generation. When moderately produced, they function as physiological signaling molecules. Thus, mitochondrial reactive oxygen species trigger hypoxia-dependent gene expression. Therefore the present study tested the implication of mitochondrial reactive oxygen species in adipocyte differentiation and their putative role in the hypoxia-dependent effect on this differentiation. Pharmacological manipulations of mitochondrial reactive oxygen species generation demonstrate a very strong and negative correlation between changes in mitochondrial reactive oxygen species and adipocyte differentiation of 3T3-F442A preadipocytes. Moreover, mitochondrial reactive oxygen species positively and specifically control expression of the adipogenic repressor CHOP-10/GADD153. Hypoxia (1% O2) strongly increased reactive oxygen species generation, hypoxia-inducible factor-1 and CHOP-10/GADD153 expression, and inhibited adipocyte differentiation. All of these hypoxia-dependent effects were partly prevented by antioxidants. By using hypoxia-inducible factor-1alpha (HIF-1alpha)-deficient mouse embryonic fibroblasts, HIF-1alpha was shown not to be required for hypoxia-mediated CHOP-10/GADD153 induction. Moreover, the comparison of hypoxia and CoCl2 effects on adipocyte differentiation of wild type or HIF-1alpha deficient mouse embryonic fibroblasts suggests the existence of at least two pathways dependent or not on the presence of HIF-1alpha. Together, these data demonstrate that mitochondrial reactive oxygen species control CHOP-10/GADD153 expression, are antiadipogenic signaling molecules, and trigger hypoxia-dependent inhibition of adipocyte differentiation.     

LC/MS/NMR analysis of isomeric divanilloylquinic acids from the root bark of Fagara zanthoxyloides Lam

Gradient HPLC coupled to DAD/UV, MS/MS and NMR has been applied to the rapid structure determination of three new isomeric divanilloylquinic acids from Fagara zanthoxyloides collected in Burkina Faso: 3,4-O-divanilloylquinic acid, 3,5-O-divanilloylquinic acid and 4,5-O-divanilloylquinic acid. Furthermore these new compounds named burkinabins A-C could play a useful role in sickle cell disease, as the active agents of Fagara zanthoxylo?des are said to be unidentified aromatic compounds with carboxylic acid grouping (Adesanya, S.A., Sofowora, A., 1983. Biological standardisation of Zanthoxylum roots for antisickling activity. Planta Med. 48, 27-33).     

Herbaceous plant richness and vegetation cover in Mediterranean grasslands and shrublands

Different types of relationship between herbaceous species richness and several parameters indicating abundance of plant material (herbaceous, woody plants, litter and bare ground cover) are presented. The data were obtained from 50 sites along a 300 km strip running from E to W within Spain and Portugal. Each site was representative of the silvo-pastoral landscape of the Mediterranean type ecosystems of the Iberian peninsula, and contained two neighboring patches, one of grassland and the other of shrubland. 3,600 20 × 20 cm subplots were randomly located (72 per site, 36 per patch) crossing the boundary grassland/shrubland. This approach allowed us to analyze the richness-occupation relationship of the space from different points of view: among and within the sites, and among and within the grassland and shrubland plant communities. We found a unimodal relationship between richness-cover similar to the one generally accepted between richness and biomass. Our results show that the dependence of this relationship varies depending on the spatial scale of the analysis and on the type of data used. When the whole region is taken into account, significant unimodal relationships are found between richness and herbaceous cover, litter and bare ground, and a negative linear relationship with woody plant cover. Within the sites there are mainly linear or non-significant relationships. But the results also depend on the type of communities analyzed. In pastures, the unimodal relationship represents the combination of positive and negative linear responses for low and high cover values, respectively. The value for herbaceous cover in which maximum richness occurs is around 60%. In shrublands, this value for cover also corresponds to maximum species richness, although the possibilities of reaching it are limited by other variables, such as woody plant cover. This implies that, on not considering variability at local scale, the relationship is linear and positive. This paper shows the existence of a common model related to herbaceous cover, but this model has multiple controlling factors that act differently in each type of community.     

Characterization of a Novel Angular Dioxygenase from Fluorene-Degrading Sphingomonas sp. Strain LB126

In this study, the genes involved in the initial attack on fluorene by Sphingomonas sp. strain LB126 were investigated. The α and β subunits of a dioxygenase complex (FlnA1-FlnA2), showing 63 and 51% sequence identity, respectively, to the subunits of an angular dioxygenase from the gram-positive dibenzofuran degrader Terrabacter sp. strain DBF63, were identified. When overexpressed in Escherichia coli, FlnA1-FlnA2 was responsible for the angular oxidation of fluorene, 9-hydroxyfluorene, 9-fluorenone, dibenzofuran, and dibenzo-p-dioxin. Moreover, FlnA1-FlnA2 was able to oxidize polycyclic aromatic hydrocarbons and heteroaromatics, some of which were not oxidized by the dioxygenase from Terrabacter sp. strain DBF63. The quantification of resulting oxidation products showed that fluorene and phenanthrene were the preferred substrates of FlnA1-FlnA2.     

Functional implication of neuronal calcium sensor-1 and phosphoinositol 4-kinase-beta interaction in regulated exocytosis of PC12 cells

Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-beta (PI4K-beta) regulate the exocytotic process of nerve and neuroendocrine cells. The aim of our study was to investigate their possible interaction at rest and during stimulation in living cells and to decipher the role of this interaction in the secretory process. In PC12 cells, we observed a stimulation-induced recruitment of NCS-1 and PI4K-beta from the intracellular compartment toward the plasma membrane. This recruitment was highly correlated to the intracellular Ca(2+) rise induced by secretagogues. Using fluorescence resonance energy transfer between PI4K-beta-ECFP and NCS-1-EYFP, we show that both proteins are interacting in resting cells and that this interaction increases with stimulation. It appears that the membrane insertion of NCS-1 is necessary for the interaction with PI4K-beta, since a mutation that prevented the membrane insertion of NCS-1 abolished NCS-1-PI4K-beta interaction, as revealed by fluorescence resonance energy transfer analysis. Additionally, the overexpression of mutated NCS-1 prevents the stimulatory effect on secretion induced by PI4K-beta, suggesting that the interaction of the two proteins on a membrane compartment is necessary for the secretory function. Moreover, extinction of endogenous PI4K-beta by small interfering RNA inhibits secretion and completely prevents the stimulatory effect of NCS-1 on calcium-evoked exocytosis from permeabilized PC12 cells, showing directly for the first time the functional implication of a NCS-1.PI4K-beta complex in regulated exocytosis.     

Structural and thermodynamical properties of CuII amyloid-β16/28 complexes associated with Alzheimer’s disease

The aggregation of the peptide amyloid-β (Aβ) to form amyloid plaques is a key event in Alzheimer’s disease. It has been shown that Cu^II can bind to soluble Aβ and influence its aggregation properties. Three histidines and the N-terminal amine have been proposed to be involved in its coordination. Here, for the first time, we show isothermal titration calorimetry (ITC) measurements of the Cu^II binding to Aβ16 and Aβ28, models of the soluble Aβ. Moreover, different spectroscopic methods were applied. The studies revealed new insights into these Cu^II–Aβ complexes: (1) ITC showed two Cu^II binding sites, with an apparent K _d of 10⁻⁷ and 10⁻⁵ M, respectively; (2) the high-affinity site has a smaller enthalpic contribution but a larger entropic contribution than the low-affinity binding site; (3) azide did not bind to Cu^II in the higher-affinity binding site, suggesting the absence of a weak, labile ligand; (4) azide could bind to the Cu^II in the low-affinity binding site in Aβ28 but not in Aβ16; (5) ¹H-NMR suggests that the carboxylate of aspartic acid in position 1 is involved in the ligation to Cu^II in the high-affinity binding site; (6) the pK _a of 11.3 of tyrosine in position 10 was not influenced by the binding of 2 equivalents of Cu^II.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells

Human Rhinovirus (HRV) infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups) that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C) able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.