Arbuscular mycorrhiza-specific signaling in rice transcends the common symbiosis signaling pathway.

Knowledge about signaling in arbuscular mycorrhizal (AM) symbioses is currently restricted to the common symbiosis (SYM) signaling pathway discovered in legumes. This pathway includes calcium as a second messenger and regulates both AM and rhizobial symbioses. Both monocotyledons and dicotyledons form symbiotic associations with AM fungi, and although they differ markedly in the organization of their root systems, the morphology of colonization is similar. To identify and dissect AM-specific signaling in rice (Oryza sativa), we developed molecular phenotyping tools based on gene expression patterns that monitor various steps of AM colonization. These tools were used to distinguish common SYM-dependent and -independent signaling by examining rice mutants of selected putative legume signaling orthologs predicted to be perturbed both upstream (CASTOR and POLLUX) and downstream (CCAMK and CYCLOPS) of the central, calcium-spiking signal. All four mutants displayed impaired AM interactions and altered AM-specific gene expression patterns, therefore demonstrating functional conservation of SYM signaling between distant plant species. In addition, differential gene expression patterns in the mutants provided evidence for AM-specific but SYM-independent signaling in rice and furthermore for unexpected deviations from the SYM pathway downstream of calcium spiking.

Local selection rules that can determine specific pathways of DNA unknotting by type II DNA topoisomerases.

We performed numerical simulations of DNA chains to understand how local geometry of juxtaposed segments in knotted DNA molecules can guide type II DNA topoisomerases to perform very efficient relaxation of DNA knots. We investigated how the various parameters defining the geometry of inter-segmental juxtapositions at sites of inter-segmental passage reactions mediated by type II DNA topoisomerases can affect the topological consequences of these reactions. We confirmed the hypothesis that by recognizing specific geometry of juxtaposed DNA segments in knotted DNA molecules, type II DNA topoisomerases can maintain the steady-state knotting level below the topological equilibrium. In addition, we revealed that a preference for a particular geometry of juxtaposed segments as sites of strand-passage reaction enables type II DNA topoisomerases to select the most efficient pathway of relaxation of complex DNA knots. The analysis of the best selection criteria for efficient relaxation of complex knots revealed that local structures in random configurations of a given knot type statistically behave as analogous local structures in ideal geometric configurations of the corresponding knot type.

Is carbohydrate-deficient transferrin a specific marker for alcohol abuse? A study in patients with chronic viral hepatitis.

Carbohydrate-deficient transferrin, a transferrin isoform, is hailed as a new marker of chronic alcohol abuse, but its specificity is, however, not unequivocally accepted. The aim of the present study was therefore to determine carbohydrate-deficient transferrin levels in patients with chronic hepatitis B and C with or without documented chronic alcohol intake. Carbohydrate-deficient transferrin was measured using a double-antibody radioimmunoassay (CDTect, Pharmacia) in serum samples from 66 patients (45 males and 21 females; mean age: 39 years) with chronic viral hepatitis B (n = 20) or C (n = 46). Diagnosis of the underlying liver disease was established by liver biopsy. Carbohydrate-deficient transferrin levels were raised in 15 patients [23%; hepatitis B (n = 2) and hepatitis C (n = 13)]. In patients with chronic hepatitis B, the carbohydrate-deficient transferrin level was raised in two abstainers. In the 46 patients with chronic hepatitis C, 10 (22%) patients with an alcohol consumption of < 60 g/day for the men and 30 g/day for the women had raised carbohydrate-deficient transferrin levels. The overall specificity of carbohydrate-deficient transferrin for chronic alcohol abuse was thus 78%, suggesting an association between elevated carbohydrate-deficient transferrin levels and the presence of chronic viral hepatitis. Carbohydrate-deficient transferrin levels were not correlated with the histological grading or staging of chronic hepatitis B and C, or with biological markers of hepatic synthesis and cellular damage. Thus, an increased carbohydrate-deficient transferrin level may occur in patients with chronic viral hepatitis in the absence of chronic alcohol abuse. This fact should be kept in mind by physicians when using this marker to detect alcohol abuse.

Allergen-specific antibody and cytokine responses, mast cell reactivity and intestinal permeability upon oral challenge of sensitized and tolerized mice.

BACKGROUND: Food allergy has reached an epidemic level in westernized countries and although central mechanisms have been described, the variability associated with genetic diversity underscores the still unresolved complexity of these disorders. OBJECTIVE: To develop models of food allergy and oral tolerance, both strictly induced by the intestinal route, and to compare antigen-specific responses. METHODS: BALB/c mice were mucosally sensitized to ovalbumin (OVA) in the presence of the mucosal adjuvant cholera toxin, or tolerized by intra-gastric administrations of OVA alone. Antibody titres and cytokines were determined by ELISA, and allergic status was determined through several physiologic parameters including decline in temperature, diarrhoea, mast cell degranulation and intestinal permeability. RESULTS: OVA-specific antibodies (IgE, IgGs and IgA in serum and feces) were produced in sensitized mice exclusively. Upon intra-gastric challenge with OVA, sensitized mice developed anaphylactic reactions associated with a decline of temperature, diarrhoea, degranulation of mast cells, which were only moderately recruited in the small intestine, and increased intestinal permeability. Cytokines produced by immune cells from sensitized mice included T-helper type 2 cytokines (IL-5, IL-13), but also IL-10, IFN-gamma and IL-17. In contrast, all markers of allergy were totally absent in tolerized animals, and yet the latter were protected from subsequent sensitization, demonstrating that oral tolerance took place efficiently. CONCLUSION: This work allows for the first time an appropriate comparison between sensitized and tolerized BALB/c mice towards OVA. It highlights important differences from other models of allergy, and thus questions some of the generally accepted notions of allergic reactions, such as the protective role of IFN-gamma, the importance of antigen-specific secretory IgA and the role of mucosal mast cells in intestinal anaphylaxis. In addition, it suggests that IL-17 might be an effector cytokine in food allergy. Finally, it demonstrates that intestinal permeability towards the allergen is increased during challenge.

Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3

Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of alpha- and gamma-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCD(cl1) cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC

The dexamethasone-induced inhibition of proliferation, migration, and invasion in glioma cell lines is antagonized by macrophage migration inhibitory factor (MIF) and can be enhanced by specific MIF inhibitors.

Glioblastomas (GBMs) are the most frequent and malignant brain tumors in adults. Glucocorticoids (GCs) are routinely used in the treatment of GBMs for their capacity to reduce the tumor-associated edema. Few in vitro studies have suggested that GCs inhibit the migration and invasion of GBM cells through the induction of MAPK phosphatase 1 (MKP-1). Macrophage migration inhibitory factor (MIF), an endogenous GC antagonist is up-regulated in GBMs. Recently, MIF has been involved in tumor growth and migration/invasion and specific MIF inhibitors have been developed on their capacity to block its enzymatic tautomerase activity site. In this study, we characterized several glioma cell lines for their MIF production. U373 MG cells were selected for their very low endogenous levels of MIF. We showed that dexamethasone inhibits the migration and invasion of U373 MG cells, through a glucocorticoid receptor (GR)- dependent inhibition of the ERK1/2 MAPK pathway. Oppositely, we found that exogenous MIF increases U373 MG migration and invasion through the stimulation of the ERK1/2 MAP kinase pathway and that this activation is CD74 independent. Finally, we used the Hs 683 glioma cells that are resistant to GCs and produce high levels of endogenous MIF, and showed that the specific MIF inhibitor ISO-1 could restore dexamethasone sensitivity in these cells. Collectively, our results indicate an intricate pathway between MIF expression and GC resistance. They suggest that MIF inhibitors could increase the response of GBMs to corticotherapy.

DNA structure-specific priming of ATR activation by DNA-PKcs.

Three phosphatidylinositol-3-kinase-related protein kinases implement cellular responses to DNA damage. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia-telangiectasia mutated respond primarily to DNA double-strand breaks (DSBs). Ataxia-telangiectasia and RAD3-related (ATR) signals the accumulation of replication protein A (RPA)-covered single-stranded DNA (ssDNA), which is caused by replication obstacles. Stalled replication intermediates can further degenerate and yield replication-associated DSBs. In this paper, we show that the juxtaposition of a double-stranded DNA end and a short ssDNA gap triggered robust activation of endogenous ATR and Chk1 in human cell-free extracts. This DNA damage signal depended on DNA-PKcs and ATR, which congregated onto gapped linear duplex DNA. DNA-PKcs primed ATR/Chk1 activation through DNA structure-specific phosphorylation of RPA32 and TopBP1. The synergistic activation of DNA-PKcs and ATR suggests that the two kinases combine to mount a prompt and specific response to replication-born DSBs.

Mice carrying ubiquitin-specific protease 2 (Usp2) gene inactivation maintain normal sodium balance and blood pressure.

Ubiquitylation plays an important role in the control of Na⁺ homeostasis by the kidney. It is well established that the epithelial Na⁺ channel ENaC is regulated by the ubiquitin-protein ligase NEDD4-2, limiting ENaC cell surface expression and activity. Ubiquitylation can be reversed by the action of deubiquitylating enzymes (DUBs). One such DUB, USP2-45, was identified previously as an aldosterone-induced protein in the kidney and is also a circadian output gene. In heterologous expression systems, USP2-45 binds to ENaC, deubiquitylates it, and enhances channel density and activity at the cell surface. Because the role of USP2-45 in renal Na⁺ transport had not been studied in vivo, we investigated here the effect of Usp2 gene inactivation in this process. We demonstrate first that USP2-45 protein has a rhythmic expression with a peak at ZT12. Usp2-KO mice did not show any differences from wild-type littermates with respect to the diurnal control of Na⁺ or K⁺ urinary excretion and plasma levels either on a standard diet or after acute and chronic changes to low- and high-Na⁺ diets, respectively. Moreover, they had similar aldosterone levels on either a low- or high-Na⁺ diet. Blood pressure measurements using telemetry did not reveal variations compared with control mice. Usp2-KO mice did not display alterations in expression of genes involved in sodium homeostasis or the ubiquitin system, as evidenced by transcriptome analysis in the kidney. Our data suggest that USP2 does not play a primary role in the control of Na⁺ balance or blood pressure.

High frequency of functionally active Melan-a-specific T cells in a patient with progressive immunoproteasome-deficient melanoma.

Tumor-reactive T cells play an important role in cancer immunosurveillance. Applying the multimer technology, we report here an unexpected high frequency of Melan-A-specific CTLs in a melanoma patient with progressive lymph node metastases, consisting of 18 and 12.8% of total peripheral blood and tumor-infiltrating CD8+ T cells, respectively. Melan-A-specific CTLs revealed a high cytolytic activity against allogeneic Melan-A-expressing target cells but failed to kill the autologous tumor cells. Loading of the tumor cells with Melan-A peptide reversed the resistance to killing, suggesting impaired function of the MHC class I antigen processing and presentation pathway. Mutations of the coding region of the HLA-A2 binding Melan-A26-35 peptide or down-regulation of the MHC class I heavy chain, the antigenic peptide TAP, and tapasin could be excluded. However, PCR and immunohistochemical analysis revealed a deficiency of the immunoproteasomes low molecular weight protein 2 and low molecular weight protein 7 in the primary tumor cells, which affects the quantity and quality of generated T-cell epitopes and might explain the resistance to killing. This is supported by our data, demonstrating that the resistance to killing can be partially reversed by pre-exposure of the tumor cells to IFN-gamma, which is known to induce the immunoproteasomes. Overall, this is the first report of an extremely high frequency of tumor-specific CTLs that exhibit competent T-cell-effector functions but fail to lyse the autologous tumor cells. Immunotherapeutic approaches should not only focus on the induction of a robust antitumor immune response, but should also have to target tumor immune escape mechanisms.

Maximum-likelihood estimation of specific differential phase and attenuation in rain

Precise estimation of propagation parameters inprecipitation media is of interest to improve the performanceof communications systems and in remote sensing applications.In this paper, we present maximum-likelihood estimators ofspecific attenuation and specific differential phase in rain. Themodel used for obtaining the cited estimators assumes coherentpropagation, reflection symmetry of the medium, and Gaussianstatistics of the scattering matrix measurements. No assumptionsabout the microphysical properties of the medium are needed.The performance of the estimators is evaluated through simulateddata. Results show negligible estimators bias and variances closeto Cramer–Rao bounds.

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51.

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.

Homeostasis and in vivo effector function of antigen-specific CD4+CD25+ regulatory T cells in experimental transplantation.

CD4+CD25+ regulatory T cells (Tregs) play a critical role in the prevention of autoimmune diseases as well as in the induction and maintenance of dominant tolerance in transplantation models. While their suppressive function has been extensively studied in vitro, their homeostasis and mechanisms of immunoregulation still remain to be clarifi ed in vivo. Using a murine adoptive transfer and skin allograft model, we analysed the expansion, effector function and traffi cking of effector T cells in the presence or absence of donor-specifi c Tregs. Although hyporesponsive to allogeneic and polyclonal stimulation in vitro, transferred Tregs survived and expanded, in response to an allograft in vivo. When co-transferred with naive CD4+CD25- effector T cells, they specifi cally prevented donor but not 3rd party allograft rejection by inhibiting the production of effector cytokines rather than the proliferation of effector T cells in response to alloantigens. The co-transfer of donor-specifi c Tregs did not affect the homing of effector T cells towards the graft draining lymph nodes, but it markedly reduced the infi ltration of the allograft by these pathogenic cells. Furthermore, in recipients where donor-specifi c transplantation tolerance was induced, Tregs preferentially accumulated in the allograft draining lymph nodes and within the grafted skin itself. Taken together, our results suggest that the suppression of graft rejection is an active process that involves the persistent presence of Tregs at the site of antigenic challenge.

A sex-specific marker reveals male heterogamety in European tree frogs.

Most amphibians examined so far show undifferentiated sex chromosomes. The heterogametic sex's identity, usually revealed through indirect means, often varies among closely related species or even populations (as do sex-linkage groups), suggesting great evolutionary instability of the sex-determining genes. Here we take advantage of a sex-specific marker that amplifies in several related species of European tree frogs (Hyla arborea group) to disclose a homogeneous pattern of male heterogamety. Besides relevance for evolutionary studies of sex determination in amphibians, our results have potential for addressing practical issues in conservation biology because sex reversal by anthropogenic endocrine disruptors is considered one possible cause of amphibian decline.

Características geológicas y geotécnicas de los lodos de flotación de la Sierra Minera de Cartagena-La Unión (SE España)

We present the results of a geological and geotechnical characterization of the metallurgic waste from the Sierra Minera de Cartagena-La Union. We have studied eight tailings dams from which we collected and analysed 42 samples of metallurgic waste. We measured grainsize distribution, the specific gravity of solid particles, plasticity index, permeability, both in situ and in the laboratory, direct shear characteristicsand moisture content. According to size distribution the tailings can be classified as sandy silt. Their plasticity index ranges from medium to nil. The internal friction angle varies between 28 and 42 degrees. Cohesion is between 0 and 2.2 t/m2. The specific gravity of the solid particles ranges widely from 1.8 to 4 g/cm3. The saturated hydraulic conductivity values vary between 1.3x 10-5 and 3.2x 10-9 m/s.The water content measured in situ shows that the degree of saturation remains relatively high despite low rainfall and high evaporation rates. Several tailings dams have failed. The leading causes of tailings-dam failure are: 1) slope instability; 2) overflow; 3) erosion; and 4) subsidence or collapse. The main factor leading to dam failure is that the tailings stored in the ponds are highly saturated