Using a physiological framework for improving the detection of quantitative trait loci related to nitrogen nutrition in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Medicago truncatula is used as a model plant for exploring the genetic and molecular determinants of nitrogen (N) nutrition in legumes. In this study, our aim was to detect quantitative trait loci (QTL) controlling plant N nutrition using a simple framework of carbon/N plant functioning stemming from crop physiology. This framework was based on efficiency variables which delineated the plant’s efficiency to take up and process carbon and N resources. A recombinant inbred line population (LR4) was grown in a glasshouse experiment under two contrasting nitrate concentrations. At low nitrate, symbiotic N₂ fixation was the main N source for plant growth and a QTL with a large effect located on linkage group (LG) 8 affected all the traits. Significantly, efficiency variables were necessary both to precisely localize a second QTL on LG5 and to detect a third QTL involved in epistatic interactions on LG2. At high nitrate, nitrate assimilation was the main N source and a larger number of QTL with weaker effects were identified compared to low nitrate. Only two QTL were common to both nitrate treatments: a QTL of belowground biomass located at the bottom of LG3 and another one on LG6 related to three different variables (leaf area, specific N uptake and aboveground:belowground biomass ratio). Possible functions of several candidate genes underlying QTL of efficiency variables could be proposed. Altogether, our results provided new insights into the genetic control of N nutrition in M. truncatula. For instance, a novel result for M. truncatula was identification of two epistatic interactions in controlling plant N₂ fixation. As such this study showed the value of a simple conceptual framework based on efficiency variables for studying genetic determinants of complex traits and particularly epistatic interactions.     

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Specific immuno capturing of the Staphylococcal superantigen toxic‐shock syndrome toxin‐1 in plasma

Toxic‐shock syndrome is primarily caused by the Toxic‐shock syndrome toxin 1 (TSST‐1), which is secreted by the Gram‐positive bacterium Staphylococcus aureus. The toxin belongs to a family of superantigens (SAgs) which exhibit several shared biological properties, including the induction of massive cytokine release and V_β‐specific T‐cell proliferation. In this study we explored the possibility to use monoclonal Variable domains of Llama Heavy‐chain antibodies (VHH) in the immuno capturing of TSST‐1 from plasma. Data is presented that the selected VHHs are highly specific for TSST‐1 and can be efficiently produced in large amounts in yeast. In view of affinity chromatography, the VHHs are easily coupled to beads, and are able to deplete TSST‐1 from plasma at very low, for example, pathologically relevant, concentrations. When spiked with 4 ng/mL TSST‐1 more than 96% of TSST‐1 was depleted from pig plasma. These data pave the way to further explore application of high‐affinity columns in the specific immuno depletion of SAgs in experimental sepsis models and in sepsis in humans. Biotechnol. Bioeng. 2009; 104: 143–151 © 2009 Wiley Periodicals, Inc.     

Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4⁺ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4⁺ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure.     

Diversity of Halimeda (Bryopsidales,Chlorophyta) in New Caledonia: a Combined Morphological and Molecular Study

Halimeda is a genus of calcified and segmented green macroalgae in the order Bryopsidales. In New Caledonia, the genus is abundant and represents an important part of the reef flora. Previous studies recorded 19 species that were identified using morphological criteria. The aim of this work was to reassess the diversity of the genus in New Caledonia using morpho‐anatomical examinations and molecular analyses of the plastid tufA and rbcL genes. Our results suggest the occurrence of 22 species. Three of these are reported for the first time from New Caledonia: Halimeda kanaloana, H. xishaensis, and an entity resembling H. stuposa. DNA analyses revealed that the species H. fragilis exhibits cryptic or pseudocryptic diversity in New Caledonia. We also show less conclusive evidence for cryptic species within H. taenicola     

Exercise training prevents oxidative stress and ubiquitin-proteasome system overactivity and reverse skeletal muscle atrophy in heart failure

Background

Heart failure (HF) is known to lead to skeletal muscle atrophy and dysfunction. However, intracellular mechanisms underlying HF-induced myopathy are not fully understood. We hypothesized that HF would increase oxidative stress and ubiquitin-proteasome system (UPS) activation in skeletal muscle of sympathetic hyperactivity mouse model. We also tested the hypothesis that aerobic exercise training (AET) would reestablish UPS activation in mice and human HF.

Methods/Principal Findings

Time-course evaluation of plantaris muscle cross-sectional area, lipid hydroperoxidation, protein carbonylation and chymotrypsin-like proteasome activity was performed in a mouse model of sympathetic hyperactivity-induced HF. At the 7^th month of age, HF mice displayed skeletal muscle atrophy, increased oxidative stress and UPS overactivation. Moderate-intensity AET restored lipid hydroperoxides and carbonylated protein levels paralleled by reduced E3 ligases mRNA levels, and reestablished chymotrypsin-like proteasome activity and plantaris trophicity. In human HF (patients randomized to sedentary or moderate-intensity AET protocol), skeletal muscle chymotrypsin-like proteasome activity was also increased and AET restored it to healthy control subjects’ levels.

Conclusions

Collectively, our data provide evidence that AET effectively counteracts redox imbalance and UPS overactivation, preventing skeletal myopathy and exercise intolerance in sympathetic hyperactivity-induced HF in mice. Of particular interest, AET attenuates skeletal muscle proteasome activity paralleled by improved aerobic capacity in HF patients, which is not achieved by drug treatment itself. Altogether these findings strengthen the clinical relevance of AET in the treatment of HF.