Patterns of linkage disequilibrium and association mapping in diploid alfalfa (M. sativa L.)

Association mapping enables the detection of marker-trait associations in unstructured populations by taking advantage of historical linkage disequilibrium (LD) that exists between a marker and the true causative polymorphism of the trait phenotype. Our first objective was to understand the pattern of LD decay in the diploid alfalfa genome. We used 89 highly polymorphic SSR loci in 374 unimproved diploid alfalfa (Medicago sativa L.) genotypes from 120 accessions to infer chromosome-wide patterns of LD. We also sequenced four lignin biosynthesis candidate genes (caffeoyl-CoA 3-O-methyltransferase (CCoAoMT), ferulate-5-hydroxylase (F5H), caffeic acid-O-methyltransferase (COMT), and phenylalanine amonialyase (PAL 1)) to identify single nucleotide polymorphisms (SNPs) and infer within gene estimates of LD. As the second objective of this study, we conducted association mapping for cell wall components and agronomic traits using the SSR markers and SNPs from the four candidate genes. We found very little LD among SSR markers implying limited value for genomewide association studies. In contrast, within gene LD decayed within 300 bp below an r (2) of 0.2 in three of four candidate genes. We identified one SSR and two highly significant SNPs associated with biomass yield. Based on our results, focusing association mapping on candidate gene sequences will be necessary until a dense set of genome-wide markers is available for alfalfa.     

Protection of peritoneal macrophages by granulocyte/macrophage colony-stimulating factor (GM-CSF) against dexamethasone suppression of killing of Aspergillus, and the effect of human GM-CSF

Murine peritoneal macrophages in vitro could kill Aspergillus fumigatus conidia, and this activity could be suppressed with dexamethasone. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) alone did not boost killing, but GM-CSF treatment concurrently with dexamethasone reversed the dexamethasone suppression. Both recombinant human and recombinant murine GM-CSF were equivalent in this activity, even though the human reagent reportedly does not stimulate differentiation of murine stem cells. Recombinant human GM-CSF could also reverse dexamethasone suppression of bronchoalveolar macrophage conidiacidal activity. Sequential studies with peritoneal macrophages indicated that recombinant human GM-CSF pretreatment also blocked dexamethasone suppression, but the GM-CSF treatment given after dexamethasone did not block the suppressive effect. Recombinant human GM-CSF did not boost spleen cell proliferation to a mitogenic stimulus, and did not reverse dexamethasone suppression of proliferation. These studies suggest GM-CSF treatment prior to and concurrent with steroid immunosuppression may ameliorate the steroid effect on tissue macrophage antifungal activity, but does not affect steroid suppression of T-cell immunity.     

Biomass Yield of Naturalized Populations and Cultivars of Reed Canary Grass

Reed canary grass is a widely adapted temperate grass with a circumglobal distribution in the northern hemisphere. Because it has relatively high biomass yields under relatively infrequent harvest systems, this species is receiving increasing attention as a bioenergy feedstock. The objective of this study was to conduct a comparative biomass yield evaluation of reed canary grass accessions from a wide range of habitats in the north central and northeastern USA. Eight cultivars and 72 accessions were evaluated for biomass yield over 2 years at five locations in Iowa, New York, and Wisconsin. Accessions produced, on average, 6.7% higher biomass yield than the cultivars. Cultivars ranked from 50th to 77th in biomass yield out of a total of 80 cultivars and accessions. Genetic expression for biomass yield was highly consistent across locations and years. Accessions from southern and western collection sites tended to have the highest biomass yield. Reed canary grass populations in rural landscapes of the central and northeastern USA have value for increasing biomass yield potential of this bioenergy feedstock candidate species. The high biomass yield of many of these populations, combined with the large amount of genetic variability among these populations, offers potential for both short-term gains by selecting superior accessions and long-term gains by selection and breeding.     

New method to study oxidative damage and antioxidants in the human small bowel: effects of iron application

Iron may induce oxidative damage to the intestinal mucosa by its catalyzing role in the formation of highly reactive hydroxyl radicals. This study aimed to determine iron-induced oxidative damage provoked by a single clinical dosage of ferrous sulfate and to elucidate the antioxidant defense mechanisms in the human small intestine in vivo. A double-lumen perfusion tube was positioned orogastrically into a 40-cm segment of the proximal small intestine in six healthy volunteers (25 +/- 5 yr). The segment was perfused with saline and subsequently with saline containing 80 mg iron as ferrous sulfate at a rate of 10 ml/min. Intestinal fluid samples were collected at 15-min intervals. Thiobarbituric acid reactive substances concentrations as an indicator of lipid peroxidation increased significantly from 0.07 microM (range, 0-0.33 microM) during saline perfusion to 3.35 microM (range, 1.19-7.27 microM) during iron perfusion (P < 0.05). Nonprotein antioxidant capacity increased significantly from 474 microM (range, 162-748 microM) to 1,314 microM (range, 674-1,542 microM) (P < 0.05). These data show that a single dosage of ferrous sulfate induces oxidative damage and the subsequent release of an antioxidant in the small intestine in vivo in healthy volunteers.     

Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

Engagement of the B-cell antigen receptor (BCR) leads to activation of the Raf-MEK-ERK pathway and Raf kinases play an important role in the modulation of ERK activity. B lymphocytes express two Raf isoforms, Raf-1 and B-Raf. Using an inducible deletion system in DT40 cells, the contribution of Raf-1 and B-Raf to BCR signalling was dissected. Loss of Raf-1 has no effect on BCR-mediated ERK activation, whereas B-Raf-deficient DT40 cells display a reduced basal ERK activity as well as a shortened BCR-mediated ERK activation. The Raf-1/B-Raf double deficient DT40 cells show an almost complete block both in ERK activation and in the induction of the immediate early gene products c-Fos and Egr-1. In contrast, BCR-mediated activation of nuclear factor of activated T cells (NFAT) relies predominantly on B-Raf. Furthermore, complementation of Raf-1/B-Raf double deficient cells with various Raf mutants demonstrates a requirement for Ras-GTP binding in BCR-mediated activation of both Raf isoforms and also reveals the important role of the S259 residue for the regulation of Raf-1. Our study shows that BCR-mediated ERK activation involves a cooperation of both B-Raf and Raf-1, which are activated specifically in a temporally distinct manner.     

Immunological basis for susceptibility and resistance to pulmonary blastomycosis in mouse strains

The immunological basis for a >10-fold resistance of outbred CD-1 mice compared to inbred BALB/c mice to pulmonary blastomycosis was investigated. Bronchoalveolar macrophages (BAM) from CD-1 mice killed yeast cells of Blastomyces dermatitidis (Bd) by 25% and this increased to 59% when activated by IFN-gamma. In contrast, BAM from BALB/c mice lacked significant killing (5%) of Bd but could be activated by IFN-gamma for enhanced killing (19%). Peritoneal macrophages (PM) from CD-1 mice had significant fungicidal activity for Bd (43%) and this increased to 63% with IFN-gamma treatment. By contrast, PM from BALB/c mice did not significantly kill Bd (14%) but were activated by IFN-gamma for significant killing (24%). Fungicidal activity of peripheral blood polymorphonuclear neutrophils (PMN) from CD-1 (87%) was greater than that of BALB/c (75%) (P<0.05). Macrophage inflammatory protein-1alpha (MIP-1alpha) production by BAM from BALB/c was significantly less than that from CD-1 in response to co-culture with Bd. IFN-gamma production by CD-1 spleen cells in response to concanavalin A (Con A, 1microg/ml) was 8-fold greater than that by BALB/c spleen cells. In contrast, BAM and PM from BALB/c mice in co-culture with Bd secreted several-fold more TNFalpha than BAM or PM from CD-1 mice. IL-2 production by BALB/c spleen cells in response to Con A was 3- to 4-fold greater than that by CD-1 spleen cells. Depressed IL-2 production by Con A stimulated CD-1 spleen cells correlated with depressed proliferative responses. Resistance of CD-1 mice to pulmonary blastomycosis correlates with enhanced fungicidal activity of BAM, PM, PMN, and IFN-gamma production by Con A stimulated spleen cells, compared to BALB/c mice. Consistent with the in vitro enhancement of effector cell function by IFN-gamma, in vivo therapy with IFN-gamma significantly (P<0.0001) improved survival of BALB/c mice with pulmonary blastomycosis.     

In vivo and in vitro activation of pulmonary macrophages by IFN-gamma for enhanced killing of Paracoccidioides brasiliensis or Blastomyces dermatitidis

The fungicidal capacity of murine pulmonary macrophages (PuM) activated in vitro with IFN or lymphokines or in vivo with IFN was studied. PuM treated overnight with IFN (1000 U/ml), Con A-stimulated spleen cell culture supernatants, or lymph node cells plus Con A significantly killed yeast cells of the Gar w isolate of Paracoccidioides brasiliensis 45.5 +/- 2.1%, 72.0 +/- 4.2%, and 51.5 +/- 0.7% respectively. Two other isolates of P. brasiliensis (Ru and LA) were also killed (45 and 34%) by PuM activated by lymph node cells plus Con A. Control PuM had lesser but significant capacity for killing of P. brasiliensis isolates, ranging from 15 to 22%. Killing of P. brasiliensis by PuM activated by Con A-stimulated spleen cell culture supernatants could not be significantly inhibited by superoxide dismutase, catalase, or azide. When mice were treated in vivo with 4 X 10(5) IFN U i.p. and PuM isolated 24 h later, the PuM had significantly enhanced ability to kill P. brasiliensis (47.0 +/- 6.3%) compared with PuM from control mice (25.0 +/- 4.2%). PuM thus activated also showed enhanced killing (43%) of a second isolate compared with control PuM (22%). PuM from IFN-treated mice were able to significantly kill Blastomyces dermatitidis (37.5 +/- 0.7%) compared with control PuM (4.5 +/- 6.3%). These results show that PuM can be activated in vitro and in vivo by IFN for enhanced fungicidal activity against two pulmonary fungal pathogens and suggests that immunologic production of IFN could be an important factor in host defenses against these diseases.