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A visible spectral survey (3675-6744 Å) from the Alcator C-Mod tokamak has been performed using a high-resolution visible spectrometer constructed at the Lawrence Livermore National Laboratory. The Alcator C-Mod deuterium plasma is shown to have visible emission from numerous atomic species and low charge state ions including, D I, B II-III, B V, C II-III, N II-III, O II-IV, F II-III, Ne I, Na X, Al II-III, Si I-II, Cl II-III, Ar I-III, Ar X, Ti I and III, Fe I-III, Cu I, and III, Mo I, and W I. Nearly all of the emission is thought to come from the cooler edge of the plasma including the scrape-off layer, outside of the last closed magnetic flux surface. However, there is at least one example, included here, where intrinsic visible emission persists deeper into the plasma. [ABSTRACT FROM AUTHOR]

The Farm Scale Evaluations (FSEs) showed that genetically modified herbicide-tolerant (GMHT) cropping systems could influence farmland biodiversity because of their effects on weed biomass and seed production. Recently published results for winter oilseed rape showed that a switch to GMHT crops significantly affected weed seedbanks for at least 2 years after the crops were sown, potentially causing longer-term effects on other taxa. Here, we seek evidence for similar medium-term effects on weed seedbanks following spring-sown GMHT crops, using newly available data from the FSEs. Weed seedbanks following GMHT maize were significantly higher than following conventional varieties for both the first and second years, while by contrast, seedbanks following GMHT spring oilseed rape were significantly lower over this period. Seedbanks following GMHT beet were smaller than following conventional crops in the first year after the crops had been sown, but this difference was much reduced by the second year for reasons that are not clear. These new data provide important empirical evidence for longer-term effects of GMHT cropping on farmland biodiversity.

Farmland biodiversity and food webs were compared in conventional and genetically modified herbicide-tolerant (GMHT) crops of beet (Beta vulgaris L.), maize (Zea mays L.) and both spring and winter oilseed rape (Brassica napus L.). GMHT and conventional varieties were sown in a split-field experimental design, at 60-70 sites for each crop, spread over three starting years beginning in 2000. This paper provides a background to the study and the rationale for its design and interpretation. It shows how data on environment, field management and the biota are used to assess the current state of the ecosystem, to define the typical arable field and to devise criteria for selecting, sampling and auditing experimental sites in the Farm Scale Evaluations. The main functional and taxonomic groups in the habitat are ranked according to their likely sensitivity to GMHT cropping, and the most responsive target organisms are defined. The value of the seedbank as a baseline and as an indicator of historical trends is proposed. Evidence from experiments during the twentieth century is analysed to show that large changes in field management have affected sensitive groups in the biota by ca. 50% during a year or short run of years--a figure against which to assess any positive or negative effects of GMHT cropping. The analysis leads to a summary of factors that were, and were not, examined in the first 3 years of the study and points to where modelling can be used to extrapolate the effects to the landscape and the agricultural region.

The Farm Scale Evaluations of genetically modified herbicide-tolerant crops (GMHT) were conducted in the UK from 2000 to 2002 on beet (sugar and fodder), spring oilseed rape and forage maize. The management of the crops studied is described and compared with current conventional commercial practice. The distribution of field sites adequately represented the areas currently growing these crops, and the sample contained sites operated at a range of management intensities, including low intensity. Herbicide inputs were audited, and the active ingredients used and the rates and the timings of applications compared well with current practice for both GMHT and conventional crops. Inputs on sugar beet were lower than, and inputs on spring oilseed rape and forage maize were consistent with, national averages. Regression analysis of herbicide-application strategies and weed emergence showed that inputs applied by farmers increased with weed densities in beet and forage maize. GMHT crops generally received only one herbicide active ingredient per crop, later and fewer herbicide sprays and less active ingredient (for beet and maize) than the conventional treatments. The audit of inputs found no evidence of bias.

A new, non-destructive assay is described to quantify cytoplasmic glutathione (GSH) levels in vivo in single cells or populations of cells from Arabidopsis suspension cultures. Cytoplasmic GSH was labelled with monochlorobimane (MCB) in situ to give a fluorescent GSH-bimane (GSB) conjugate. At low (10-100 microM) concentrations of MCB, labelling was mediated by a glutathione S-transferase, which confers specificity for GSH. HPLC analysis of MCB-labelled low molecular-weight thiols showed that the assay measures the total GSH pool, including the oxidized glutathione. The progress curve for the labelling could be described using Michaelis-Menten kinetics with an apparent KM of 40 microM and Vmax of 470 micromol lcyt -1 min-1. There was no evidence for de novo synthesis of GSH during the labelling period of 2 h, suggesting that control of GSH synthesis is not mediated by feedback control of gamma-glutamylcysteine synthetase in this system. The total cellular level of GSH was calculated from the plateau value of the progress curve, after appropriate calibration, as 830-942 nmol g-1 FW. The volume fraction of cytoplasm was measured from serial optical sections of bimane-labelled cells collected by confocal laser scanning microscopy (CLSM) with excitation 442 nm, or two-photon laser scanning microscopy (TPLSM) with excitation 770 nm. A value of 42 +/- 3% cytoplasm was determined by manual segmentation, and a value of 37 +/- 2% using stereological techniques. Using these figures, values for cytoplasmic [GSH] were estimated to be between 2.7 +/- 0.3 and 3.2 +/- 0.3 mM for cell populations. In addition, measurement of GSH levels in individual cells using CLSM and TPLSM gave values of 3.0 +/- 0.5 and 3.5 +/- 0.7 mM, respectively.

Activation of the transcription factor nuclear factor (NF)-kappaB by proinflammatory stimuli leads to increased expression of genes involved in inflammation. Activation of NF-kappaB requires the activity of an inhibitor of kappaB (IkappaB)-kinase (IKK) complex containing two kinases (IKKalpha and IKKbeta) and the regulatory protein NEMO (NF-kappaB essential modifier). An amino-terminal alpha-helical region of NEMO associated with a carboxyl-terminal segment of IKKalpha and IKKbeta that we term the NEMO-binding domain (NBD). A cell-permeable NBD peptide blocked association of NEMO with the IKK complex and inhibited cytokine-induced NF-kappaB activation and NF-kappaB-dependent gene expression. The peptide also ameliorated inflammatory responses in two experimental mouse models of acute inflammation. The NBD provides a target for the development of drugs that would block proinflammatory activation of the IKK complex without inhibiting basal NF-kappaB activity.

Activation of cell division in the root apical meristem after germination is essential for postembryonic root development. Arabidopsis plants homozygous for a mutation in the ROOT MERISTEMLESS1 (RML1) gene are unable to establish an active postembryonic meristem in the root apex. This mutation abolishes cell division in the root but not in the shoot. We report the molecular cloning of the RML1 gene, which encodes the first enzyme of glutathione (GSH) biosynthesis, gamma-glutamylcysteine synthetase, and which is allelic to CADMIUM SENSITIVE2. The phenotype of the rml1 mutant, which was also evident in the roots of wild-type Arabidopsis and tobacco treated with an inhibitor of GSH biosynthesis, could be relieved by applying GSH to rml1 seedlings. By using a synchronized tobacco cell suspension culture, we showed that the G(1)-to-S phase transition requires an adequate level of GSH. These observations suggest the existence of a GSH-dependent developmental pathway essential for initiation and maintenance of cell division during postembryonic root development.

This paper reports that the glutathione (GSH)-deficient mutant, cad2-1, of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, gamma-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, gamma-glutamylcysteine. In vitro enzyme assays showed that the cad2-1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA, identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2-1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2-1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2-1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.

Glutathione (GSH) is a key component of plant antioxidant defenses. We have sought to determine how the rate-limiting step in GSH biosynthesis, catalyzed by gamma-glutamylcysteine synthase (gammaECS) is regulated in Arabidopsis. Functional complementation of a yeast mutant deficient in this enzyme with an Arabidopsis expression library yielded two cDNAs with sequence identical to the previously described AtgammaECS. Nevertheless, the cellular concentration of GSH in these transformants was only 10% of wild-type concentrations and this was not a result of Cys availability. To explore the possibility that Arabidopsis gammaECS requires additional factors for full catalytic activity, we analyzed the GSH levels and the enzyme activities and transcript levels of both enzymes of the GSH biosynthetic pathway in Arabidopsis suspension cultures subjected to a variety of stresses that raise GSH levels. Our results demonstrate rapid posttranscriptional activation of Arabidopsis gammaECS. The implications of these findings for the mechanisms by which GSH concentrations are regulated during plant-stress responses are discussed.

We provide evidence that the tripeptide thiol glutathione (GSH) participates in the regulation of cell division in the apical meristem of Arabidopsis roots. Exogenous application of micromolar concentrations of GSH raised the number of meristematic cells undergoing mitosis, while depletion of GSH had the opposite effect. A role for endogenous GSH in the control of cell proliferation is also provided by mapping of GSH levels in the root meristem using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. High levels of GSH were associated with the epidermal and cortical initials and markedly lower levels in the quiescent center. The mechanisms controlling cell division could also be triggered by other reducing agents: ascorbic acid and dithiothreitol. Our data also reveal significant plasticity in the relationship between the trichoblast cell length and the hair it subtends in response to alterations in intracellular redox homeostasis. While mechanisms that control trichoblast elongation are influenced by nonspecific redox couples, root hair tip growth has a more specific requirement for sulfhydryl groups. The responses we describe here may represent the extremes of redox control of root plasticity and would allow the root to maintain exploration of the soil under adverse conditions with minimal cell divisions and root hair production or capitalize on a favorable environment by production of numerous long hairs. Redox sensing of the environment and subsequent redox-dependent modulation of growth and development may be crucial components in the strategies plants have evolved for survival in a fluctuating environment.

1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNF alpha was dose-dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E-selectin and VCAM-1 but after 24 h was only inhibited by anti-VCAM-1. 6. Sodium orthovanadate had no effect on TNF alpha-induced U937 adhesion but dose-dependently enhanced adhesion to unstimulated HUVEC. Vanadate-induced adhesion was inhibited by an antibody against VCAM-1. 7. These results demonstrate that PTK-mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine-induced adhesion molecule expression.

The rapid synthesis and release of prostacyclin (PGI2) and the exocytotic secretion of von Willebrand Factor (vWF) elicited by activation of G-protein-coupled receptors on endothelium occur via signaling mechanisms which are incompletely defined. Activation of protein tyrosine kinases (PTKs) and modulation of the tyrosine-phosphorylation state of endogenous proteins have been implicated in several cellular processes including arachidonate release and exocytosis. In the present study we have examined the regulatory role of PTKs in agonist-stimulated release of PGI2 and vWF from human umbilical vein endothelial cells (HUVECs) using two chemically and mechanistically dissimilar PTK inhibitors (genistein and ST271). Genistein, but not the less active analogue daidzein, dose-dependently attenuated PGI2 release in response to thrombin and histamine (IC50 approx. 20 microM), and to the thrombin-receptor-activating peptide. A more potent inhibition of thrombin- and histamine-induced PGI2 synthesis was observed in cells exposed to ST271. In contrast, neither genistein nor ST271 modulated agonist-drive vWF secretion. At concentrations that abolished PGI2 release, genistein blocked thrombin- or histamine-evoked tyrosine phosphorylation of a 42 kDa protein. Ca2+ ionophore-induced PGI2 generation, but not vWF secretion, was also inhibited by both genistein and ST271, suggesting that these agents modulate PGI2 synthesis by acting at, or distal to, agonist-induced changes in intracellular CA2+ ([Ca2+]i). In fura-2-loaded HUVECs genistein partially reduced the histamine-induced peak [Ca2+]i but had no effect on the thrombin response. Ca(2+)-induced PGI2 release from electrically permeabilized HUVECs was abolished in the presence of ST271 or genistein, but not diadzein. The generation of PGI2 in response to exogenous arachidonic acid was not modulated by genistein or ST271, suggesting that PTK inhibitors do not directly inhibit cyclo-oxygenase activity. Taken together, these results suggest that PTKs regulate PGI2 synthesis and release in HUVECs by modulating, directly or indirectly, a CA(2+)-sensitive step upstream of cyclo-oxygenase.

An Arabidopsis thaliana cDNA (AtGSHS) encoding a protein with high primary sequence identity to cDNAs previously isolated from Xenopus laevis (42%), Schizosaccharomyces pombe (40%), Rattus norvegicus (40%) and Homo sapiens (37%) encoding glutathione synthetase (EC 6.3.2.3) has been isolated by functional complementation of an Escherichia coli mutant deficient in this enzyme. AtGSHS is encoded by a single gene, GSHB, as determined by Southern blot analysis and the corresponding mRNA is abundant in both roots and leaves of Arabidopsis.

Developments in finite-difference time-domain (FD-TD) computational modeling of Maxwell's equations, super-computer technology, and computed tomography (CT) imagery open the possibility of accurate numerical simulation of electromagnetic (EM) wave interactions with specific, complex, biological tissue structures. One application of this technology is in the area of treatment planning for EM hyperthermia. In this paper, we report the first highly automated CT image segmentation and interpolation scheme applied to model patient-specific EM hyperthermia. This novel system is based on sophisticated tools from the artificial intelligence, computer vision, and computer graphics disciplines. It permits CT-based patient-specific hyperthermia models to be constructed without tedious manual contouring on digitizing pads or CRT screens. The system permits in principle near real-time assistance in hyperthermia treatment planning. We apply this system to interpret actual patient CT data, reconstructing a 3-D model of the human thigh from a collection of 29 serial CT images at 10 mm intervals. Then, using FD-TD, we obtain 2-D and 3-D models of EM hyperthermia of this thigh due to a waveguide applicator. We find that different results are obtained from the 2-D and 3-D models, and conclude that full 3-D tissue models are required for future clinical usage.