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The Opacity Platform on the National Ignition Facility (NIF) has been developed to measure iron opacities at varying densities and temperatures relevant to the solar interior and to verify recent experimental results obtained at the Sandia Z-machine, that diverge from theory. The first set of NIF experiments collected iron opacity data at ∼150 eV to 160 eV and an electron density of ∼7 × 10 21  cm -3 , with a goal to study temperatures up to ∼210 eV, with electron densities of up to ∼3 × 10 22  cm -3 . Among several techniques used to infer the temperature of the heated Fe sample, the absolutely calibrated DANTE-2 filtered diode array routinely provides measurements of the hohlraum conditions near the sample. However, the DANTE-2 temperatures are consistently low compared to pre-shot LASNEX simulations for a range of laser drive energies. We have re-evaluated the estimated uncertainty in the reported DANTE-2 temperatures and also the error generated by varying channel participation in the data analysis. An uncertainty of ±5% or better can be achieved with appropriate spectral coverage, channel participation, and metrology of the viewing slot.

Sentinel is a 16-channel, filtered x-ray diode array spectrometer that has been developed to measure ∼1 keV-20 keV x-ray emission generated by the National Ignition Facility (NIF) laser. Unlike the large, fixed-port versions of this diagnostic that currently exist on the NIF (known as Dante), Sentinel is a Diagnostic Instrument Manipulator compatible such that it can be fielded along the polar or equatorial lines-of-sight-an essential new capability for characterizing the often anisotropic x-ray emission from laser-driven sources. We present the diagnostic design along with preliminary diode calibrations and performance results. The novel, small-form-factor x-ray diode design allows for ≳5×-25× increased channel areal density over that of Dante, simultaneously enabling improved diagnostic robustness and fidelity of spectral reconstructions. While the Sentinel diagnostic is anticipated to improve line-of-sight spectral characterization of x-ray sources for a wide variety of programs on the NIF, the compact and portable design is also attractive to small- and mid-scale facilities with limited diagnostic real estate.

Filtered diode array spectrometers are routinely employed to infer the temporal evolution of spectral power from x-ray sources, but uniquely extracting spectral content from a finite set of broad, spectrally overlapping channel spectral sensitivities is decidedly nontrivial in these under-determined systems. We present the use of genetic algorithms to reconstruct a probabilistic spectral intensity distribution and compare to the traditional approach most commonly found in the literature. Unlike many of the previously published models, spectral reconstructions from this approach are neither limited by basis functional forms nor do they require a priori spectral knowledge. While the original intent of such measurements was to diagnose the temporal evolution of spectral power from quasi-blackbody radiation sources-where the exact details of spectral content were not thought to be crucial-we demonstrate that this new technique can greatly enhance the utility of the diagnostic by providing more physical spectra and improved robustness to hardware configuration for even strongly non-Planckian distributions.

The one-dimensional imager of neutrons (ODIN) at the Sandia Z facility consists of a 10-cm block of tungsten with rolled edges, creating a slit imager with slit widths of either 250, 500, or 750 μ m. Designed with a 1-m neutron imaging line of sight, we achieve about 4:1 magnification and 500- μ m axial spatial resolution. The baseline inertial confinement fusion concept at Sandia is magnetized liner inertial fusion, which nominally creates a 1-cm line source of neutrons. ODIN was designed to determine the size, shape, and location of the neutron producing region, furthering the understanding of compression quality along the cylindrical axis of magnetized liner implosions. Challenges include discriminating neutrons from hard x-rays and gammas with adequate signal-to-noise in the 2 × 10 12 deuterium-deuterium (DD) neutron yield range, as well as understanding the point spread function of the imager to various imaging detectors (namely, CR-39). Modeling efforts were conducted with MCNP6.1 to determine neutron response functions for varying configurations in a clean DD neutron environment (without x-rays or gammas). Configuration alterations that will be shown include rolled-edge slit orientation and slit width, affecting the resolution and response function. Finally, the experiment to determine CR-39 neutron sensitivity, with and without a high density polyethylene (n, p) converter, an edge spread function, and resolution will be discussed.

Direct drive implosions of plastic capsules have been performed at the National Ignition Facility to provide a broad-spectrum (500-2000 eV) X-ray continuum source for X-ray transmission spectroscopy. The source was developed for the high-temperature plasma opacity experimental platform. Initial experiments using 2.0 mm diameter polyalpha-methyl styrene capsules with ∼20  μ m thickness have been performed. X-ray yields of up to ∼1 kJ/sr have been measured using the Dante multichannel diode array. The backlighter source size was measured to be ∼100  μ m FWHM, with ∼350 ps pulse duration during the peak emission stage. Results are used to simulate transmission spectra for a hypothetical iron opacity sample at 150 eV, enabling the derivation of photometrics requirements for future opacity experiments.

The Dante is an 18 channel filtered diode array used at the National Ignition Facility (NIF) to measure the spectrally and temporally resolved radiation flux between 50 eV and 20 keV from various targets. The absolute flux is determined from the radiometric calibration of the x-ray diodes, filters, and mirrors and a reconstruction algorithm applied to the recorded voltages from each channel. The reconstructed spectra are very low resolution with features consistent with the instrument response and are not necessarily consistent with the spectral emission features from the plasma. Errors may exist between the reconstructed spectra and the actual emission features due to assumptions in the algorithm. Recently, a high resolution convex crystal spectrometer, VIRGIL, has been installed at NIF with the same line of sight as the Dante. Spectra from L-shell Ag and Xe have been recorded by both VIRGIL and Dante. Comparisons of these two spectroscopic measurements yield insights into the accuracy of the Dante reconstructions.

Filtered x-ray diode (XRD) arrays are often used to measure x-ray spectra vs. time from spectrally continuous x-ray sources such as hohlraums. A priori models of the incident x-ray spectrum enable a more accurate unfolding of the x-ray flux as compared to the standard technique of modifying a thermal Planckian with spectral peaks or dips at the response energy of each filtered XRD channel. A model x-ray spectrum consisting of a thermal Planckian, a Gaussian at higher energy, and (in some cases) a high energy background provides an excellent fit to XRD-array measurements of x-ray emission from laser heated hohlraums. If high-resolution measurements of part of the x-ray emission spectrum are available, that information can be included in the a priori model. In cases where the x-ray emission spectrum is not Planckian, candidate x-ray spectra can be allowed or excluded by fitting them to measured XRD voltages. Examples are presented from the filtered XRD arrays, named Dante, at the National Ignition Facility and the Laboratory for Laser Energetics.

A basic tenet of material science is that the flow stress of a metal increases as its grain size decreases, an effect described by the Hall-Petch relation. This relation is used extensively in material design to optimize the hardness, durability, survivability, and ductility of structural metals. This Letter reports experimental results in a new regime of high pressures and strain rates that challenge this basic tenet of mechanical metallurgy. We report measurements of the plastic flow of the model body-centered-cubic metal tantalum made under conditions of high pressure (>100  GPa) and strain rate (∼10(7)  s(-1)) achieved by using the Omega laser. Under these unique plastic deformation ("flow") conditions, the effect of grain size is found to be negligible for grain sizes >0.25  μm sizes. A multiscale model of the plastic flow suggests that pressure and strain rate hardening dominate over the grain-size effects. Theoretical estimates, based on grain compatibility and geometrically necessary dislocations, corroborate this conclusion.

Photons in the MeV range emitted from the dense plasma focus (DPF) at the NSTec North Las Vegas Facility have been measured with both neutron-damaged GaAs and natural diamond photoconductive detectors (PCDs). The DPF creates or "pinches" plasmas of various gases (e.g., H2, D2, Ne, Ar., etc.) that have enough energy to create MeV photons from either bremsstrahlung and/or (n,n(')) reactions if D2 gas is used. The high bandwidth of the PCDs enabled the first ever measurement of the fast micro-pinches present in DPF plasmas. Comparisons between a slower more conventional scintillator/photomultiplier tube based nuclear physics detectors were made to validate the response of the PCDs to fast intense MeV photon signals. Significant discrepancies in the diamond PCD responses were evident.

The NF-κB transcription factor has a central role in diverse processes, including inflammation, proliferation and cell survival, and its activity is dysregulated in diseases such as autoimmunity and cancer. We recently identified the TRE17/ubiquitin-specific protease 6 (USP6) oncogene as the first de-ubiquitinating enzyme to activate NF-κB. TRE17/USP6 is translocated and overexpressed in aneurysmal bone cyst (ABC), a pediatric tumor characterized by extensive bone degradation and inflammatory recruitment. In the current study, we explore the mechanism by which TRE17 induces activation of NF-κB, and find that it activates the classical NF-κB pathway through an atypical mechanism that does not involve IκB degradation. TRE17 co-precipitates with IκB kinase (IKK), and IKK activity is augmented in stable cell lines overexpressing TRE17, in a USP-dependent manner. Optimal activation of NF-κB by TRE17 requires both catalytic subunits of IKK, distinguishing its mechanism from the classical and non-canonical pathways, which require either IKKβ or IKKα, respectively. TRE17 stimulates phosphorylation of p65 at serine 536, a modification that has been associated with enhanced transcriptional activity and nuclear retention. Induction of S536 phosphorylation by TRE17 requires both IKKα and IKKβ, as well as the IKKγ/NEMO regulatory subunit of IKK. We further demonstrate that TRE17(long) is highly tumorigenic when overexpressed in NIH3T3 fibroblasts, and that inhibition of NF-κB significantly attenuates tumor formation. In summary, these studies uncover an unexpected signaling mechanism for activation of classical NF-κB by TRE17. They further reveal a critical role for NF-κB in TRE17-mediated tumorigenesis, and suggest that NF-κB inhibitors may function as effective therapeutic agents in the treatment of ABC.

The oncogenic fusion protein RET/PTC3 (RP3) that is expressed in papillary thyroid carcinoma (PTC) and thyroid epithelia in Hashimoto's thyroiditis activates nuclear factor-kappa B (NF-κB) and induces pro-inflammatory gene expression; however, the mechanism of this activation is unknown. To address this, we expressed RP3 in murine embryonic fibroblasts (MEFs) lacking key classical and noncanonical NF-κB signaling components. In wild-type MEFs, RP3 upregulated CCL2, CXCL1, granulocyte-macrophage colony-stimulating factor and tumor necrosis factor expression and activated classical but not noncanonical NF-κB. RP3-activated NF-κB in IκB kinase (IKK)β(-/-) MEFs but not IKKα- or NF-κB essential modulator (NEMO)-deficient cells and activation was inhibited by a peptide that blocks NEMO binding to the IKKs. RP3 increased the levels of NF-κB-inducing kinase (NIK) and did not activate NF-κB in NIK-deficient MEFs. Notably, NIK stabilization was not accompanied by TRAF3 degradation demonstrating that RP3 disrupts normal basal NIK regulation. Dominant-negative NIK blocked RP3-induced NF-κB activation and an RP3 signaling mutant (RP3(Y588F)) did not stabilize NIK. Finally, examination of PTC specimens revealed strong positive staining for NIK. We therefore conclude that RP3 activates classical NF-κB via NIK, NEMO and IKKα. Importantly, our findings reveal a novel mechanism for oncogene-induced NF-κB activation via stabilization of NIK.

The nuclear factor kappa B (NF-kappaB) transcription factors are activated by a range of stimuli including pro-inflammatory cytokines. Active NF-kappaB regulates the expression of genes involved in inflammation and cell survival and aberrant NF-kappaB activity plays pathological roles in certain types of cancer and diseases characterized by chronic inflammation. NF-kappaB signaling is an attractive target for the development of novel anti-inflammatory or anti-cancer drugs and we discuss here how the method of peptide transduction has been used to specifically target NF-kappaB. Peptide transduction relies on the ability of certain small cell-penetrating peptides (CPPs) to enter cells, and a panel of CPP-linked inhibitors (CPP-Is) has been developed to directly inhibit NF-kappaB signaling. Remarkably, several of these NF-kappaB-targeting CPP-Is are effective in vivo and therefore offer exciting potential in the clinical setting.

Currently, the UK has no procedure for the approval of novel agricultural practices that is based on environmental risk management principles. Here, we make a first application of the 'bow-tie' risk management approach in agriculture, for assessment of land use changes, in a case study of the introduction of genetically modified herbicide tolerant (GMHT) sugar beet. There are agronomic and economic benefits, but indirect environmental harm from increased weed control is a hazard. The Farm Scale Evaluation (FSE) trials demonstrated reduced broad-leaved weed biomass and seed production at the field scale. The simplest mitigation measure is to leave a proportion of rows unsprayed in each GMHT crop field. Our calculations, based on FSE data, show that a maximum of 2% of field area left unsprayed is required to mitigate weed seed production and 4% to mitigate weed biomass production. Tilled margin effects could simply be mitigated by increasing the margin width from 0.5 to 1.5 m. Such changes are cheap and simple to implement in farming practices. This case study demonstrates the usefulness of the bow-tie risk management approach and the transparency with which hazards can be addressed. If adopted generally, it would help to enable agriculture to adopt new practices with due environmental precaution.

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.

The UK Farm-Scale Evaluations (FSE) compared the effects on biodiversity of management of genetically modified herbicide-tolerant (GMHT) spring-sown crops with conventional crop management. The FSE reported larger weed abundance under GMHT management for fodder maize, one of three crops studied. Increased seed production may be important for the long-term persistence of these arable weeds and may benefit invertebrates, small mammals and seed-eating birds. In three-quarters of FSE maize fields, growers used atrazine on the conventionally managed half, reflecting contemporary commercial practice. Withdrawal of the triazine herbicides atrazine, simazine and cyanazine from approved lists of EU chemicals could therefore reduce or even reverse the reported benefits of GMHT maize. Here we analyse effects of applications of triazine herbicides in conventional maize regimes on key indicators, using FSE data. Weed abundances were decreased greatly relative to all other regimes whenever atrazine was applied before weeds emerged. Here, we forecast weed abundances in post-triazine herbicide regimes. We predict weed abundances under future conventional herbicide management to be considerably larger than that for atrazine used before weeds emerged, but still smaller than for the four FSE sites analysed that used only non-triazine herbicides. Our overall conclusion is that the comparative benefits for arable biodiversity of GMHT maize cropping would be reduced, but not eliminated, by the withdrawal of triazines from conventional maize cropping.

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.

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55-63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes- safeners- in monocotyledonous species) revealed that the members of this gene family are differentially regulated.

Work from this and other laboratories has identified a role for protein tyrosine kinases in interleukin-1 alpha (IL-1 alpha)- and tumor necrosis factor-alpha (TNF-alpha)-induced responses in endothelial cells. In this study, we show that activation of human umbilical vein endothelial cells (HUVEC) by IL-1 alpha leads to increased tyrosine phosphorylation of several proteins including one with a molecular mass of approximately 42 kDa. This protein was identified as p42mapk by Western blot analysis. Tyrosine phosphorylation and catalytic activation of p42mapk by IL-1 alpha was transient, reaching maximal levels after 30 min and returning to basal levels by 120-300 min. Activation of p42mapk in HUVEC was also observed in response to TNF-alpha or to the protein kinase C (PKC)-activating phorbol ester phorbol 12-myristate 13-acetate (PMA). Pretreatment of HUVEC with IL-1 alpha or TNF-alpha prevented reactivation of p42mapk by either cytokine but did not affect subsequent activation in response to PMA. Activation of p42mapk by PMA was significantly reduced by the PKC inhibitor Ro-31-8220 and completely inhibited by the protein tyrosine kinase inhibitor genistein. Genistein, but not Ro-31-8220, attenuated IL-1 alpha- and TNF-alpha-induced p42mapk activation. Taken together, the results of this study demonstrate 1) that p42mapk is transiently activated in HUVEC by IL-1 alpha and TNF-alpha, 2) that this activation is PKC independent, and 3) that a genistein-inhibitable tyrosine kinase may be an upstream regulator of cytokine-induced p42mapk activation in human endothelium.

The transcription factor NF-kappa B, more than a decade after its discovery, remains an exciting and active area of study. The involvement of NF-kappa B in the expression of numerous cytokines and adhesion molecules has supported its role as an evolutionarily conserved coordinating element in the organism's response to situations of infection, stress, and injury. Recently, significant advances have been made in elucidating the details of the pathways through which signals are transmitted to the NF-kappa B:I kappa B complex in the cytosol. The field now awaits the discovery and characterization of the kinase responsible for the inducible phosphorylation of I kappa B proteins. Another exciting development has been the demonstration that in certain situations NF-kappa B acts as an anti-apoptotic protein; therefore, elucidation of the mechanism by which NF-kappa B protects against cell death is an important goal. Finally, the generation of knockouts of members of the NF-kappa B/I kappa B family has allowed the study of the roles of these proteins in normal development and physiology. In this review, we discuss some of these recent findings and their implications for the study of NF-kappa B.

Rel/NF-kappa B proteins constitute a family of structurally-related transcription factors that are regulated by interaction with a family of regulatory proteins, the I kappa B proteins. In this review, we will familiarize the reader with the known Rel/NF-kappa B and I kappa B molecules, and will discuss the types of interactions that occur between these proteins to regulate their function. In addition, we will describe what is known about the structure of Rel/NF-kappa B transcription complexes on DNA. Lastly, we will discuss the wide variety of biological stimuli and conditions which activate NF-kappa B and the genes whose expression are regulated by this family of transcription factors.

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.

The adhesion of lymphocytes to cytokine-treated high endothelium was studied using cultured high endothelial cells (HEC). Pretreatment of the HEC layer with a variety of cytokines caused up-regulation of lymphocyte adhesion with the effects ordered interferon gamma (IFN-gamma) greater than tumor necrosis factor-alpha (TNF-alpha) greater than or equal to interleukin 1 beta (IL 1 beta). Increased lymphocyte adhesion was found to be independent of ICAM-1 as expression by HEC was not increased by cytokines and antibodies against ICAM-1 did not block adhesion. The peptide CS1 and anti-beta 1 integrin subunit antibodies, however, caused partial inhibition of lymphocyte adhesion thus indicating a role for fibronectin on HEC and alpha 4 beta 1 on lymphocytes. Study of the kinetics of lymphocyte adhesion showed that the effects of IFN-gamma and TNF-alpha were persistent and remained detectable 2.5 h after removal of the cytokines whereas the effects of IL 1 beta were transient and were not sustained beyond 1 h. All of the cytokines used caused transient increases in the number of surface-bound lymphocytes with IFN-gamma greater than TNF-alpha greater than or equal to IL 1 beta, however, the most dramatic effect was on the transmigration of lymphocytes across the HEC. Both IFN-gamma and TNF-alpha caused sustained increased transmigration with IFN-gamma having the greater effect. IL 1 beta had little effect on transmigration. This model demonstrates that the binding and transmigration of lymphocytes across HEC can be differentially regulated by the actions of individual cytokines. These results support the concept that locally produced cytokines regulate HEC function within the lymph node.