Brief Report: Anti-Eukaryotic Initiation Factor 2B Autoantibodies Are Associated With Interstitial Lung Disease in Patients With Systemic Sclerosis.

OBJECTIVE: To investigate novel systemic sclerosis (SSc) autoantibodies in autoantibody-negative patients and establish clinical associations. METHODS: Serum samples and clinical data for 548 patients with SSc were collected. Routine serologic techniques were used to test the serum samples for known SSc autoantibodies, and samples with negative results were further investigated by radiolabeled-protein immunoprecipitation assay. Sera that immunoprecipitated a novel 30-kd band were analyzed by indirect immunofluorescence and immunoprecipitation, using depleted cell extracts to establish a common reactivity. Mass spectrometry was performed to identify the novel autoantigen, and the results were confirmed using commercial antibodies. Sera from 426 patients with other forms of connective tissue disease, 103 with rheumatoid arthritis, 114 with idiopathic interstitial lung disease (ILD), and 150 healthy subjects were serotyped as controls. RESULTS: A novel autoantigen with a molecular weight of ∼30 kd was recognized by 7 sera from patients with SSc, 6 of whom had ILD, and by no controls. Six of the patients had diffuse cutaneous involvement, and 4 had overlap features with other autoimmune diseases. Immunodepletion experiments indicated that all samples targeted the same autoantigen, and mass spectrometry identified the novel autoantigen as eukaryotic initiation factor 2B (eIF2B). CONCLUSION: We report the identification of a novel autoantibody (anti-eIF2B) in a small number of patients with SSc (∼1%); this autoantibody is closely associated with diffuse cutaneous manifestations and the presence of ILD.

The effects of flooding on dioxin and PCB levels in food produced on industrial river catchments.

This research examined the effect of regular flooding upon PCDD/F and PCB levels in milk, beef and lamb, produced on the floodplains of industrial river catchments. Our unique dataset included more than 200 samples analysed for PCDD/Fs and PCBs over two data collection phases (1998-1999 & 2008-2010) from working farms. A robust paired study design was adopted with samples taken from flood-prone farms and nearby control farms not subject to flooding. On industrial river catchments regular flooding is associated with higher PCDD/F and PCB levels in soils and grass. This contamination may be transferred to food but the impact varied by food type. These contrasts may be due to physiological differences between animals, the ages at which they are sent to market and differences in animal husbandry. To minimise the risks of producing food on flood-prone land in industrial river catchments, as well as on any land with elevated PCDD/F and PCB levels, this research suggests a number of options. The choice of livestock may be important and as an example in our study beef cattle accumulated PCDD/Fs to a higher degree than sheep. Land management may also play a role and could include minimising the time that livestock spend on such land or feeding commercial feed, low in PCDD/Fs and PCBs, where appropriate.

The effects of river flooding on dioxin and PCBs in beef.

In 2008-2010, samples of meat from 40 beef cattle, along with grass, soil and commercial feed, taken from ten matched pairs of flood-prone and control farms, were analysed for PCDD/Fs and PCBs. Concentrations were higher in soil and grass from flood-prone farms. The beef samples from flood-prone farms had total TEQ levels about 20% higher than on control farms. A majority of flood-prone farms (7/10) had higher median levels in beef than on the corresponding control farm. This first controlled investigation into PCDD/F and PCB contamination in beef produced on flood-prone land, presents robust evidence that flooding is a contaminant transfer mechanism to cattle raised on river catchments with a history of urbanisation and industrialisation. PCDD/F and PCB sources in these river systems are likely to be a result of the legacy of contamination from previous industrialisation, as well as more recent combustion activity or pollution events.

Seasonal variations in the levels of PCDD/Fs, PCBs and PBDEs in cows' milk.

The first detailed investigation into seasonal variations in PCDD/F, PCB and PBDE concentrations in cows' milk from individual farms was conducted. From August 2009 milk samples were taken at 6 weeks intervals from the bulk tank of 2 farms over a period of one year. Samples of dietary inputs including commercial feed, grass, silage and soil were also collected at 6 weekly intervals from each farm. Detailed animal husbandry information was additionally obtained. For all these samples total TEQ, ∑ICES6 and the ∑7 PBDE congeners was calculated. The results demonstrated that the concentrations of these sets of compounds fluctuate notably over short periods in cows' milk. This variability was such that the highest observed concentrations were nearly double the lowest concentrations detected for both PCDD/Fs and PCBs and PBDEs. Fluctuations between extremes were observed over time periods as short as 6 weeks. Some, but not all, of these variations may be explained by changes in the contaminant concentrations of dietary inputs consumed by the cattle. Changes in contaminant inputs from grass and silage were identified as being the most important source of these fluctuations. Given this variability, the results from PCDD/F and PCB and PBDE monitoring studies may be highly dependent upon when the individual samples were taken. The results have important implications for total diet studies and sampling design.

Effects of river flooding on polybrominated diphenyl ether (PBDE) levels in cows' milk, soil, and grass.

The first investigation into PBDE levels in food produced from flood-prone land on industrial river catchments was conducted. In August 2008 samples of cows' milk, along with grass and soil were taken from 5 pairs of flood-prone and control farms on the River Trent (Central UK). The sum of 7 BDE congeners (28, 47, 99, 100, 153, 154, and 183) was calculated. Higher PBDE levels occurred in soil on flood-prone compared to control farms (median 770 vs 280 ng/kg dry weight). These higher levels were not reflected in the grass samples indicating that PBDE contamination on soils is not transferred efficiently to grass. This observation alongside the fact that cows on flood-prone farms spend time on non-flood-prone land and are fed substantial quantities of commercial feed are reasons why higher PBDE levels were not found in milk from flood-prone farms (median 300 vs 250 ng/kg fat weight). Similar BDE47/BDE99 ratios were observed in soil and grass samples compared to the PBDE product commonly used in the UK, indicating few differences in source-pathway transfer efficiencies between congeners. The BDE47/BDE99 ratio in the milk samples was greater than those in the grass and feed indicating differential food to milk transfer efficiencies between congeners.

Proteomic identification of glyceraldehyde 3-phosphate dehydrogenase as an inhibitory target of hydrogen peroxide in Arabidopsis.

Hydrogen peroxide (H2O2) is now recognised as a key signalling molecule in eukaryotes. In plants, H2O2 is involved in regulating stomatal closure, gravitropic responses, gene expression and programmed cell death. Although several kinases, such as oxidative signal-inducible 1 (OXI1) kinase and mitogen-activated protein kinases are known to be activated by exogenous H2O2, little is known about the proteins that directly react with H2O2. Here, we utilised a proteomic approach, using iodoacetamide-based fluorescence tagging of proteins in conjunction with mass spectrometric analysis, to identify several proteins that might be potential targets of H2O2 in the cytosolic fraction of Arabidopsis thaliana, the most prominent of which was cytosolic glyceraldehyde 3-phosphate dehydrogenase (cGAPDH; EC 1.2.1.12). cGAPDH from Arabidopsis is inactivated by H2O2 in vitro, and this inhibition is reversible by the subsequent addition of reductants such as reduced glutathione (GSH). It has been suggested recently that Arabidopsis GAPDH has roles outside of its catalysis as part of glycolysis, while in other systems this includes that of mediating reactive oxygen species (ROS) signalling. Here, we suggest that cGAPDH in Arabidopsis might also have such a role in mediating ROS signalling in plants.

Quantitative analysis of indigo and indigo precursors in leaves of Isatis spp. and Polygonum tinctorium.

Analysis of extracts from two woad species (Isatis tinctoria and Isatis indigotica) and Polygonum tinctorium revealed that only one indigo precursor (indican) was present in Polygonum, but two precursors were found in Isatis spp. This was done using high performance liquid chromatography (HPLC), coupled to an evaporative light scattering detector (ELSD). In Isatis spp., the indigo precursors indican and a fraction representing isatan B were identified. The proportion of indican and isatan B was different between the two Isatis spp. tested. For the first time, it was possible to quantify the precursors in woad plant species, and the results were found to be in good agreement with those made from total indigo quantification using two different spectrophotometric methods or a derivatization technique.

(+)-(10R)-Germacrene A synthase from goldenrod, Solidago canadensis; cDNA isolation, bacterial expression and functional analysis.

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.

Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.).

Sesquiterpenoid lactones (SLs) from lettuce (Lactuca sativa L.) include constitutive components of latex such as lactucin and the induced phytoalexin, lettucenin A. A redundant primer strategy was used to recover two full length cDNA clones (LTC1 and LTC2) encoding sesquiterpene synthases from a cDNA library derived from seedlings with the red spot disorder, which accumulate phytoalexins. Recombinant enzymes produced from LTC1 and LTC2 in Escherichia coli catalysed the cyclisation of farnesyl diphosphate to germacrene A, potentially an early step in the biosynthesis of SLs. RT-PCR analysis showed LTC1 and LTC2 were expressed constitutively in roots, hypocotyls and true leaves but not in cotyledons. Expression in cotyledons was induced by challenge with the downy mildew pathogen Bremia lactucae in the disease resistant cultivar Diana. Southern hybridisation experiments showed that LTC1 and LTC2 were not part of a multigene family. The germacrene A synthases provide targets for modified expression to generate beneficial modifications to the SL profile in lettuce.

Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties.

Multiple molecular forms of inhibitors of trypsin (TI) and chymotrypsin (CI), which are typical digestive enzymes of insects, mammals and micro-organisms, and subtilisin (SI), a proteinase of many bacteria and phytopathogenic fungi, were identified in seeds and vegetative organs of the majority of 128 wild and cultivated species representing 65 genera of three of the subfamilies of the Compositae. Inhibitors with M(r) ranging from 7450 to 7800 and combining activities towards subtilisin and trypsin and/or chymotrypsin (T/C/SI) had the widest distribution and may be involved in plant defense mechanisms. They were found in many species of the subfamilies Carduoideae (genera Carthamus, Centaurea, Cirsium), Cichorioideae (Lactuca, Taraxacum) and Asteroideae (Helianthus, Cosmos, Bidens). Partial amino acid sequencing showed that the safflower (Carthamus tinctorius) T/C/SI and Cosmos bipinnatus T/C/SI, T/SI and C/SI belonged to the potato I inhibitor family. The most active, variable and heterogeneous inhibitors were found in species of the tribe Heliantheae, which is placed in the evolutionary advanced subfamily Asteroideae. Seeds of Helianthus species, Eclipta prostrata, Gailardia aristata, Zinnia elegans and Silphium perfoliatum contained various TI with M(r) ranging from 1500 to 14,750, with some also containing SI. H. annuus seeds contain a unique cyclic TI of M(r) 1514 and similar TI were also present in other Helianthus spp. and the related species Tithonia diversifolia. Zinnia elegans contained a TI with M(r) 11,350 which appeared to represent a novel type of inhibitor distantly related to the cereal subgroup of Bowman-Birk inhibitors. TI and T/SI varied widely in H. annuus lines and wild Helianthus species in their presence or absence and composition. Similar T/SI components were found in the cultivated diploid H. annuus and annual diploid species with the B genome but not in perennials with the A genome. Some T/SI, SI and TI were detected in vegetative organs of sunflower and other Compositae. Studies of the polymorphism and distribution of proteinase inhibitors are relevant to the evolution of protective protein systems and the mechanisms of resistance to pathogenic organisms in the Compositae and other plants.

Fungicides and sterol-deficient mutants of Ustilago maydis: plasma membrane physico-chemical characteristics do not explain growth inhibition.

Plasma membrane vesicles from erg11 and erg2 sterol-deficient mutants and from wild-type Ustilago maydis sporidia treated with and without inhibitors of sterol 14α-demethylase or sterol ∆8-∆7 isomerase (triadimenol and fenpropimorph fungicides, respectively) were purified by aqueous two-phase partitioning. Changes in plasma membrane lipid composition were mostly restricted to sterols and complex lipid-bound fatty acids (CLB fatty acids). There was a greater accumulation of abnormal sterols (14α-methyl-or ∆ 8-unsaturated sterols) in plasma membranes from sterol-deficient mutants than from those treated with their fungicide counterparts. However, greater growth inhibition was observed on fungicide-treated wild-type than on mutants. Changes in CLB fatty acids were restricted to alterations in the relative proportion of linoleic acid (18:2) with respect to oleic acid (18:1). The 18:2 to 18:1 ratio found in CLB fatty acids in plasma membranes could be correlated to rates of sporidial growth but not to accumulation of a particular abnormal sterol or to the extent of sterol replacement. Plasma membrane permeability to protons was increased moderately in the mutants only. No changes were observed in plasma membrane fluidity. Plasma membrane H+-ATPase activity was increased up to twofold in those cases with lower growth rate. It was concluded that fungicide-induced growth inhibition in U. maydis was not due to accumulation of abnormal sterols in plasma membranes but probably due to intracellular ATP depletion by the H+-ATPase and that changes in 18:2 to 18:1 ratio in CLB fatty acids were not directly dependent on the plasma membrane physical state or lipid composition but were possibly part of a stress adaptation mechanism.