A Polyketide Synthase Gene Required for Biosynthesis of the Aflatoxin-like Toxin, Dothistromin

Dothistromin is a polyketide toxin, produced by a fungal forest pathogen, with structural similarity to the aflatoxin precursor versicolorin B. Biochemical and genetic studies suggested that there are common steps in the biosynthetic pathways for these metabolites and showed similarities between some of the genes. A polyketide synthase gene (pksA) was isolated from dothistromin-producing Dothistroma septosporum by hybridization with an aflatoxin ortholog from Aspergillus parasiticus. Inactivation of this gene in D. septosporum resulted in mutants that could not produce dothistromin but that could convert exogenous aflatoxin precursors, including norsolorinic acid, into dothistromin. The mutants also had reduced asexual sporulation compared to the wild type. So far four other genes are known to be clustered immediately alongside pksA. Three of these (cypA, moxA, avfA) are predicted to be orthologs of aflatoxin biosynthetic genes. The other gene (epoA), located between avfA and moxA, is predicted to encode an epoxide hydrolase, for which there is no homolog in either the aflatoxin or sterigmatocystin gene clusters. The pksA gene is located on a small chromosome of ~1.3 Mb in size, along with the dothistromin ketoreductase (dotA) gene.     

Prion protein interacts with BACE1 protein and differentially regulates its activity toward wild type and Swedish mutant amyloid precursor protein

In Alzheimer disease amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP) accumulate in the brain. Cleavage of APP by the β-secretase BACE1 is the rate-limiting step in the production of Aβ. We have reported previously that the cellular prion protein (PrP(C)) inhibited the action of BACE1 toward human wild type APP (APP(WT)) in cellular models and that the levels of endogenous murine Aβ were significantly increased in PrP(C)-null mouse brain. Here we investigated the molecular and cellular mechanisms underlying this observation. PrP(C) interacted directly with the prodomain of the immature Golgi-localized form of BACE1. This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves APP(WT) but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APP(Swe)). In transgenic mice expressing human APP with the Swedish and Indiana familial mutations (APP(Swe,Ind)), PrP(C) deletion had no influence on APP proteolytic processing, Aβ plaque deposition, or levels of soluble Aβ or Aβ oligomers. In cells, although PrP(C) inhibited the action of BACE1 on APP(WT), it did not inhibit BACE1 activity toward APP(Swe). The differential subcellular location of the BACE1 cleavage of APP(Swe) relative to APP(WT) provides an explanation for the failure of PrP(C) deletion to affect Aβ accumulation in APP(Swe,Ind) mice. Thus, although PrP(C) exerts no control on cleavage of APP(Swe) by BACE1, it has a profound influence on the cleavage of APP(WT), suggesting that PrP(C) may be a key protective player against sporadic Alzheimer disease.     

Transforming Growth Factor-β, Macrophage Colony-Stimulating Factor and C-Reactive Protein Levels Correlate with CD14highCD16+ Monocyte Induction and Activation in Trauma Patients

Severe injury remains a leading cause of death and morbidity in patients under 40, with the number of annual trauma-related deaths approaching 160,000 in the United States. Patients who survive the initial trauma and post-traumatic resuscitation are at risk for immune dysregulation, which contributes to late mortality and accounts for approximately 20% of deaths after traumatic injury. This post-traumatic immunosuppressed state has been attributed to over-expression of anti-inflammatory mediators in an effort to restore host homeostasis. We measured a panel of monocyte markers and cytokines in 50 severely injured trauma patients for 3 days following admission. We made the novel observation that the subpopulation of monocytes expressing high levels of CD14 and CD16 was expanded in the majority of patients. These cells also expressed CD163 consistent with differentiation into alternatively activated macrophages with potential regulatory or wound-healing activity. We examined factors in trauma plasma that may contribute to the generation and activation of these cells. The percentage of CD14^highCD16⁺ monocytes after trauma correlated strongly with plasma C-reactive protein (CRP) transforming growth factor-β (TGF-β), and macrophage colony-stimulating factor (M-CSF) levels. We demonstrate a role for TGF-β and M-CSF, but not CRP in generating these cells using monocytes from healthy volunteers incubated with plasma from trauma patients. CD16 is a receptor for CRP and IgG, and we showed that monocytes differentiated to the CD14^highCD16⁺ phenotype produced anti-inflammatory cytokines in response to acute phase concentrations of CRP. The role of these cells in immunosuppression following trauma is an area of ongoing investigation.     

Age-specific responses to climate identified in the growth of Quercus alba

The objective of this research was to determine whether the dendroclimatic responses of young Quercus alba (aged 29–126 years) differ from those of old Q. alba (149–312 years). We collected Q. alba increment cores across a range of size classes from Buffalo Mountain Natural Area Preserve, an oak-hickory forest in southcentral Virginia, USA. Tree cores were crossdated and raw ring widths were detrended to remove the influence of increasing circumference with age, microsite, and local stand dynamics. Standardized ring widths were averaged to develop two master chronologies from the 20 oldest and youngest trees. Ring-width indices were correlated with temperature, precipitation, and Palmer Drought Severity Index (PDSI). Annual tree-ring growth in old and young Q. alba was significantly correlated with precipitation from the previous growing season, but was not significantly correlated with temperature. Only the old trees showed a significant correlation between annual ring width and PDSI. These results may indicate that growth in old trees is more sensitive to drought than in young trees. If future climate change includes the predicted increase in mid-growing season droughts, tree-level responses are likely to be age-dependent with older trees experiencing relatively greater reductions in growth.     

Phylogenetics and taxonomy of the New World leafy spurges,Euphorbia section Tithymalus (Euphorbiaceae)

The 480 species of leafy spurges, Euphorbia subgenus Esula, represent the main temperate radiation in the large genus Euphorbia. This group is distributed primarily in temperate Eurasia, but with smaller, disjunct centres of diversity in the mountains of the Old World tropics, in temperate southern Africa and in the New World. The majority of New World diversity (32 species) occurs in a single section, section Tithymalus. We analysed sequences of the nrITS and plastid ndhF, trnH‐psbA, trnS‐trnG and trnD‐trnT regions to reconstruct the phylogeny of section Tithymalus and to examine the origins and diversification of the species native to the New World. Our results indicate that the New World species of section Tithymalus form a clade that is sister to the widespread, weedy E. peplus. The New World species fall into two primary groups: a ‘northern annual clade’ from eastern North America and a diverse clade of both annual and perennial species that is divided into three subgroups. Within the second group, there is a small ‘southern annual clade’ from Texas and northern Mexico, a perennial ‘Brachycera clade’ from the western United States and northern Mexico, and a perennial ‘Esuliformis clade’ from montane areas of Mexico, Guatemala, Honduras and the Caribbean island of Hispaniola. Ancestral state reconstructions indicate that the annual habit probably evolved in the ancestor of E. peplus and the New World clade, with a subsequent reversal to the perennial habit. In conjunction with this phylogenetic framework, the New World species of section Tithymalus are comprehensively reviewed. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 191–228.     

The Number and Morphology of Moss Chromosomes

The time that suitable cytological material is available can be extended by bringing mosses into the laboratory in late winter and keeping them in covered glass dishes placed in strong diffuse light. Best growth of the gametophyte occurs at 68-70° F. and at a relative humidity just high enough to prevent the leaves from curling. Fruiting plants can be maintained in the laboratory in the same way but at a slightly lower relative humidity. Tissue for the study of the gametophytic chromosome complement is obtained from the stem apex and embryonic leaves. Sporophytic mitoses are obtained from the meristematic tip of developing sporophytes. For the study of meiosis, the columella, to which the spore mother cells adhere, is removed from the capsule. The tissues are fixed in acetic alcohol for 1-3 hours and then stained and squashed. Ways in which moss tissue reacts differently to the conventional squash methods are discussed and special directions are given.     

Cellular reactions of O6-methylguanine, a product of some alkylating carcinogens

Cultures of a purine-requiring mutant of Chinese hamster ovary cells (CHO-104b), randomly bred hamster embryo cells, or Escherichia coli B_s−1 were treated with non-toxic doses of ³H-labelled O⁶-methylguanine. DNA and RNA were isolated and subjected to enzymic digestion to nucleosides at pH8. The products of digestion were analysed by ion-exchange chromatography on columns of Dowex 50 (NH₄⁺ form) at pH8.9. No ³H-labelled O⁶-methylguanosine was detected in nucleic acid digests. ³H-labelled O⁶-methylguanine was O-demethylated yielding [³H]guanine in CHO-104b cells. Radioactivity in nucleic acid digests was associated with thymidine, guanosine, deoxyguanosine and an unidentified early-eluting product. Reports of similar unidentified products from nucleic acids labelled with various agents are discussed.     

Conformational changes of yeast plasma membrane H(+)-ATPase during activation by glucose: role of threonine-912 in the carboxy-terminal tail

Yeast Pma1 H(+)-ATPase, which belongs to the P-type family of cation-transporting ATPases, is activated up to 10-fold by growth on glucose, and indirect evidence has linked the activation to Ser/Thr phosphorylation within the C-terminal tail. We have now used limited trypsinolysis to map glucose-induced conformational changes throughout the 100 kDa ATPase. In the wild-type enzyme, trypsin cleaves first at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavages occur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6-M7 cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At the C-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes from glucose-starved cells (GS) but becomes accessible in membranes from glucose-metabolizing cells (GM). In the presence of orthovanadate, Lys-174 at the boundary between M2 and the A domain also becomes open to cleavage in GM but not GS samples (site T5). Significantly, this global conformational change can be suppressed by mutations at Thr-912, a consensus phosphorylation site near the C-terminus. Substitution by Ala at position 912 leads to a GS-like (trypsin-resistant) state, while substitution by Asp leads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecular movements that result in glucose activation.     

Symptoms of nitrogen saturation in an aggrading forested watershed in western Maryland

The objective of this study was to evaluate the nitrogen (N) biogeochemistry of an 18–22 year old forested watershed in western Maryland. We hypothesized that this watershed should not exhibit symptoms of N saturation. This watershed was a strong source of nitrate (NO₃ ⁻) to the stream in all years, with a mean annual export of 9.5 kg N ha⁻¹ year⁻¹ and a range of 4.4–18.4 kg N ha⁻¹ year⁻¹. During the 2001 and 2002 water years, wet deposition of inorganic N was 9.0 kg N ha⁻¹ year⁻¹ and 6.3 kg N ha⁻¹ year⁻¹, respectively. Watershed N export rates in 2001 and 2002 water years were 4.2 kg N ha⁻¹ year⁻¹ and 5.3 kg N ha⁻¹ year⁻¹, respectively. During the wetter water years of 2003 and 2004, the watershed exported 15.0 kg N ha⁻¹ year⁻¹ and 18.4 kg N ha⁻¹ year⁻¹, rates that exceeded annual wet deposition of N by a factor of two (7.5 kg N ha⁻¹ year⁻¹ in 2003) and three (5.5 kg N ha⁻¹ year⁻¹ in 2004). Consistent with the high rates of N export, were high concentrations (2.1–3.3%) of N in foliage, wood (0.3%) and fine roots, low C:N ratios in the forest floor (17–24) and mineral soil (14), high percentages (83–96%) of the amount of mineralized N that was nitrified and elevated N concentrations (up to 3 mg N l⁻¹) in soil solution. Although this watershed contained a young aggrading forest, it exhibited several symptoms of N saturation commonly observed in more mature forests.     

Fucose, placental evolution and the glycocode

A family of 13 fucosyltransferase genes has evolved to catalyze the addition of fucose in various linkage positions to nascent glycoproteins. Null mutations in mice are unearthing unsuspected functions for glycoprotein fucosylation that affect embryo implantation and growth of the conceptus. Furthermore, as we show here, histological studies demonstrate that a variety of fucosylated structures are found within the glycan-rich interface between trophectoderm and uterine epithelium. We suggest that conservation or change in fucosyltransferase gene expression over evolutionary time has played a role in determining the stability of the maternal-fetal interface and therefore in shaping reproductive compatibility and, in turn, speciation.