Effects of Phragmites australis removal on marsh nutrient cycling

In the northeast US removal of exotic and invasive plant species is a common wetland restoration activity and the invasive common reed (Phragmites australis) is often the target of control efforts. We examined effects of reed removal on sediment nutrient pools and denitrification potential in a tidal freshwater marsh on the Connecticut River. In the first year after herbicide application and cutting of a reed stand, porewater ammonium concentrations in the removal area were about 4× higher relative to extant reed or cattail. Denitrification potentials were 50% lower than in a reference stand of reed. Denitrification activity had recovered by the second growing season after reed removal but porewater ammonium continued to accumulate. By the third growing season following reed removal, plant regrowth had occurred over approximately half the experimental plot and porewater ammonium had declined to pre-manipulation levels. Sediment organic content, moisture and porewater phosphate showed no significant response to reed removal over the four-year course of this study. Reed removal allowed regrowth of a more diverse plant community thereby achieving one of the goals of this restoration effort but patterns in ammonium accumulation and denitrification suggest a reduction in the capacity of this site to act as a sink for nitrogen.     

Plasma membrane display of anti-viral single chain Fv fragments confers resistance to tobacco mosaic virus

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor -domain (TcR) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcR in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcR transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcR were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in planta and are a novel approach for engineering disease-resistant crops.     

Trace analysis of SN-38 in human plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method with fluorescence detection was developed and validated for the quantitation of SN-38, the active metabolite of irinotecan (CPT-11), a new anticancer drug. This method uses solid-phase extraction with a C₁₈ column for sample clean-up and concentration following acidification of human plasma with two volumes of 0.1 M HCl. Using blank plasma spiked with SN-38, we found the assay to be linear over the concentration range of 10–500 pM (3.9–195 pg/ml) with acceptable total and within-day imprecision. The recovery of SN-38 ranged from 48.3% (10 pM) to 91.5% (500 pM) whereas that of the internal standard, 20-(S)-camptothecin, was 96.9% (500 pM). This method represents a sizeable increase in sensitivity over other published methods and is shown to be suitable for the measurement of ‘trough' concentrations of SN-38 during the treatment of patients with a weekly regimen of irinotecan.     

Relationships between aromatase activity, follicular fluid oestradiol-17 beta and testosterone concentrations, and diameter and atresia of individual ovine follicles

Aromatase activity was measured in granulosa cells using a 1-h in-vitro assay. This activity correlated with the concentration of oestradiol-17 beta and the ratio of oestradiol-17 beta to testosterone in follicular fluid of individual follicles ranging from 1.5 to 7.0 mm diameter. These data show an 8-10-fold difference in aromatase activity between small and large follicles and that aromatase activity per cell increased in small non-atretic follicles (less than 3.5 mm) whereas it remained relatively constant in large nonatretic follicles (greater than or equal to 3.5 mm). Aromatase activity was much lower in follicles at more advanced stages of atresia. Atresia was assessed using the morphological and the morphometric methods (% of maximum number of granulosa cells/follicle). Although the morphological method of assessment was preferable to the morphometric method, it did not differentiate a decrease in aromatase activity as a very early event in the atretic process. We believe this is due to the inability of these methods to detect follicles in the initial stages of atresia.     

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.     

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3.     

Break-induced loss of heterozygosity in fission yeast: dual roles for homologous recombination in promoting translocations and preventing de novo telomere addition

Loss of heterozygosity (LOH), a causal event in tumorigenesis, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms leading to such extensive LOH are poorly understood. We investigated the mechanisms of DNA double-strand break (DSB)-induced extensive LOH by screening for auxotrophic marker loss approximately 25 kb distal to an HO endonuclease break site within a nonessential minichromosome in Schizosaccharomyces pombe. Extensive break-induced LOH was infrequent, resulting from large translocations through both allelic crossovers and break-induced replication. These events required the homologous recombination (HR) genes rad32(+), rad50(+), nbs1(+), rhp51(+), rad22(+), rhp55(+), rhp54(+), and mus81(+). Surprisingly, LOH was still observed in HR mutants, which resulted predominantly from de novo telomere addition at the break site. De novo telomere addition was most frequently observed in rad22Delta and rhp55Delta backgrounds, which disrupt HR following end resection. Further, levels of de novo telomere addition, while increased in ku70Delta rhp55Delta strains, were reduced in exo1Delta rhp55Delta and an rhp55Delta strain overexpressing rhp51. These findings support a model in which HR prevents de novo telomere addition at DSBs by competing for resected ends. Together, these results suggest that the mechanisms of break-induced LOH may be predicted from the functional status of the HR machinery.     

Engineering a twist in 9,10-diethynylanthracenes by steric interactions.

A series of 9,10-bis(phenylethynyl)anthracenes decorated with sterically demanding tert-butyl substituents have been prepared and spectroscopically characterised. We demonstrate that the introduction of two bulky substituents in the ortho position of the phenyl rings effectively locks the ground state into a conformation in which the three rings are orthogonal. Fluorescence spectroscopy reveals evidence for partial planarisation of this compound in the excited state at ambient temperature, but this is prevented in low temperature solvent glasses.