High-throughput screening for norepinephrine transporter inhibitors using the FLIPRTetra

Monoamine transporters regulate the concentration of neurotransmitters in the synapse following neurotransmission and are very important drug targets in the pharmaceutical industry. Because of the labor-intensive nature of functional uptake assays using radioactive substrates, high-throughput screening for monoamine transporter inhibitors has been limited to radioligand binding assays. In this article, the authors describe the development of a 384-well, high-throughput functional screening assay for norepinephrine transporter inhibitors using the FLIPR(Tetra) and a recently identified fluorescent substrate, 4-(4-dimethylaminostyryl)- N-methyl-pyridinium (ASP(+)).     

QTLs for shelf life in lettuce co-locate with those for leaf biophysical properties but not with those for leaf developmental traits

Developmental and biophysical leaf characteristics that influence post-harvest shelf life in lettuce, an important leafy crop, have been examined. The traits were studied using 60 informative F9 recombinant inbed lines (RILs) derived from a cross between cultivated lettuce (Lactuca sativa cv. Salinas) and wild lettuce (L. serriola acc. UC96US23). Quantitative trait loci (QTLs) for shelf life co-located most closely with those for leaf biophysical properties such as plasticity, elasticity, and breakstrength, suggesting that these are appropriate targets for molecular breeding for improved shelf life. Significant correlations were found between shelf life and leaf size, leaf weight, leaf chlorophyll content, leaf stomatal index, and epidermal cell number per leaf, indicating that these pre-harvest leaf development traits confer post-harvest properties. By studying the population in two contrasting environments in northern and southern Europe, the genotype by environment interaction effects of the QTLs relevant to leaf development and shelf life were assessed. In total, 107 QTLs, distributed on all nine linkage groups, were detected from the 29 traits. Only five QTLs were common in both environments. Several areas where many QTLs co-located (hotspots) on the genome were identified, with relatively little overlap between developmental hotspots and those relating to shelf life. However, QTLs for leaf biophysical properties (breakstrength, plasticity, and elasticity) and cell area correlated well with shelf life, confirming that the ideal ideotype lettuce should have small cells with strong cell walls. The identification of QTLs for leaf development, strength, and longevity will lead to a better understanding of processability at a genetic and cellular level, and allow the improvement of salad leaf quality through marker-assisted breeding.     

Hawaiian skirt: an F-box gene that regulates organ fusion and growth in Arabidopsis

A fast neutron-mutagenized population of Arabidopsis (Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt (hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein (At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, Pro(PGAZAT):beta-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using Pro(HWS):beta-glucuronidase or Pro(HWS):green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro(35S):HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.     

Molecular dissection of an inhibitor targeting the HIV integrase dependent preintegration complex nuclear import

Human immunodeficiency virus (HIV) continues to be a major contributor to morbidity and mortality worldwide, particularly in developing nations where high cost and logistical issues severely limit the use of current HIV therapeutics. This, combined HIV's high propensity to develop resistance, means that new antiviral agents against novel targets are still urgently required. We previously identified novel anti‐HIV agents directed against the nuclear import of the HIV integrase (IN) protein, which plays critical roles in the HIV lifecycle inside the cell nucleus, as well as in transporting the HIV preintegration complex (PIC) into the nucleus. Here we investigate the structure activity relationship of a series of these compounds for the first time, including a newly identified anti‐IN compound, budesonide, showing that the extent of binding to the IN core domain correlates directly with the ability of the compound to inhibit IN nuclear transport in a permeabilised cell system. Importantly, compounds that inhibited the nuclear transport of IN were found to significantly decrease HIV viral replication, even in a dividing cell system. Significantly, budesonide or its analogue flunisolide, were able to effect a significant reduction in the presence of specific nuclear forms of the HIV DNA (2‐LTR circles), suggesting that the inhibitors work though blocking IN, and potentially PIC, nuclear import. The work presented here represents a platform for further development of these specific inhibitors of HIV replication with therapeutic and prophylactic potential.     

A new subdivision of the Albian spore-pollen zonation of Australia

The Albian spore-pollen zonation of Australia is used to date terrestrial sequences from Antarctica to New Zealand. A detailed qualitative and quantitative study of the Albian Crybelosporites striatus and Coptospora paradoxa Zones in the Gippsland Basin confirms many of the published zone indicator taxa are reliable. The first and last appearance datums can be further refined quantitatively based on the upward reduction in the abundance of seed fern produced pollen. Three subzones are defined in the C. paradoxa Zone using a combination of presence/absence range and abundance data. These are (from oldest to youngest): the Trilobosporites trioreticulosus, Pilosisporites grandis and Cicatricosisporites cuneiformis Subzones. This latter subzone presents a new subdivision of this zone in the Gippsland Basin in southeast Australia. All these subzonal divisions have sufficient resolution to facilitate reservoir-scale correlation of fluvial strata confined to the C. paradoxa Zone in the Gippsland Basin. This study has the first record of Taxodiaceaepollenites hiatus in the Early Cretaceous of Australia and a new species Aequitriradites burgerii is described. A re-examination of the chronology of the zones in the Cretaceous marine basins confirms a 103.5 Ma age for the top of the C. paradoxa Zone, however the chronology of the base of this zone is unclear in the Albian.     

Global enhancement of nuclear localization-dependent nuclear transport in transformed cells

Fundamental to eukaryotic cell function, nucleocytoplasmic transport can be regulated at many levels, including through modulation of the importin/exportin (Imp/Exp) nuclear transport machinery itself. Although Imps/Exps are overexpressed in a number of transformed cell lines and patient tumor tissues, the efficiency of nucleocytoplasmic transport in transformed cell types compared with nontransformed cells has not been investigated. Here we use quantitative live cell imaging of 3 isogenic nontransformed/transformed cell pairs to show that nuclear accumulation of nuclear localization signal (NLS)-containing proteins, but not their NLS-mutated derivatives, is increased up to 7-fold in MCF10CA1h human epithelial breast carcinoma cells and in simian virus 40 (SV40)-transformed fibroblasts of human and monkey origin, compared with their nontransformed counterparts. The basis for this appears to be a significantly faster rate of nuclear import in transformed cell types, as revealed by analysis using fluorescence recovery after photobleaching for the human MCF10A/MCF10CA1h cell pair. Nuclear accumulation of NLS/nuclear export signal-containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp-1-mediated nuclear export was not impaired in transformed compared with nontransformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2- to 4-fold higher expression of specific Imps/Exps in transformed cells, as indicated by quantitative Western blot analysis, with ectopic expression of Imps able to enhance NLS nuclear accumulation levels up to 5-fold in nontransformed MCF10A cells. The findings indicate that transformed cells possess altered nuclear transport properties, most likely due to the overexpression of Imps/Exps. The findings have important implications for the development of tumor-specific drug nanocarriers in anticancer therapy.     

Manipulating resource allocation in plants

The distribution of nutrients and assimilates in different organs and tissues is in a constant state of flux throughout the growth and development of a plant. At key stages during the life cycle profound changes occur, and perhaps one of the most critical of these is during seed filling. By restricting the competition for reserves in Arabidopsis plants, the ability to manipulate seed size, seed weight, or seed content has been explored. Removal of secondary inflorescences and lateral branches resulted in a stimulation of elongation of the primary inflorescence and an increase in the distance between siliques. The pruning treatment also led to the development of longer and larger siliques that contained fewer, bigger seeds. This seems to be a consequence of a reduction in the number of ovules that develop and an increase in the fatty acid content of the seeds that mature. The data show that shoot architecture could have a substantial impact on the partitioning of reserves between vegetative and reproductive tissues and could be an important trait for selection in rapid phenotyping screens to optimize crop performance.     

A phase I study of recombinant interferon gamma administered by s.c. injection three times per week in patients with solid tumours

Summary Human recombinant DNA interferon gamma (IFN-G), with a specific activity of 2×10⁶ IU/mg protein, was administered s.c. 3 days per week for 2 months to patients with solid tumors. The maximum tolerated dose (MTD) was 10×10⁶ IU/m² (5.0 mg/m²) per injection, and six patients were treated at the MTD. Two of these ceased treatment because of severe subjective toxity (headache, rigors and pyrexia) and three patients developed WHO grade 3 leucopenia. Subjective toxicity varied considerably between patients and some patients at low dose levels experienced severe constitutional symptoms whilst others treated at the MTD had few side effects. These differences were unrelated to pharmacokinetic parameters. Bioavailability of this IFN-G administered s.c. was very variable from one patient to another at the same dose level. We therefore counsel caution in using this IFN-G preparation s.c. in phase II studies.     

Successional trajectories of a grazed salt desert shrubland

Successional trajectories through the statistical space of ordinations were used to examine response to grazing in salt desert shrub communities of western Utah, USA. Relative cover data were periodically collected over a 53 year period from grazing exclosures and pastures grazed with light or heavy stocking rates in fall or spring (4 grazing treatments). Two-way indicator species analysis was used to select the 98 most similar plots (24 m²) from among 358 plots in 1935. Foliar cover of 23 species on those 98 plots was followed at irregular intervals over 53 years, resulting in 709 plot-time samples. Successional trajectories were developed for each grazing treatment with ordinations from detrended correspondence analysis. Grazing season had a more pronounced influence on floristic trajectories than did grazing intensity. These ordinations suggest grazing season was an important factor regulating response to grazing and identify annual March-April grazing as an important cause of retrogression in the salt desert shrub ecosystem.Abbreviations (DER) Desert Experimental Range