Ploidity and cell cycle progression during treatment with gold chloride, auranofin and sodium aurothiomalate. Studies on a human epithelial cell line and its sub-strains made resistant to the antiproliferative effects of these gold compounds

The possible influence of gold(III) chloride and the two gold(I)-containing anti-arthritic drugs, auranofin and sodium aurothiomalate, on cellular ploidity and cell cycle progression was investigated on cultured human epithelial cells. Four different cell lines were used: the parent line (HE) and three sub-strains which previously had acquired resistance to the antiproliferative effects of either 350 mumol gold chloride/l culture medium (HEAu350), 2 mumol auranofin/l (HEAF) or 300 mumol sodium aurothiomalate/l (HEMyo). DNA-histograms were obtained by flow cytometry examinations during a 9-days' exposure to either of these gold-containing compounds and concentrations. The HE, HEAF and HEMyo cells had similar ploidities, close to tetraploid. The HEAu350 cells had altered ploidity to distinct tetraploid. The distribution of the resistant cells with the cell cycle phases was not different from that of untreated HE cells. The HE cells, when treated with auranofin or sodium aurothiomalate, accumulated in the G2-phase of the cell cycle. In addition, a new cedecoploid peak appeared. No such changes were observed on gold chloride exposure or in HE controls grown without drug supplement. The effects of auranofin and sodium aurothiomalate on cell cycle progression of the HE cells possibly indicate a tendency to polyploidity, and furthermore that inhibition of cellular mitosis is one mechanism of the antiproliferative effect common to the two drugs.     

Lectin Binding Assays for In-Process Monitoring of Sialylation in Protein Production

Many therapeutic proteins require appropriate glycosylation for their biological activities and plasma half life. Coagulation factor VIII (FVIII) is a glycoprotein which has extensive post-translational modification by N-linked glycosylation. The terminal sialic acid in the N-linked glycans of FVIII is required for maximal circulatory half life. The extent of FVIII sialylation can be determined by high pH anion-exchange chromatography coupled with a pulse electrochemical detector (HPAEC-PED), but this requires a large amount of purified protein. Using FVIII as a model, the objective of the present study was to develop assays that enable detection and prediction of sialylation deficiency at an early stage in the process and thus prevent downstream product quality excursions. Lectin ECA (Erythrina Cristagalli) binds to unsialylated Galβ1-4 GlcNAc and the ECA-binding level (i.e., terminal Gal(β1-4) exposure) is inversely proportional to the level of sialylation. By using ECA, a cell-based assay was developed to measure the global sialylation profile in FVIII producing cells. To examine the Galβ1-4 exposure on the FVIII molecule in bioreactor tissue culture fluid (TCF), an ELISA-based ECA-FVIII binding assay was developed. The ECA-binding specificity in both assays was assessed by ECA-specific sugar inhibitors and neuraminidase digestion. The ECA-binding specificity was also independently confirmed by a ST3GAL4 siRNA knockdown experiment. To establish the correlation between Galβ1-4 exposure and the HPAEC-PED determined FVIII sialylation value, the FVIII containing bioreactor TCF and the purified FVIII samples were tested with ECA ELISA binding assay. The results indicated an inverse correlation between ECA binding and the corresponding HPAEC-PED sialylation value. The ECA-binding assays are cost effective and can be rapidly performed, thereby making them effective for in-process monitoring of protein sialylation.     

Genome scans and elusive candidate genes: detecting the variation that matters for speciation

Next‐generation sequencing is providing us with vast amounts of genetic data, yet we are currently struggling in our attempts to make sense of them. In particular, it has proven difficult to link phenotypic divergence and speciation to genome level divergence. In the current issue of Molecular Ecology, Ruegg et al. ( 2014 ) present new empirical results from two closely related bird taxa. They use a promising approach combining genome scan and candidate gene analysis. Their results suggest that we may have been looking in vain for candidate speciation genes by focusing only on genes found within genomic islands of divergence. This is because genes important in divergence and speciation may not be detected by genome scans and because features of the genomic architecture per se may have a large effect on the pattern of genome divergence.     

Premature expression of cyclin B sensitizes human HT1080 cells to caffeine-induced premature mitosis

Eukaryotic cells do not normally initiate mitosis when DNA replication is blocked. This cell cycle checkpoint can be bypassed in some cells, however, by treatment with caffeine and certain other chemicals. Although S-phase arrested hamster cells undergo mitosis-specific events such as premature chromosome condensation (PCC) and nuclear envelope disassembly when exposed to caffeine, human cells show little response under the same conditions. To further investigate the molecular basis of this cell type specificity, a panel of hamster/human whole cell hybrids was created. The frequency of caffeine-induced PCC and the level of cyclin B-associated H1 kinase activity in the various hybrids were directly correlated with the extent of cyclin B synthesis during S-phase arrest. To determine whether expression of cyclin B alone could sensitize human cells to caffeine, cyclin B1 was transiently overexpressed in S-phase arrested HT1080 cells. The transfected cell population displayed a 5-fold increase in the frequency of caffeine-induced PCC when compared with normal HT1080 cells, roughly equivalent to the frequency of cells expressing exogenous epitope-tagged cyclin B1. In addition, immunofluorescent microscopy showed that individual cells overexpressing cyclin B1 during S phase arrest underwent PCC when exposed to caffeine. These results provide direct evidence that premature expression of cyclin B1 can make cells more vulnerable to chemically-induced uncoupling of mitosis from the completion of DNA replication. © 1995 Wiley-Liss, Inc.     

Analysis of the embryonic lineage of vertebrate taste buds

In all vertebrates, taste buds are the last sensory receptorsto appear late in embryonic development. They are thought toarise locally from the oropharyngeal epithelium, although thishypothesis has not been tested experimentally. Alternatively,taste buds have been proposed to arise from neurocctodermalcells that migrate from peripheral neurogenic sources to theoropharyngeal epithelium and give rise to taste bud precursorcells. In order to determine the exact embryonic lineage ofthe cells of vertebrate taste buds, we have employed a combinationof endogenous and exogenous cell marking techniques to followneuroectodermal and endodermal cells through development. Wefind, in the ambystomatid salamander used in our studies, tastebuds arise locally within the endodermally-derived epitheliumlining the oropharyngeal cavity, and do not receive a contributionfrom neuroectodermal sources, i.e. ectodermal placodes or cephalicneural crest.     

Linking behavioral states to landscape features for improved conservation management

1. A central theme for conservation is understanding how animals differentially use, and are affected by change in, the landscapes they inhabit. However, it has been challenging to develop conservation schemes for habitat‐specific behaviors.
2. Here we use behavioral change point analysis to identify behavioral states of golden eagles (Aquila chrysaetos) in the Sonoran and Mojave Deserts of the southwestern United States, and we identify, for each behavioral state, conservation‐relevant habitat associations.
3. We modeled behavior using 186,859 GPS points from 48 eagles and identified 2,851 distinct segments comprising four behavioral states. Altitude above ground level (AGL) best differentiated behavioral states, with two clusters of short‐distance movement behaviors characterized by low AGL (state 1 AGL = 14 m (median); state 2 AGL = 11 m) and two associated with longer‐distance movement behaviors and characterized by higher AGL (state 3 AGL = 108 m; state 4 AGL = 450 m).
4. Behaviors such as perching and low‐altitude hunting were associated with short‐distance movements in updraft‐poor environments, at higher elevations, and over steeper and more north‐facing terrain. In contrast, medium‐distance movements such as hunting and transiting were over gentle and south‐facing slopes. Long‐distance transiting occurred over the desert habitats that generate the best updraft.
5. This information can guide management of this species, and our approach provides a template for behavior‐specific habitat associations for other species of management concern.

     

Early vigour in wheat: Could it lead to more severe terminal drought stress under elevated atmospheric [CO2] and semi‐arid conditions?

Early vigour in wheat is a trait that has received attention for its benefits reducing evaporation from the soil surface early in the season. However, with the growth enhancement common to crops grown under elevated atmospheric CO₂ concentrations (e[CO₂]), there is a risk that too much early growth might deplete soil water and lead to more severe terminal drought stress in environments where production relies on stored soil water content. If this is the case, the incorporation of such a trait in wheat breeding programmes might have unintended negative consequences in the future, especially in dry years. We used selected data from cultivars with proven expression of high and low early vigour from the Australian Grains Free Air CO₂ Enrichment (AGFACE) facility, and complemented this analysis with simulation results from two crop growth models which differ in the modelling of leaf area development and crop water use. Grain yield responses to e[CO₂] were lower in the high early vigour group compared to the low early vigour group, and although these differences were not significant, they were corroborated by simulation model results. However, the simulated lower response with high early vigour lines was not caused by an earlier or greater depletion of soil water under e[CO₂] and the mechanisms responsible appear to be related to an earlier saturation of the radiation intercepted. Whether this is the case in the field needs to be further investigated. In addition, there was some evidence that the timing of the drought stress during crop growth influenced the effect of e[CO₂] regardless of the early vigour trait. There is a need for FACE investigations of the value of traits for drought adaptation to be conducted under more severe drought conditions and variable timing of drought stress, a risky but necessary endeavour.     

A process‐based metacommunity framework linking local and regional scale community ecology

The metacommunity concept has the potential to integrate local and regional dynamics within a general community ecology framework. To this end, the concept must move beyond the discrete archetypes that have largely defined it (e.g. neutral vs. species sorting) and better incorporate local scale species interactions and coexistence mechanisms. Here, we present a fundamental reconception of the framework that explicitly links local coexistence theory to the spatial processes inherent to metacommunity theory, allowing for a continuous range of competitive community dynamics. These dynamics emerge from the three underlying processes that shape ecological communities: (1) density‐independent responses to abiotic conditions, (2) density‐dependent biotic interactions and (3) dispersal. Stochasticity is incorporated in the demographic realisation of each of these processes. We formalise this framework using a simulation model that explores a wide range of competitive metacommunity dynamics by varying the strength of the underlying processes. Using this model and framework, we show how existing theories, including the traditional metacommunity archetypes, are linked by this common set of processes. We then use the model to generate new hypotheses about how the three processes combine to interactively shape diversity, functioning and stability within metacommunities.