Greenhouse tests on resistance management of Bt transgenic plants using refuge strategies

Experimental evaluation of the effectiveness of resistance management tactics is vital to help provide guidelines for the deployment of transgenic insecticidal crops. Transgenic broccoli expressing a Cry1Ac gene of Bacillus thuringiensis (Bt) and the diamondback moth, Plutella xylostella (L.), were used in greenhouse tests to evaluate the influence of size and placement of nontransgenic refuge plants on changes in resistance allele frequency and pest population growth. In the first test with an initial Cry1Ac-resistance (R) allele frequency of 0.007, P. xylostella were introduced into cages with the following treatments: 0, 3.3, 10, 20, and 100% refuge plants. Results after four generations showed that resistance could be delayed by increasing the proportion of refuge plants in the cage. Population growth was also influenced by refuge size with the highest populations occurring in treatments that had either no refuge plants or all refuge plants. In the second test, we evaluated the effect of refuge placement by comparing 20% separate and 20% mixed refuges. P. xylostella with an initial frequency of resistant alleles at 0.0125 were introduced into cages and allowed to cycle; later generations were evaluated for resistance and population growth. Separating the refuge had a pronounced effect on delaying resistance and slowing establishment of resistant larvae on Bt plants. Combining information from both trials, we found a strong negative correlation between the number of larvae on Bt plants and the mortality of the population in leaf dip bioassays. Results from larval movement studies showed that separate refuges delayed resistance better than mixed refuges because they conserved relatively more susceptible alleles than R alleles and did not increase the effective dominance of resistance.     

Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

Normal development and fertility of knockout mice lacking the tumor suppressor gene LRP1b suggest functional compensation by LRP1

LRP1b and the closely related LRP1 are large members of the low-density lipoprotein receptor family. At the protein level LRP1b is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cargo transport and cellular signaling. Somatic LRP1b mutations frequently occur in non-small cell lung cancer and urothelial cancers, suggesting a role in the modulation of cellular growth. In contrast to LRP1, LRP1b-deficient mice develop normally, most likely due to its restricted expression pattern and functional compensation by LRP1 or other receptors. LRP1b is expressed predominantly in the brain, and a differentially spliced form is present in the adrenal gland and in the testis. Despite the presence of a potential furin cleavage site and in contrast to LRP1, immunoblotting for LRP1b reveals the presence of a single 600-kDa polypeptide species. Using a yeast two-hybrid approach, we have identified two intracellular proteins, the postsynaptic density protein 95 and the aryl hydrocarbon receptor-interacting protein, that bind to the intracellular domain of LRP1b. In addition, we have found several potential ligands that bind to the extracellular domain. Analysis of LRP1b knockout mice may provide further insights into the role of LRP1b as a tumor suppressor and into the mechanisms of cancer development.     

alpha3beta1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

We show that alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.     

B-raf and insulin synergistically prevent apoptosis and induce cell cycle progression in hematopoietic cells

FDC-P1 hematopoietic cells were conditionally transformed to grow in response to (delta)B Raf:ER, (delta)Raf-1:ER or DA-Raf:ER in which the hormone binding domain of the estrogen receptor (ER) was linked to the N-terminal truncated (delta) Raf genes. When these cells were deprived of IL-3 or beta-estradiol for 24 hrs, they exited the cell cycle and underwent apoptosis. FD/(delta)Raf-1:ER and FD/(delta)A-Raf:ER, but not FD/(delta)B-Raf:ER cells, were readily induced to re-enter the cell cycle after addition of beta-estradiol or IL-3. Deprived FD/(delta)Raf-1:ER, but not FD/(delta)B-Raf:ER cells, expressed activated forms of MEK1 and ERK after beta-estradiol or IL-3 stimulation. Insulin or beta-estradiol alone did not induce FD/(delta)B-Raf:ER cells to re-enter the cell cycle, whereas cell cycle entry was observed upon their co-addition. Apoptosis was prevented in FD/(delta)B-Raf:ER cells when they were cultured in the presence of IL-3 or beta-estradiol, whereas they underwent apoptosis in their absence. Insulin by itself did not prevent apoptosis, however, upon DB-Raf:ER or DRaf-1:ER activation and addition of insulin, more than an additive effect was observed in both lines indicating that these path- ways synergized to prevent apoptosis. Raf isoforms differ in their abilities to control apoptosis and cell cycle progression and B-Raf requires insulin-activated pathways for full antiapoptotic and proliferative activity.     

Synergy between PI3K/Akt and Raf/MEK/ERK pathways in IGF-1R mediated cell cycle progression and prevention of apoptosis in hematopoietic cells

The insulin like growth factor-1 (IGF-1) receptor (R) induced PI3K/Akt signal transduction cascade has critical roles in prevention of apoptosis and regulation of cell cycle progression. Here, we discuss the effects of IGF-1R-mediated signal transduction on hematopoietic cells which normally require interleukin-3 (IL-3) for growth and prevention of apoptosis. Cytokine-dependent FDC-P1 hematopoietic cells were conditionally transformed to grow in response to overexpression of IGF-1R in the presence of IGF-1. When these cells were deprived of IL-3 or IGF-1 for 24 hrs, they exited the cell cycle, activated caspase 3 and underwent apoptosis. The effects of inhibitors which targeted the PI3K/Akt and Raf/MEK/ERK pathways were determined. When the cells were cultured with IGF-1 and either PI3K or MEK inhibitors, cell cycle progression and DNA synthesis were inhibited and caspase 3 activity and apoptosis were induced. Coinhibition of both pathways synergized to prevent cell cycle progression, inhibit DNA synthesis and induce apoptosis. These inhibitors had more apoptotic inducing effects when the cells were grown in response to IGF-1 than IL-3, indicating that IL-3 can induce additional anti-apoptotic pathways. These results demonstrate that the PI3K/Akt and Raf/MEK/ERK pathways are intimately involved in IGF-1R-mediated cell cycle progression and prevention of apoptosis in hematopoietic cells.     

The monarch butterfly controversy: scientific interpretations of a phenomenon

The future development and use of agricultural biotechnology has been challenged by two preliminary studies indicating potential risk to monarch butterfly populations by pollen from corn engineered to express proteins from Bacillus thuringiensis. Likewise, these studies have also challenged the way in which science should be performed, published in scientific journals and communicated to the public at large. Herein, we provide a history of the monarch controversy to date. We believe a retrospective view may be useful for providing insights into the proper roles and responsibilities of scientists, the media and public agencies and the consequences when they go awry.     

POCUS: mining genomic sequence annotation to predict disease genes

Here we present POCUS (prioritization of candidate genes using statistics), a novel computational approach to prioritize candidate disease genes that is based on over-representation of functional annotation between loci for the same disease. We show that POCUS can provide high (up to 81-fold) enrichment of real disease genes in the candidate-gene shortlists it produces compared with the original large sets of positional candidates. In contrast to existing methods, POCUS can also suggest counterintuitive candidates.