SB203580 Induces Prolonged B-Raf Activation and Promotes Neuronal Differentiation upon EGF Treatment of PC12 Cells

SB203580 is a p38 MAPK inhibitor that has been implicated in the activation of c-Raf. This study shows that the addition of SB203580 to PC12 cells causes the sustained activation of B-Raf but not of ERK. The addition of SB203580 prolonged the transient activation of both B-Raf and ERK by EGF alone. No significant change was detected in MAPKAPK-2 activity at low concentrations of SB203580, which induced neurite outgrowth in the EGF-stimulated PC12 cells. Therefore, these results indicate that SB203580 influences not only c-Raf as previously reported, but can also induce the activation of B-Raf, which in conjunction with EGF causes the sustained activation of ERK and differentiation in PC12 cells.     

Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis

This study focuses on the specific problems of protein extraction from recalcitrant plant tissues and evaluates several methods to bypass them. Sample preparation is a critical step in a two-dimensional gel electrophoresis proteome approach and is absolutely essential for good results. We evaluated four methods: the classical trichloroacetic acid (TCA)/acetone precipitation, TCA/acetone precipitation and fractionation, an alternative based on fractionation and without precipitation, and phenol extraction methanol/ammonium acetate precipitation. We optimized the phenol extraction protocol for small amounts of tissue, which is essential when the study material is limited. The protocol was optimized for banana (Musa spp.) and was subsequently applied to two other plant species: apple (Malus domestica L.) and potato (Solanum tuberosum L.). Banana (Musa spp.) is a good representative of a "difficult" plant species since it contains many interfering metabolites. Only classical TCA/acetone precipitation and phenol extraction methods proved useful as standard methods. Both methods are associated with a minor but reproducible loss of proteins. Every extraction method and the subsequent analytical procedure have their physicochemical limitations; both methods should be investigated before selecting an appropriate protocol. The study, which is presented in this paper, is useful for guiding the experimental setup of many other nonmodel species, containing various interfering elements.     

Extracellular signal-regulated kinase 1c (ERK1c), a novel 42-kilodalton ERK, demonstrates unique modes of regulation, localization, and function

Extracellular signal-regulated kinases (ERKs) are signaling molecules that regulate many cellular processes. We have previously identified an alternatively spliced 46-kDa form of ERK1 that is expressed in rats and mice and named ERK1b. Here we report that the same splicing event in humans and monkeys causes, due to sequence differences in the inserted introns, the production of an ERK isoform that migrates together with the 42-kDa ERK2. Because of the differences of this isoform from ERK1b, we named it ERK1c. We found that its expression levels are about 10% of ERK1. ERK1c seems to be expressed in a wide variety of tissues and cells. Its activation by MEKs and inactivation by phosphatases are slower than those of ERK1, which is probably the reason for its differential regulation in response to extracellular stimuli. Unlike ERK1, ERK1c undergoes monoubiquitination, which is increased with elevated cell density concomitantly with accumulation of ERK1c in the Golgi apparatus. Elevated cell density also causes enhanced Golgi fragmentation, which is facilitated by overexpression of native ERK1c and is prevented by dominant-negative ERK1c, indicating that ERK1c mediates cell density-induced Golgi fragmentation. The differential regulation of ERK1c extends the signaling specificity of MEKs after stimulation by various extracellular stimuli.     

Optimisation of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment

Conditions for microparticle bombardment were optimised for four explant types of sorghum Sorghum bicolor (L.) Moench based on transient expression of the uidA reporter gene. The tested physical parameters included acceleration pressure, target distance, gap width and macroprojectile travel distance. The sorghum explants studied were immature and mature embryos, shoot tips and embryogenic calli. In addition, the activity of four heterologous promoters was determined both by GUS histochemical staining and enzymatic activity assay in immature embryos and shoot tips. The strength of these promoters could be placed in the following order: ubi1> act1D> adh1>CaMV 35S. The optimised bombardment conditions were applied for selecting phosphinothricin- or geneticin-resistant in vitro cultures in order to generate transgenic plants. Production of transgenic plants via phosphinothricin-selection was not successful due to the release of phenolic substances from the herbicide-resistant cultures during the regeneration process. After selection on geneticin, however, fertile transgenic sorghum plants were regenerated from immature embryos as well as from shoot tips. Stable integration and Mendelian inheritance of the neo selectable marker gene was demonstrated in all transgenic plants.     

DO AID AGENCIES HAVE AN ETHICAL DUTY TO COMPLY WITH RESEARCHERS? A RESPONSE TO RENNIE1

Medical AID organisations such as Médecins Sans Frontières receive several requests from individuals and international academic institutions to conduct research at their implementation sites in Africa. Do AID agencies have an ethical duty to comply with research requests? In this paper we respond to the views and constructed theories (albeit unfounded) of one such researcher, whose request to conduct research at one of our sites in the Democratic Republic of Congo was turned down.     

High‐throughput physiological phenotyping and screening system for the characterization of plant–environment interactions

We present a simple and effective high‐throughput experimental platform for simultaneous and continuous monitoring of water relations in the soil–plant–atmosphere continuum of numerous plants under dynamic environmental conditions. This system provides a simultaneously measured, detailed physiological response profile for each plant in the array, over time periods ranging from a few minutes to the entire growing season, under normal, stress and recovery conditions and at any phenological stage. Three probes for each pot in the array and a specially designed algorithm enable detailed water‐relations characterization of whole‐plant transpiration, biomass gain, stomatal conductance and root flux. They also enable quantitative calculation of the whole plant water‐use efficiency and relative water content at high resolution under dynamic soil and atmospheric conditions. The system has no moving parts and can fit into many growing environments. A screening of 65 introgression lines of a wild tomato species (Solanum pennellii) crossed with cultivated tomato (S. lycopersicum), using our system and conventional gas‐exchange tools, confirmed the accuracy of the system as well as its diagnostic capabilities. The use of this high‐throughput diagnostic screening method is discussed in light of the gaps in our understanding of the genetic regulation of whole‐plant performance, particularly under abiotic stress.     

From crossbreeding to biotechnology-facilitated improvement of banana and plantain

The annual harvest of banana and plantain (Musa spp.) is approximately 145 million tons worldwide. About 85% of this global production comes from small plots and kitchen or backyard gardens from the developing world, and only 15% goes to the export trade. Musa acuminata and Musa balbisiana are the ancestors of several hundreds of parthenocarpic Musa diploid and polyploid cultivars, which show multiple origins through inter- and intra-specific hybridizations from these two wild diploid species. Generating hybrids combining host plant resistance to pathogens and pests, short growth cycles and height, high fruit yield, parthenocarpy, and desired quality from the cultivars remains a challenge for Musa crossbreeding, which started about one century ago in Trinidad. The success of Musa crossbreeding depends on the production of true hybrid seeds in a crop known for its high levels of female sterility, particularly among polyploid cultivars. All banana export cultivars grown today are, however, selections from somatic mutants of the group Cavendish and have a very narrow genetic base, while smallholders in sub-Saharan Africa, tropical Asia and Latin America use some bred-hybrids (mostly cooking types). Musa improvement goals need to shift to address emerging threats because of the changing climate. Innovative cell and molecular biology tools have the potential to enhance the pace and efficiency of genetic improvement in Musa. Micro-propagation has been successful for high throughput of clean planting materials while in vitro seed germination assists in obtaining seedlings after inter-specific and across ploidy hybridization. Flow cytometry protocols are used for checking ploidy among genebank accessions and breeding materials. DNA markers, the genetic maps based on them, and the recent sequencing of the banana genome offer means for gaining more insights in the genetics of the crops and to identifying genes that could lead to accelerating Musa betterment. Likewise, DNA fingerprinting has been useful to characterize Musa diversity. Genetic engineering provides a complementary tool to Musa breeders who can introduce today transgenes that may confer resistance to bacteria, fungi and nematodes, or enhance pro-vitamin A fruit content. In spite of recent advances, the genetic improvement of Musa depends on a few crossbreeding programs (based in Brazil, Cameroon, Côte d'Ivoire, Guadeloupe, Honduras, India, Nigeria, Tanzania and Uganda) or a handful of genetic engineering endeavors (Australia, Belgium, India, Kenya, Malaysia and Uganda). Development investors (namely international aid and philanthropy) should therefore increase their funding to genetically enhance this crop that ranks among the 10-top staple foods of the developing world.     

Induction of Enhanced Acoustic Startle Response by Noise Exposure: Dependence on Exposure Conditions and Testing Parameters and Possible Relevance to Hyperacusis

There has been a recent surge of interest in the development of animal models of hyperacusis, a condition in which tolerance to sounds of moderate and high intensities is diminished. The reasons for this decreased tolerance are likely multifactorial, but some major factors that contribute to hyperacusis are increased loudness perception and heightened sensitivity and/or responsiveness to sound. Increased sound sensitivity is a symptom that sometimes develops in human subjects after acoustic insult and has recently been demonstrated in animals as evidenced by enhancement of the acoustic startle reflex following acoustic over-exposure. However, different laboratories have obtained conflicting results in this regard, with some studies reporting enhanced startle, others reporting weakened startle, and still others reporting little, if any, change in the amplitude of the acoustic startle reflex following noise exposure. In an effort to gain insight into these discrepancies, we conducted measures of acoustic startle responses (ASR) in animals exposed to different levels of sound, and repeated such measures on consecutive days using a range of different startle stimuli. Since many studies combine measures of acoustic startle with measures of gap detection, we also tested ASR in two different acoustic contexts, one in which the startle amplitudes were tested in isolation, the other in which startle amplitudes were measured in the context of the gap detection test. The results reveal that the emergence of chronic hyperacusis-like enhancements of startle following noise exposure is highly reproducible but is dependent on the post-exposure thresholds, the time when the measures are performed and the context in which the ASR measures are obtained. These findings could explain many of the discrepancies that exist across studies and suggest guidelines for inducing in animals enhancements of the startle reflex that may be related to hyperacusis.