Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Bacterially Induced Weathering of Ultramafic Rock and Its Implications for Phytoextraction

The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two Arthrobacter strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO₄ solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator Alyssum serpyllifolium subsp. malacitanum was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.     

Delayed post-conditioning reduces post-ischemic glutamate level and improves protein synthesis in brain

In the clinic delayed post-conditioning would represent an attractive strategy for the survival of vulnerable neurons after an ischemic event. In this paper we studied the impact of ischemia and delayed post-conditioning on blood and brain tissue concentrations of glutamate and protein synthesis. We designed two groups of animals for analysis of brain tissues and blood after global ischemia and post-conditioning, and one for analysis of blood glutamate after transient focal ischemia.     

Development of a pattern in biochemical parameters in the core and penumbra during infarct evolution after transient MCAO in rats

The period from stroke initiation to the cessation of penumbra damage spread represents a therapeutic window when expansion can be alleviated. In the present work, we studied some biochemical parameters helpful for the estimation of infarct progression and thus for the application of interventions.     

The function of Notch signalling in segment formation in the crustacean Daphnia magna (Branchiopoda)

Ten years ago we showed for the first time that Notch signalling is required in segmentation in spiders, indicating the existence of similar mechanisms in arthropod and vertebrate segmentation. However, conflicting results in various arthropod groups hampered our understanding of the ancestral function of Notch in arthropod segmentation. Here we fill a crucial data gap in arthropods and analyse segmentation in a crustacean embryo. We analyse the expression of homologues of the Drosophila and vertebrate segmentation genes and show that members of the Notch signalling pathway are expressed at the same time as the pair-rule genes. Furthermore, inactivation of Notch signalling results in irregular boundaries of the odd-skipped-like expression domains and affects the formation of segments. In severe cases embryos appear unsegmented. We suggest two scenarios for the function of Notch signalling in segmentation. The first scenario agrees with a segmentation clock involving Notch signalling, while the second scenario discusses an alternative mechanism of Notch function which is integrated into a hierarchical segmentation cascade.     

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes

Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein–protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.     

Effete,a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.     

Recent Dinoflagellate cyst distribution in the North Canary Basin,NW Africa

The North Canary Basin (NW Africa) falls within a major eastern boundary upwelling system. This part of the coastal upwelling system is seasonal and is characterised by the development of large filaments migrating seawards. Hence, 16 samples from this location were selected to identify an “upwelling signal” in the composition of the dinoflagellate cyst assemblages.

Samples closest to the most intense upwelling cells are dominated by L. machaerophorum and G. catenatum and Protoperidinium spp. These make up the “upwelling signal” characteristic for the system. Moreover, the “upwelling signal” can be advected offshore, with filaments that may extend as far as 300 km. Finally, the finding of cysts from G. catenatum, a toxic dinoflagellate, raises the need for a better understanding of the relationship between its presence and distribution in the region, and the coastal upwelling system.     

Pollen morphology of the Pavetteae (Rubiaceae,Ixoroideae) and its taxonomic significance

Pollen grains of the tribe Pavetteae (Rubiaceae, subfamily Ixoroideae) are examined using LM and SEM. Grains are 3‐ or 4‐colporate and (semi‐) tectate (in one Versteegia species atectate). Sexine patterns vary between perforate, microreticulate, reticulate, rugulate and striato‐reticulate. Supratectal elements are sometimes present. The variation in pollen morphology in the Pavetteae allows to recognize seven pollen types, the distribution of which is useful to evaluate generic delimitations and relationships within the tribe. Pollen characters corroborate the close relationships between the genera Coleactina, Dictyandra and Leptactina and between Homollea, Homolliella and Paracephaelis. All the genera of the tribe proved to be stenopalynous (the species examined possess the same pollen type), except Pavetta, Rutidea, Versteegia and Tarenna which are eurypalynous. In the huge genus Pavetta the existing infrageneric classification is supported pollen morphologically. Pollen morphology further indicates that the genus Tarenna is badly delimited and strongly in need of a revision. The small genus Versteegia is in need of further taxonomic and palynological study to understand the pollen morphological variation encountered here. At a higher rank, pollen morphology also does not contradict the recent division of the Pavetteae in the Ixoreae (a stenopalynous tribe with presumably primitive pollen) and the Pavetteae sensu stricto (eurypalynous).