Stable transformation and reverse genetic analysis of Penium margaritaceum: a platform for studies of charophyte green algae,the immediate ancestors of land plants

The charophyte green algae (CGA, Streptophyta, Viridiplantae) occupy a key phylogenetic position as the immediate ancestors of land plants but, paradoxically, are less well‐studied than the other major plant lineages. This is particularly true in the context of functional genomic studies, where the lack of an efficient protocol for their stable genetic transformation has been a major obstacle. Observations of extant CGA species suggest the existence of some of the evolutionary adaptations that had to occur for land colonization; however, to date, there has been no robust experimental platform to address this genetically. We present a protocol for high‐throughput Agrobacterium tumefaciens‐mediated transformation of Penium margaritaceum, a unicellular CGA species. The versatility of Penium as a model for studying various aspects of plant cell biology and development was illustrated through non‐invasive visualization of protein localization and dynamics in living cells. In addition, the utility of RNA interference (RNAi) for reverse genetic studies was demonstrated by targeting genes associated with cell wall modification (pectin methylesterase) and biosynthesis (cellulose synthase). This provided evidence supporting current models of cell wall assembly and inter‐polymer interactions that were based on studies of land plants, but in this case using direct observation in vivo. This new functional genomics platform has broad potential applications, including studies of plant organismal biology and the evolutionary innovations required for transition from aquatic to terrestrial habitats.     

Dysbalance of Astrocyte Calcium under Hyperammonemic Conditions

Increased brain ammonium (NH₄⁺/NH₃) plays a central role in the manifestation of hepatic encephalopathy (HE), a complex syndrome associated with neurological and psychiatric alterations, which is primarily a disorder of astrocytes. Here, we analysed the influence of NH₄⁺/NH₃ on the calcium concentration of astrocytes in situ and studied the underlying mechanisms of NH₄⁺/NH₃-evoked calcium changes, employing fluorescence imaging with Fura-2 in acute tissue slices derived from different regions of the mouse brain. In the hippocampal stratum radiatum, perfusion with 5 mM NH₄⁺/NH₃ for 30 minutes caused a transient calcium increase in about 40% of astrocytes lasting about 10 minutes. Furthermore, the vast majority of astrocytes (∼90%) experienced a persistent calcium increase by ∼50 nM. This persistent increase was already evoked at concentrations of 1–2 mM NH₄⁺/NH₃, developed within 10–20 minutes and was maintained as long as the NH₄⁺/NH₃ was present. Qualitatively similar changes were observed in astrocytes of different neocortical regions as well as in cerebellar Bergmann glia. Inhibition of glutamine synthetase resulted in significantly larger calcium increases in response to NH₄⁺/NH₃, indicating that glutamine accumulation was not a primary cause. Calcium increases were not mimicked by changes in intracellular pH. Pharmacological inhibition of voltage-gated sodium channels, sodium-potassium-chloride-cotransporters (NKCC), the reverse mode of sodium/calcium exchange (NCX), AMPA- or mGluR5-receptors did not dampen NH₄⁺/NH₃-induced calcium increases. They were, however, significantly reduced by inhibition of NMDA receptors and depletion of intracellular calcium stores. Taken together, our measurements show that sustained exposure to NH₄⁺/NH₃ causes a sustained increase in intracellular calcium in astrocytes in situ, which is partly dependent on NMDA receptor activation and on release of calcium from intracellular stores. Our study furthermore suggests that dysbalance of astrocyte calcium homeostasis under hyperammonemic conditions is a widespread phenomenon, which might contribute to the disturbance of neurotransmission during HE.     

Sediment distribution and accumulation in lagoons of the Southern Mediterranean Region (the MELMARINA Project) with special reference to environmental change and aquatic ecosystems

Surface sediments and sediment cores were collected from coastal lagoons and lakes located in the Southern Mediterranean Region (SMR) as part of the MELMARINA Project which involved integrated eco-hydrological monitoring and modelling. This study uses surface sediments and sediment cores to infer spatial characteristics and temporal changes at the MELMARINA primary sites, Merja Zerga in Morocco, Ghar El Melh in Tunisia and Lake Manzala in Egypt. In addition, surface sediment sampling was undertaken at Egyptian Lake Bardawil and sediment cores were collected from the Lagune de Nador (Morocco). Sediment distribution patterns are investigated using GIS with georeferenced sample locations to facilitate display and resurvey. Major variations in sedimentary organic matter and, particularly, carbonate content, occur within and between sites. Local landscapes combined with hydrological and biogeochemical processes influence the distributions of sediment bulk components (carbonates, organic material and clastic matter) and molluscan shells and shell debris are an important source of sedimentary carbonate at all three primary sites. Sediment cores were dated using natural (²¹⁰Pb) and artificial (¹³⁷Cs) radionuclides, and sediment accumulation rate changes indicate that sources of sediment supply varied markedly through the twentieth century but have generally diminished after the mid-1960s. Sedimentary siliceous microfossils (diatoms) were generally poorly preserved, but mollusc shell remains were well represented. Sediment chronologies and sediment bulk composition allow discussion of some recent changes in bulk, minerogenic and biogenic sediment accumulation patterns in the SMR lagoons. Sediment accumulation rates also varied between sites and multiple cores from Lake Manzala indicated that rates showed considerable spatial variability. Low-level sediment contamination by fossil fuel combustion particulates and trace metals was demonstrated for Ghar El Melh and Lagune de Nador where Pb and Zn accumulation rates were highest in twentieth century sediment. It is emphasized that sediment quality and quantity have strong influences on lagoon ecosystem function and sedimentation is relevant to hydromorphology and to concepts of ecological quality. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: J. R. Thompson & R. J. Flower Hydro-ecological Monitoring and Modelling of North African Coastal Lagoons     

PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia

Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.     

SUB-CELLULAR LOCALISATION OF ENHANCED LYSINE INCORPORATION INTO CEREBRAL CORTEX PROTEINS IN DARK-REARED AND LIGHT-EXPOSED RATS

Incorporation of lysine into acid-insoluble material from subcellular fractions of rat cerebral cortex has been studied using double and single-labelling techniques, in littermates reared for 50 days in the dark and then dark-maintained or light-exposed for 1 h. When light-exposed animals were compared to dark controls the only subcellular fraction from the whole cortex in which lysine incorporation shows a significant elevation (168%, P < 0.05) was located in the ribosomal pellet of the cerebral cortex. A similar comparison of subcellular fractions from visual and motor cortices showed that the elevation was again in the ribosomes and confined to visual cortex only. Motor cortex of light-exposed animals showed a small depression of incorporation in ribosomes as compared to dark controls. Sub-fractionation of nuclei from whole cortex preparations showed varying, but non-significant elevations in light-exposed animals in all but the histone fraction in which there was negligible incorporation of precursor. It is concluded that enhancement of incorporation of precursor into proteins of the cerebral cortex, which accompanies first exposure to light, is a complex response. At exposure for 1 h it involves a number of particular protein species located in the visual cortex, a major proportion of which are ribosomally bound.     

A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface.

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.