Single-Step Purification of Two Functional Human Apolipoprotein E Variants Hyperexpressed inEscherichia coli

We have cloned, from total human liver RNA, the cDNA encoding apolipoprotein E3 (apoE3). Site-directed mutagenesis was used to obtain the cDNA encoding the apoE4 isoform, a major variant of this apolipoprotein in man. These two cDNAs were subcloned into the procaryotic expression vector pAHRS. A polyhistidine tag was added at the NH₂-termini of the recombinant proteins (apoE3 and apoE4) to enable rapid purification. The resulting plasmids (pAHRS-apoE3 and pAHRS-apoE4) were introduced into theEscherichia colistrain BL21(DE3). Recombinant strains were grown at 37°C in a Luria and Bertani medium and the addition of isopropyl β-thiogalactoside resulted in the expression of large amounts of apoE protein (40.5 kDa), representing at least 15% of cellular proteins. The recombinant apoE isoforms were purified, under denaturating conditions, in one step by affinity chromatography on a Ni-chelated agarose column, yielding to about 20 mg of 96% pure protein per liter of culture. Compared to plasma apoE3 purified from human very low density lipoproteins, the two renatured recombinant apoE isoforms have the same secondary structure content, as revealed by circular dichroism measurement. Moreover, the recombinant apoE3 isoform shares similar properties for the association with lipids, compared to the human protein, indicating that the addition of the amino-terminal polyhistidine peptide does not influence the structure and the lipid binding properties of this recombinant apoE isoform. No differences in the secondary structure of recombinant apoE4 were detected, whereas this isoform presents specific reactivity with lipids. This simple and rapid procedure for the expression and the purification of functional recombinant apoE should therefore enable structural and physiological studies requiring large amounts of these apolipoproteins.     

Analysis of Gene Evolution: the software AGE

The software AGE (Analysis of Gene Evolution) has been developedboth to study a genetic reality, i. e. the identification ofstatistical properties in genes (e.g. periodicities), and tosimulate this observed genetic reality, by models of molecularevolution. AGE has two types of models: (i) models of sequencecreation from oligonucleotides: concatenation model in seriesof an oligonucleotide, independent (or Markov) mixing modelof oligonucleotides according to given probabilities (or a Markovmatrix); (ii) models of sequence evolution from created sequences:insertion/deletion process of (mono, di, tri)nucleot-ides, basemutation process. The study of a reality and the developmentof simulation models are based on several new algorithms: approximatedsimulation and exact calculus to compute various autocorrelationfunctions, Fourier transformation of autocorrelation curves,recognition of a curve form, etc. AGE is implemented on IBMor compatible microcomputers and can be used by biologists withoutany computer knowledge to identify statistical properties intheir newly determined DNA sequence and to explain them by modelsof molecular evolution.     

Infection of brassica leaf protoplasts by turnip yellow mosaic virus

Summary Conditions optimal for the infection of Brassica leaf protoplasts by turnip yellow mosaic virus (TYMV) were found to be: pH 5.4–5.8 with citrate buffer; concentration of poly-L-ornithine, 0.8–1.0 g/ml; concentration of TYMV, 0.2–1.0 g/ml. More than 90% of Brassica rapa and B. sinensis protoplasts were infected under these conditions. TYMV replication in Brassica protoplasts was followed by quantitating the virus extracted from protoplasts and separated by sucrose density gradient centrifugation. Brassica protoplasts produced 1 to 2x10⁶ TYMV particles per cell within 48 hrs. Infection by TYMV induced the formation of polyplasts (aggregates of chloroplasts) in Brassica protoplasts. Polyplast formation paralleled the appearance of TYMV-specific immunofluorescence and could thus be used conveniently to determine the number of infected protoplasts. TYMV replication in Brassica protoplasts was resistant to actinomycin D and chloramphenicol, but was completely inhibited by cycloheximide.     

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.     

Effect of a Bacillus subtilis strain on flax protection against Fusarium oxysporum and its impact on the root and stem cell walls

In Normandy, flax is a plant of important economic interest because of its fibres. Fusarium oxysporum, a telluric fungus, is responsible for the major losses in crop yield and fibre quality. Several methods are currently used to limit the use of phytochemicals on crops. One of them is the use of plant growth promoting rhizobacteria (PGPR) occurring naturally in the rhizosphere. PGPR are known to act as local antagonists to soil‐borne pathogens and to enhance plant resistance by eliciting the induced systemic resistance (ISR). In this study, we first investigated the cell wall modifications occurring in roots and stems after inoculation with the fungus in two flax varieties. First, we showed that both varieties displayed different cell wall organization and that rapid modifications occurred in roots and stems after inoculation. Then, we demonstrated the efficiency of a Bacillus subtilis strain to limit Fusarium wilt on both varieties with a better efficiency for one of them. Finally, thermo‐gravimetry was used to highlight that B. subtilis induced modifications of the stem properties, supporting a reinforcement of the cell walls. Our findings suggest that the efficiency and the mode of action of the PGPR B. subtilis is likely to be flax variety dependent.     

Endoplasmic reticulum maintains ion homeostasis required for plasma membrane repair

Of the many crucial functions of the ER, homeostasis of physiological calcium increase is critical for signaling. Plasma membrane (PM) injury causes a pathological calcium influx. Here, we show that the ER helps clear this surge in cytoplasmic calcium through an ER-resident calcium pump, SERCA, and a calcium-activated ion channel, Anoctamin 5 (ANO5). SERCA imports calcium into the ER, and ANO5 supports this by maintaining electroneutrality of the ER lumen through anion import. Preventing either of these transporter activities causes cytosolic calcium overload and disrupts PM repair (PMR). ANO5 deficit in limb girdle muscular dystrophy 2L (LGMD2L) patient cells compromises their cytosolic and ER calcium homeostasis. By generating a mouse model of LGMD2L, we find that PM injury causes cytosolic calcium overload and compromises the ability of ANO5-deficient myofibers to repair. Addressing calcium overload in ANO5-deficient myofibers enables them to repair, supporting the requirement of the ER in calcium homeostasis in injured cells and facilitating PMR.