Phosphorylation of ADF/cofilin abolishes EGF-induced actin nucleation at the leading edge and subsequent lamellipod extension

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, we inhibited ADF/cofilin's activity in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). We report here that expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation.     

Diversity of bacteria that nodulate Prosopis juliflora in the eastern area of Morocco

A total of 274 bacterial strains were isolated from the root nodules of Prosopis juliflora, growing in two arid soils of the eastern area of Morocco. A physiological plate screening allowed the selection of 15 strains that could tolerate NaCl concentrations between 175 and 500 mM. These were compared with 15 strains chosen from among the ones which did not tolerate high salinity. The diversity of strains was first assessed by rep-PCR amplification fingerprinting using BOXA1R and ERIC primers. An analysis of the PCR-amplified 16S rDNA gene digestion profiles using five endonucleases indicated the presence of different lineages among the taxa associated with P. juliflora nodules in the soils studied. Nucleotide sequencing of the small subunit rRNA gene and BLAST analysis showed that P. juliflora could host at least six bacterial species in this region and that the identity of those associated with high salt tolerance was clearly distinct from that of the salt-sensitive ones. Among the former, the first type displayed 99% similarity with different members of the genus Sinorhizobium, the second 97% similarity with species within the genus Rhizobium, while the third ribosomal type had 100% homology to Achromobacter xylosoxidans. Within the salt-sensitive isolates the prevailing type observed showed 98% similarity with Rhizobium multihospitium and R. tropici, a second type had 98% similarity to R. giardinii, and a further case displayed 97% colinearity with the Ensifer group including E. maghrebium and E. xericitae. All of the thirty strains encompassing these types re-nodulated P. juliflora in microbiologically controlled conditions and all of them were shown to possess a copy of the nodC gene. This is the first report detecting the betaproteobacterial genus Achromobacter as nodule-forming species for legumes. The observed variability in symbiont species and the abundance of nodulation-proficient strains is in line with the observation that the plant always appears to be nodulated and efficiently fixing nitrogen in spite of a wide range of soil and environmental conditions.     

Abscisic acid and sucrose increase the protein content in date palm somatic embryos,causing changes in 2-DE profile

Various supplements (abscisic acid (ABA) or sucrose) were added to the initial embryo culture medium (M3) with the aim of improving the vigour of vitroplants deriving from date palm somatic embryogenesis. ABA (20 and 40 μM) and sucrose (90 g/l) applied for 4 and 2 weeks respectively increased embryo thickness, with no apparent difference in length. ABA (5–40 μM) increased embryo proliferation rate. Somatic embryos maintained in modified M3 (M3 supplemented with ABA and an increased sucrose concentration) contained a higher amount of protein than those maintained in initial M3 (no ABA, 30 g/l of sucrose), with a 1.5–1.7-fold increase depending on the compound and concentration assayed. The 1-D and 2-DE protein profiles showed qualitative and quantitative differences between the somatic embryos cultured in initial M3 (control) and in modified M3. Statistical analysis of spot intensity was performed by principal component analysis, yielding two accurate groups of samples and determining the most discriminating spots. Samples were also clustered using Euclidean distance with an average linkage algorithm. Thirty-four variable spots were identified using mass spectrometry analysis. Identified proteins were classified into the following functional categories: energy metabolism (five proteins); protein translation, folding and degradation (9); redox maintenance (5); cytoskeleton (3); storage protein (2); and with no assigned function as (10). While “up-regulation” of stress-related proteins and “down-regulation” of energy metabolism proteins were observed in somatic embryos matured in M3 supplemented with ABA, storage proteins (legumin) were “up-regulated” in somatic embryos matured in M3 supplemented with increased sucrose.     

Chemical Composition and Biological Screening of the Essential Oils of Micromeria macrosiphon and M. arganietorum (Lamiaceae)

The chemical composition and in vitro biological activities of the essential oil (EO) of Micromeria macrosiphon Coss. and M. arganietorum (J. Emb.) R. Morales, two Lamiaceae endemic to south Morocco, were investigated. GC/MS analysis resulted in the identification of 36 metabolites from the EO of M. macrosiphon, 45 from M. arganietorum. Borneol was the major metabolite in both oils and together with related derivatives such as camphor, accounted for 2/3 of the EO of M. macrosiphon, 1/3 of those of M. arganietorum. Pinene and terpinene derivatives were also present in high proportions. From a chemotaxonomic point of view, the composition of the examined samples may be related to those of other species endemic to Macaronesia. Both EOs showed significant toxicity towards liver HepG2 and melanoma B16 4A5 tumor cell lines at 100 μg/mL; however, they were also cytotoxic towards S17 normal cell lines, with a selectivity index <1. No antibacterial activity was noticed against 52 strains at 100 μg/mL.     

Foliar application of the triterpene derivative 24-methylen-elemo-lanosta-8,24-dien-3-one alleviates salt toxicity in grapevine

This work focused on the effect triterpene derivative 24-methylen-elemo-lanosta-8,24-dien-3-one (F3) on the induction of salt stress tolerance of the Moroccan grapevine cv. “Doukkali”. Hardwood cuttings of the grapevine from a homogeneous plant material collected in the field were grown in hydroponic medium under different salt concentrations and treated with 50 or 100 µg ml^?1 of F3. Salt stress affected several physiological and biochemical parameters including relative water content, chlorophyll a and b content, peroxidase, and polyphenol oxidase activities, which decreased along with time. Meanwhile, proline, proteins, soluble sugars, H₂O₂, and carotenoid content, as well as phenolic compound content increased, suggesting an evidence of tolerance of this local variety to salinity. An exogenous supply of the triterpenic product increased all these parameters under normal conditions. In addition, F3 at low dose was found to be successful in lowering Na⁺ content and alleviating the inhibitory effects of salt stress on relative water content as well as on chlorophyll a and b.     

Phenotypic and genotypic characterization of rhizobia associated with Acacia saligna (Labill.) Wendl. in nurseries from Algeria

Twenty seven rhizobial strains associated with Acacia saligna grown in northern and southern Algeria were characterized, including generation time, host-range, the 16S rRNA gene and 16S–23S rRNA intergenic spacer restriction patterns, 16S rRNA gene sequence analysis and tolerance to salinity and drought. Cross inoculation tests indicated that 11 slow-growing isolates from northern nurseries were able to nodulate introduced Australian acacias exclusively, whereas 16 fast-growing isolates, mainly from southern nurseries, were capable of also nodulating native acacias. Restriction patterns and sequence analysis of the 16S rRNA gene showed that strains of the first group belonged to Bradyrhizobium while strains of the second group were related to Sinorhizobium meliloti and Rhizobium gallicum. Interestingly, five strains of the first group formed a distinct cluster phylogenetically close to Bradyrhizobium betae, a non-nodulating species causing tumour-like deformations in sugar beet roots. Bradyrhizobium strains were in general more sensitive to NaCl and PEG than the S. meliloti and R. gallicum representatives. Among the latter, strains S. meliloti BEC1 and R. gallicum DJA2 were able to tolerate up to 1 M NaCl and 20% PEG. This, together with their wide host-range among Acacia species, make them good candidates for developing inoculants for A. saligna and other acacia trees growing in arid areas.     

Influence of carbon source on growth and mycotoxin production by isolates of Pyrenophora tritici-repentis from wheat

The fungus Pyrenophora tritici-repentis can infect wheat kernels, causing red smudge, and has been shown to produce the anthraquinone mycotoxins emodin, catenarin, and islandicin. The growth of 8 fungal isolates from diverse regions was evaluated on various culture media and was found to be generally slowest on the semisynthetic Fries medium. The choice of carbon source had a significant effect on mycotoxin production, as assessed by high-performance liquid chromatography. The highest emodin concentration (194.18 ± 16.26 μg/g medium) was observed for isolate Alg 3-24 on Fries medium supplemented with fructose, while the highest catenarin concentration (302.54 ±13.92 μg/g medium) was observed for TS93-71B on Fries medium supplemented with starch. Islandicin was not produced by any isolate under the conditions tested. Evaluation of the dynamics of mycotoxin production by isolate 331-2 on V8-potato dextrose agar medium revealed a rapid accumulation of emodin and catenarin during the first week of incubation, followed by a large decline by 14 days. Differences in the growth of and mycotoxin production by isolates of P. tritici-repentis likely resulted from the differential composition of the media and (or) intraspecies variability. Accordingly, the optimization of growth medium should be considered when evaluating the potential of specific isolates for mycotoxin production.     

Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.     

Mechanism of activation of methyltransferases involved in translation by the Trm112 'hub' protein

Methylation is a common modification encountered in DNA, RNA and proteins. It plays a central role in gene expression, protein function and mRNA translation. Prokaryotic and eukaryotic class I translation termination factors are methylated on the glutamine of the essential and universally conserved GGQ motif, in line with an important cellular role. In eukaryotes, this modification is performed by the Mtq2-Trm112 holoenzyme. Trm112 activates not only the Mtq2 catalytic subunit but also two other tRNA methyltransferases (Trm9 and Trm11). To understand the molecular mechanisms underlying methyltransferase activation by Trm112, we have determined the 3D structure of the Mtq2-Trm112 complex and mapped its active site. Using site-directed mutagenesis and in vivo functional experiments, we show that this structure can also serve as a model for the Trm9-Trm112 complex, supporting our hypothesis that Trm112 uses a common strategy to activate these three methyltransferases.