Mapping of Drebrin Binding Site on F-Actin

Drebrin is a filament-binding protein involved in organizing the dendritic pool of actin. Previous in vivo studies identified the actin-binding domain of drebrin (DrABD), which causes the same rearrangements in the cytoskeleton as the full-length protein. Site-directed mutagenesis, electron microscopic reconstruction, and chemical cross-linking combined with mass spectrometry analysis were employed here to map the DrABD binding interface on actin filaments. DrABD could be simultaneously attached to two adjacent actin protomers using the combination of 2-iminothiolane (Traut's reagent) and MTS1 [1,1-methanediyl bis(methanethiosulfonate)]. Site-directed mutagenesis combined with chemical cross-linking revealed that residue 238 of DrABD is located within 5.4 Å from C374 of actin protomer 1 and that native cysteine 308 of drebrin is near C374 of actin protomer 2. Mass spectrometry analysis revealed that a zero-length cross-linker, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, can link the N-terminal G-S extension of the recombinant DrABD to E99 and/or E100 on actin. Efficient cross-linking of drebrin residues 238, 248, 252, 270, and 271 to actin residue 51 was achieved with reagents of different lengths (5.4-19 Å). These results suggest that the “core” DrABD is centered on actin subdomain 2 and may adopt a folded conformation upon binding to F-actin. The results of electron microscopic reconstruction, which are in a good agreement with the cross-linking data, revealed polymorphism in DrABD binding to F-actin and suggested the existence of two binding sites. These results provide new structural insight into the previously observed competition between drebrin and several other F-actin-binding proteins.     

Tight binding of NADPH to the 39-kDa subunit of complex I is not required for catalytic activity but stabilizes the multiprotein complex

In addition to the 14 central subunits, respiratory chain complex I from the aerobic yeast Yarrowia lipolytica contains at least 24 accessory subunits, most of which are poorly characterized. Here we investigated the role of the accessory 39-kDa subunit which belongs to the heterogeneous short-chain dehydrogenase/reductase (SDR) enzyme family and contains non-covalently bound NADPH. Deleting the chromosomal copy of the gene that codes for the 39-kDa subunit drastically impaired complex I assembly in Y. lipolytica. We introduced several site-directed mutations into the nucleotide binding motif that severely reduced NADPH binding. This effect was most pronounced when the arginine at the end of the second β-strand of the NADPH binding Rossman fold was replaced by leucine or aspartate. Mutations affecting nucleotide binding had only minor or moderate effects on specific catalytic activity in mitochondrial membranes but clearly destabilized complex I. One mutant exhibited a temperature sensitive phenotype and significant amounts of three different subcomplexes were observed even at more permissive temperature. We concluded that the 39-kDa subunit of Y. lipolytica plays a critical role in complex I assembly and stability and that the bound NADPH serves to stabilize the subunit and complex I as a whole rather than serving a catalytic function.     

Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins

To obtain antibodies suitable to be used in an antigen-capture assay, we have identified, synthesized, and evaluated a series of peptides from different Plasmodium falciparum excretory-secretory proteins: glutamate-rich protein (GLURP); histidine-rich protein 2; histidine-rich protein 3; Falciparum interspersed repeat antigen and, serine-rich antigen homologous. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in rabbits. Out of the 14 peptide constructs, eight by ELISA and, six by MABA elicited antibodies that showed correspondence between the predictive study and the immunogenicity obtained in rabbits. All antipeptide (GLURP, HRP2, and FIRA) antisera were found to bind to the corresponding synthetic sequence in an ELISA assay. The binding activity and specificity of antibodies were determined by Western blot with supernatant culture from P. falciparum. Anti-GLURP (IMT-94 and IMT-200) antisera bound to five molecules present in supernatant with molecular weight of 73, 82, 116, 124, and 128 kDa. Anti-HRP2 (IMT-192) antisera recognized a band of 58 kDa. In both cases, the specific molecules were inhibited by preincubation with the homologous peptide. Anti-HRP3, anti-FIRA neither anti-SERPH antisera showed reactivity. Anti-peptides HRP2 antibodies recognized the recombinant protein present in Parasight-F test. The same way, synthetic peptides from HRPII molecule were recognized by monoclonal antibody present in the Parasight-F assay. Our results confirm the potential value of synthetic peptides when inducing monospecific polyclonal antibodies for the development of diagnostic tests based on the capture of antigens.     

Phylogenetic analysis of Culicoides species from France based on nuclear ITS1-rDNA sequences

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) play important roles in the transmission of viral diseases affecting wild and domestic ruminants and horses, including Bluetongue (BT) and African horse sickness (AHS) respectively. In southern Europe, BT has been largely transmitted by the classical Afro-Asian vector Culicoides imicola Kieffer. However, other species such as C. obsoletus Meigen, C. scoticus Downs & Kettle and C. pulicaris Linné may also be involved in BTV transmission. As a consequence of the discovery of C. imicola followed by BTV-2 outbreaks on the island of Corsica in October 2000, further studies on these biting midges have been carried out. To better characterize the evolution and phylogenetic relations of Culicoides, molecular analysis in parallel with a morphology-based taxonomic approach were performed. Phylogenetic analyses of French Culicoides species were undertaken using the ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) as a molecular target. This region was shown to be useful in understanding evolutionary and genetic relationships between species. Construction of several trees showed that molecular phylogeny within the genus Culicoides correlates not only with morphological-based taxonomy but also with ecological patterns.     

Ricerche Sulla Vegetazione dei Dintorni di Firenze

Riassunto

L'A. espone in questo lavoro i risultati di ricerche sistematiche e fitogeografiche sulla vegetazione di Monte Ferrato (presso Prato in Toscana), piccolo gruppo di colline costituite da rocce ofiolitiche (serpentino, eufotide ecc.), Dopo averne elencata la florula, dove riporta anche le diagnosi di alcune forme nuove, l'A. prende in esame la vegetazione dei principali tipi di stazione, che vi ha potuto riconoscere, in base a criteri fisionomici e statistici. Considera poi le caratteristiche morfologiche delle specie pi[ugrave] significative del serpentino, che raggruppa in cinque tipi di speciali morfosi. Sulla base dell'analisi della distribuzione geografica di alcune notevoli entità che si presentano a M. Ferrato in stazione disgiunta rispetto al loro areale, dello studio geografico e sistematico degli endemismi, e in genere poi di tutte le specie pi[ugrave] significative di questa Flora, l'A. ne discute il valore fitogeografico e climatico, rilevando e discutendo la convivenza di specie di diverso valore fitogeografico, in rapporta alle particolari condizioni microstazionali.     

Rhomboid proteins in the chloroplast envelope affect the level of allene oxide synthase in Arabidopsis thaliana

Rhomboids are intra‐membrane serine proteases whose sequences are found in nearly all organisms. They are involved in a variety of biological functions in both eukaryotes and prokaryotes. Localization assays revealed that two Arabidopsis thaliana rhomboid‐like proteases (AtRBL), AtRBL8 and AtRBL9, are targeted to the chloroplast. Using transgenic plants expressing epitope‐tagged AtRBL9, we localized AtRBL9 to the chloroplast inner envelope membrane, with both its N‐ and C‐termini facing the stroma. Mass spectrometry analyses confirmed this localization, and suggested that this is also the case for AtRBL8. Both are proteins of very low abundance. The results of size‐exclusion chromatography implied that AtRBL9 forms homo‐oligomers. In search of a putative function, a comparative proteomic analysis was performed on wild‐type and double‐knockout plants, lacking both AtRBL8 and AtRBL9, using the iTRAQ method. Of 180 envelope proteins, the level of only a few was either increased or decreased in the mutant line. One of the latter, allene oxide synthase, is involved in jasmonic acid biosynthesis. This observation provides an explanation for the recently reported aberration in flower morphology that is associated with the loss of AtRBL8.     

Mycorrhizal symbiosis affected by different genotypes of Pinus pinaster

Background and aims

Higher growth rate and morphological traits have been the major criteria for selecting trees in breeding programs. The symbiotic associations between P. pinaster and ectomycorrhizal fungi can be an effective approach to enhance plant development. The aim of this work was to assess whether the establishment of mycorrhizal symbiosis at nursery stage was affected by tree breeding.

Methods

Seeds of P. pinaster from a clonal population, designed to select for various traits, and from neighboring wild plants were inoculated with compatible ectomycorrhizal fungi: Suillus bovinus, Pisolithus tinctorius or Rhizopogon roseolus, and grown in individual cells containing forest soil, in a commercial forest nursery. Growth and nutritional traits, colonisation parameters and the fungal community established were assessed.

Results

R. roseolus and P. tinctorius were the most efficient isolates in promoting plant development. Inoculated selected saplings had an overall superior development than their wild counterparts, with up to a 4.9-fold in root dry weight and a 13.6-fold increase in the total number of ectomycorrhizal root tips. Differences in fungal community were revealed through the denaturing gradient gel electrophoresis profile of each treatment.

Conclusions

The results from our study suggest that the selected genotype benefits more from the mycorrhizal association and therefore this could be a valuable biotechnological tool for the nursery production of P. pinaster.     

Bathymonorchis polyipni (Reimer, 1985) n. g., n. comb. (Digenea: Monorchiidae) from bathypelagic fishes of the eastern mid-Atlantic Ocean

The new genus Bathymonorchis is defined and differentiated from the genera Monorchis and Allolasiotocus. B. polyipni (Reimer) n. comb., previously attributed to Monorchis, is described from Neoscopelus macrolepidotus and Polymetme corythaeola from the Atlantic off the coast of NW Africa. The new genus is characterised by its single vitelline field, multilobate ovary, spinous blind portion of Looss' organ, lack of external seminal vesicle, testicular position and uterine configuration.