Molecular architecture of leishmania EF-1alpha reveals a novel site that may modulate protein translation: a possible target for drug development

Elongation factor-1alpha plays an essential role in eukaryotic protein biosynthesis. Recently, we have shown by protein structure modeling the presence of a hairpin-loop of 12 amino acids in mammalian EF-1alpha that is absent in the leishmania homologue [D. Nandan, A. Cherkasov, R. Sabouti, T. Yi, N.E. Reiner, Molecular cloning, biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue, Biochem. Biophys. Res. Commun. 302 (2003) 646-652]. As a consequence of this deletion, an exposed region is available on the main body of leishmania EF-1alpha. Here we report the generation of an anti-EF-1alpha antibody (DN-3) which bound selectively to the exposed region of leishmania EF-1alpha, with no reactivity with human EF-1alpha. In a leishmania cell-free protein translation system, DN-3 substantially inhibited protein translation. A similar inhibitory effect was observed when a specific peptide based on the exposed region was used in the cell-free protein translation assay. The application of structure-based in silico methods to identify potential ligands to target the exposed region identified a small molecule that selectively attenuated in vitro translation using leishmania extracts. Moreover, this small molecule showed selective suppressive effect on multiplication of leishmania in culture. Taken together, these findings identify a novel, exposed region in leishmania EF-1alpha that may be involved in protein synthesis and a potential site for drug targeting.     

A recurrent and localized dinoflagellate bloom in a Mediterranean beach

A recurrent, prolonged and singular bloom of Alexandrium tayloriBalech in an open beach (La Fosca, Spain, NW Mediterranean)is described. Alexandrium taylori appears at several placesalong a wide area of the NW Mediterranean (Costa Brava) duringthe summer, reaching concentrations up to 10⁵ cells l^–1,but it only proliferates persistently, massively (densities>10⁶ cells l^–1) and recurrently during August in LaFosca beach. The A.taylori bloom can be considered a manifestationof large-scale proliferation in a restricted area, where couplingbetween resting cysts in the sediment and bloom outbreak isnot a major factor compared to the interaction of local environmentalconditions with the planktonic organism's life history. Fromobservations of environmental conditions (the environmentalwindow) and the multiscale spatio-temporal distributions andlife history of A.taylori, we describe the bloom dynamics andanswer some critical questions about the different phases ofthe bloom. Some of these answers are: (i) the source of theA.taylori population is widespread offshore and is not locateddirectly at the beach; (ii) high cell densities are reachedand maintained with a moderate in situ growth and low loss rates;(iii) temporary cysts act as a reserve of the population.     

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.     

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes)

We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution. Received September 8, 1998; accepted November 23, 1998.     

Reproductive biology of Lactoris fernandeziana (Lactoridaceae)

Lactoris fernandeziana, monotypic in its family, is endemic to the cloud forests of Robinson Crusoe Island. Although there has been considerable study of the relationships of Lactoris, as a rare species and as a putative primitive paleoherb, little is known of its reproductive biology. Knowledge of the latter is essential for effective conservation programs. The species is gynomonoecious. The overall proportion of flowers is ∼1 female:1 hermaphrodite. The inconspicuous semipendulous green flowers, usually in mixed-gender inflorescences, do not produce rewards. Hermaphrodite flowers are herkogamous and protogynous. Pollen grains are shed from the extrorse anthers in permanent dry tetrads. There is a mean of 12879 tetrads per hermaphrodite flower. Both flower types bear an average of ∼18 ovules. The P/O (pollen/ovule) ratios imply facultative or obligate xenogamy, but hand pollinations show that Lactoris is self-compatible. No floral visitors were ever observed, but stigmata of open-pollinated flowers bore tetrads, and 64% of such styles had pollen tubes. Flowers enclosed in large mesh (1 mm) bags bore similar numbers of tetrads and pollen tubes. Thus, we conclude that Lactoris is anemophilous, a syndrome perhaps reflected by the P/O ratio. Low genetic diversity (isozymes and DNA) supports selfing and implies limited distance wind pollen dispersal. The small size of the island, the ± 1000 extant Lactoris plants, coupled with anemophily, self-compatibility, and pendant flower position, have yielded a geitonogamous system with high seed set and low genetic diversity. If inbreeding depression is expressed, it is in seed germination and seedling vigor, for Lactoris is very difficult to cultivate. For this species, effective conservation practices need to focus on habitat preservation and promotion of outcrossing.     

Lipoprotein alterations, abdominal fat distribution and breast cancer.

Plasma lipid profile and abdominal obesity have been associated with breast cancer risk, however published results have been inconsistent. To clarify these associations we studied lipid and lipoprotein alterations, obesity degree and body fat distribution, in 30 newly diagnosed breast cancer patients without treatment and 30 controls matched by age and menopausal status. Both pre and postmenopausal breast cancer patients presented higher body mass index, waist/hip ratio and insulin levels than their matched controls. An increase in triglycerides and a decrease in HDL-cholesterol, especially in the HDL2 subfraction, were observed in patients with breast cancer. Besides, HDL particle from these patients showed increased apo A1/HDL-cholesterol ratio. These alterations were correlated with waist/hip ratio. The association between lipoprotein alterations and abdominal obesity independent of menopausal status, in untreated newly diagnosed breast cancer patients is reported for the first time in this study.     

Pseudomonas syringae effector HopZ3 suppresses the bacterial AvrPto1–tomato PTO immune complex via acetylation

The plant pathogen Pseudomonas syringae secretes multiple effectors that modulate plant defenses. Some effectors trigger defenses due to specific recognition by plant immune complexes, whereas others can suppress the resulting immune responses. The HopZ3 effector of P. syringae pv. syringae B728a (PsyB728a) is an acetyltransferase that modifies not only components of plant immune complexes, but also the Psy effectors that activate these complexes. In Arabidopsis, HopZ3 acetylates the host RPM1 complex and the Psy effectors AvrRpm1 and AvrB3. This study focuses on the role of HopZ3 during tomato infection. In Psy-resistant tomato, the main immune complex includes PRF and PTO, a RIPK-family kinase that recognizes the AvrPto effector. HopZ3 acts as a virulence factor on tomato by suppressing AvrPto1_Psy-triggered immunity. HopZ3 acetylates AvrPto1_Psy and the host proteins PTO, SlRIPK and SlRIN4s. Biochemical reconstruction and site-directed mutagenesis experiments suggest that acetylation acts in multiple ways to suppress immune signaling in tomato. First, acetylation disrupts the critical AvrPto1_Psy-PTO interaction needed to initiate the immune response. Unmodified residues at the binding interface of both proteins and at other residues needed for binding are acetylated. Second, acetylation occurs at residues important for AvrPto1_Psy function but not for binding to PTO. Finally, acetylation reduces specific phosphorylations needed for promoting the immune-inducing activity of HopZ3’s targets such as AvrPto1_Psy and PTO. In some cases, acetylation competes with phosphorylation. HopZ3-mediated acetylation suppresses the kinase activity of SlRIPK and the phosphorylation of its SlRIN4 substrate previously implicated in PTO-signaling. Thus, HopZ3 disrupts the functions of multiple immune components and the effectors that trigger them, leading to increased susceptibility to infection. Finally, mass spectrometry used to map specific acetylated residues confirmed HopZ3’s unusual capacity to modify histidine in addition to serine, threonine and lysine residues.