Morphologic and Aerodynamic Considerations Regarding the Plumed Seeds of Tragopogon pratensis and Their Implications for Seed Dispersal

Tragopogon pratensis is a small herbaceous plant that uses wind as the dispersal vector for its seeds. The seeds are attached to parachutes that increase the aerodynamic drag force and increase the total distance travelled. Our hypothesis is that evolution has carefully tuned the air permeability of the seeds to operate in the most convenient fluid dynamic regime. To achieve final permeability, the primary and secondary fibres of the pappus have evolved with complex weaving; this maximises the drag force (i.e., the drag coefficient), and the pappus operates in an “optimal” state. We used computational fluid dynamics (CFD) simulations to compute the seed drag coefficient and compare it with data obtained from drop experiments. The permeability of the parachute was estimated from microscope images. Our simulations reveal three flow regimes in which the parachute can operate according to its permeability. These flow regimes impact the stability of the parachute and its drag coefficient. From the permeability measurements and drop experiments, we show how the seeds operate very close to the optimal case. The porosity of the textile appears to be an appropriate solution to achieve a lightweight structure that allows a low terminal velocity, a stable flight and a very efficient parachute for the velocity at which it operates.     

Endogenous biotin‐binding proteins: an overlooked factor causing false positives in streptavidin‐based protein detection

Biotinylation is widely used in DNA, RNA and protein probing assays as this molecule has generally no impact on the biological activity of its substrate. During the streptavidin‐based detection of glycoproteins in Lactobacillus rhamnosus GG with biotinylated lectin probes, a strong positive band of approximately 125 kDa was observed, present in different cellular fractions. This potential glycoprotein reacted heavily with concanavalin A (ConA), a lectin that specifically binds glucose and mannose residues. Surprisingly, this protein of 125 kDa could not be purified using a ConA affinity column. Edman degradation of the protein, isolated via cation and anion exchange chromatography, lead to the identification of the band as pyruvate carboxylase, an enzyme of 125 kDa that binds biotin as a cofactor. Detection using only the streptavidin conjugate resulted in more false positive signals of proteins, also in extracellular fractions, indicating biotin‐associated proteins. Indeed, biotin is a known cofactor of numerous carboxylases. The potential occurence of false positive bands with biotinylated protein probes should thus be considered when using streptavidin‐based detection, e.g. by developing a blot using only the streptavidin conjugate. To circumvent these false positives, alternative approaches like detection based on digoxigenin labelling can also be used.     

Primula spectabilis Tratt. aerial parts: morphology, volatile compounds and flavonoids

The vacuolar and epicuticular flavonoids and the volatiles of the leaves and parts of flower of P. spectabilis Tratt., an endemic species in the Italian Oriental Alps, were investigated. From a MeOH extract of the leaves two flavone glycosides, 8-C-β-glucopyranosylluteolin 7-O-α-arabinofuranoside (1) and 6-C-α-arabinofuranosylapigenin (2) were isolated, in addition to a flavone and three flavonols already known from species of Primula. From an EtOH extract of leaf exudates, 7,3',4'-tri-O-methylquercetin was obtained. The structures were elucidated on the basis of their 1D 1H- and 13C NMR data and 2D NMR techniques, as well as of HPLC-MS. The volatiles emitted by the leaves were mainly constituted by non-terpene derivatives, followed by comparable proportions of hemiterpens, oxygenated monoterpenes and sesquiterpene hydrocarbons. In flowers, monoterpene hydrocarbons were the most represented chemical class followed by non-terpene derivatives. Different proportions of compounds were found when individual parts of flowers were examined separately; calyx produced a greater proportion (approx. 49.5%) of non-terpenes as its volatile metabolites. P. spectabilis has glandular trichomes in the hyaline margins of the epidermal depressions, distributed on the adaxial leaf blade. Glandular hairs were also present on the corolla. Correlations of phytochemical data with the morphological features of leaf, flower and glandular hair are discussed, and a hypothesis is proposed on the ecological roles of the flavonoids and volatile compounds on the general fitness of the species and cross-pollination strategies.     

Evidence for coupled biogenesis of yeast Gap1 permease and sphingolipids: essential role in transport activity and normal control by ubiquitination

Current models for plasma membrane organization integrate the emerging concepts that membrane proteins tightly associate with surrounding lipids and that biogenesis of surface proteins and lipids may be coupled. We show here that the yeast general amino acid permease Gap1 synthesized in the absence of sphingolipid (SL) biosynthesis is delivered to the cell surface but undergoes rapid and unregulated down-regulation. Furthermore, the permease produced under these conditions but blocked at the cell surface is inactive, soluble in detergent, and more sensitive to proteases. We also show that SL biogenesis is crucial during Gap1 production and secretion but that it is dispensable once Gap1 has reached the plasma membrane. Moreover, the defects displayed by cell surface Gap1 neosynthesized in the absence of SL biosynthesis are not compensated by subsequent restoration of SL production. Finally, we show that down-regulation of Gap1 caused by lack of SL biogenesis involves the ubiquitination of the protein on lysines normally not accessible to ubiquitination and close to the membrane. We propose that coupled biogenesis of Gap1 and SLs would create an SL microenvironment essential to the normal conformation, function, and control of ubiquitination of the permease.     

Chemical composition and intraspecific variability of the essential oils of five populations of Hypericum triquetrifolium Turra growing in North Tunisia

The chemical composition of the essential oils of five populations of Hypericum triquetrifolium Turra from Tunisia and their intraspecific variability were analyzed in detail by GC/MS. One hundred seventy-four compounds were identified, representing averages of 87.9 to 98.7% of the oil composition. The components are represented here by homologous series of monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpenes hydrocarbons, oxygenated sesquiterpenes, non-terpenic hydrocarbons, and others. Sesquiterpene hydrocarbons were the most abundant chemical compounds. Multivariate chemometric techniques, such as cluster analysis (CA) and principal-component analysis (PCA), were used to characterize the samples according to the geographical origin. By statistical analysis, the analyzed populations were classified into four chemotype groups.     

P130Cas-associated protein (p140Cap) as a new tyrosine-phosphorylated protein involved in cell spreading

Integrin-mediated cell adhesion stimulates a cascade of signaling pathways that control cell proliferation, migration, and survival, mostly through tyrosine phosphorylation of signaling molecules. p130Cas, originally identified as a major substrate of v-Src, is a scaffold molecule that interacts with several proteins and mediates multiple cellular events after cell adhesion and mitogen treatment. Here, we describe a novel p130Cas-associated protein named p140Cap (Cas-associated protein) as a new tyrosine phosphorylated molecule involved in integrin- and epidermal growth factor (EGF)-dependent signaling. By affinity chromatography of human ECV304 cell extracts on a MBP-p130Cas column followed by mass spectrometry matrix-assisted laser desorption ionization/time of flight analysis, we identified p140Cap as a protein migrating at 140 kDa. We detected its expression in human, mouse, and rat cells and in different mouse tissues. Endogenous and transfected p140Cap proteins coimmunoprecipitate with p130Cas in ECV304 and in human embryonic kidney 293 cells and associate with p130Cas through their carboxy-terminal region. By immunofluorescence analysis, we demonstrated that in ECV304 cells plated on fibronectin, the endogenous p140Cap colocalizes with p130Cas in the perinuclear region as well as in lamellipodia. In addition p140Cap codistributes with cortical actin and actin stress fibers but not with focal adhesions. We also show that p140Cap is tyrosine phosphorylated within 15 min of cell adhesion to integrin ligands. p140Cap tyrosine phosphorylation is also induced in response to EGF through an EGF receptor dependent-mechanism. Interestingly expression of p140Cap in NIH3T3 and in ECV304 cells delays the onset of cell spreading in the early phases of cell adhesion to fibronectin. Therefore, p140Cap is a novel protein associated with p130Cas and actin cytoskeletal structures. Its tyrosine phosphorylation by integrin-mediated adhesion and EGF stimulation and its involvement in cell spreading on matrix proteins suggest that p140Cap plays a role in controlling actin cytoskeleton organization in response to adhesive and growth factor signaling.     

Apple 1-aminocyclopropane-1-carboxylate synthase in complex with the inhibitor L-aminoethoxyvinylglycine. Evidence for a ketimine intermediate

The 1.6-A crystal structure of the covalent ketimine complex of apple 1-aminocyclopropane-1-carboxylate (ACC) synthase with the potent inhibitor l-aminoethoxyvinylglycine (AVG) is described. ACC synthase catalyzes the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. AVG is widely used in plant physiology studies to inhibit the activity of ACC synthase. The structural assignment is supported by the fact that the complex absorbs maximally at 341 nm. These results are not in accord with the recently reported crystal structure of the tomato ACC synthase AVG complex, which claims that the inhibitor only associates noncovalently. The rate constant for the association of AVG with apple ACC synthase was determined by stopped-flow spectrophotometry (2.1 x 10(5) m(-1) s(-1)) and by the rate of loss of enzyme activity (1.1 x 10(5) m(-1) s(-1)). The dissociation rate constant determined by activity recovery is 2.4 x 10(-6) s(-1). Thus, the calculated K(d) value is 10-20 pm.