Antialgal ent-labdane diterpenes from Ruppia maritima

Seven ent-labdane diterpenes have been isolated from Ruppia maritima. The structures 15,16-epoxy-ent-labda-8(17),13(16),14-trien-19-al; 15,16-epoxy-ent-labda-8(17),13(16),14-trien-19-ol acetate; methyl 15,16-epoxy-12-oxo-ent-labda-8(17),13(16),14-trien-19-oate; 15,16-epoxy-ent-labd-8(17),13E-dien-15-ol and 13-oxo-15,16-bis-nor-ent-labd-8(17)-ene have been assigned to the five new compounds by spectroscopic means and chemical correlations. The phytotoxicity of the diterpenes has been assessed using the alga Selenastrum capricornutum as organism test.     

Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum

Dictyoglomus turgidum is a hyperthermophilic, anaerobic, gram-negative bacterium that shows an array of putative glycoside hydrolases (GHs) encoded by its genome, a feature that makes this microorganism very interesting for biotechnological applications. The aim of this work is the characterization of a hyperthermophilic GH5, Dtur_0671, of D. turgidum, annotated as endoglucanase and herein named DturCelB in agreement to DturCelA, which was previously characterized. The synthetic gene was expressed in Escherichia coli. The purified recombinant enzyme is active as a monomer (40 kDa) and CD structural studies showed a conserved α/β structure at different temperatures (25 and 70 °C) and high thermoresistance (Tm of 88 °C). Interestingly, the enzyme showed high endo-β-1,4-mannanase activity vs various mannans, but low endo-β-1,4 glucanase activity towards carboxymethylcellulose. The K _M and V _max of DturCelB were determined for both glucomannan and CMC: they were 4.70 mg/ml and 473.1 μmol/min mg and 1.83 mg/ml and 1.349 μmol/min mg, respectively. Its optimal activity towards temperature and pH resulted to be 70 °C and pH 5.4, respectively. Further characterization highlighted good thermal stability (~ 50% of enzymatic activity after 2 h at 70 °C) and pH stability over a broad range (> 90% of activity after 1 h in buffer, ranging pH 5–9); resistance to chemicals was also observed.

     

A question of time: the land snail Murella muralis (Gastropoda: Pulmonata) reveals constraints on past ecological speciation

The lively debate about speciation currently focuses on the relative importance of factors driving population differentiation. While many studies are increasingly producing results on the importance of selection, little is known about the interaction between drift and selection. Moreover, there is still little knowledge on the spatial‐temporal scales at which speciation occurs, that is, arrangement of habitat patches, abruptness of habitat transitions, climate and habitat changes interacting with selective forces. To investigate these questions, we quantified variation on a fine geographical scale analysing morphological (shell) and genetic data sets coupled with environmental data in the land snail Murella muralis, endemic to the Mediterranean island of Sicily. Analysis of a fragment of the mitochondrial DNA cytochrome oxidase I gene (COI) and eight nuclear microsatellite loci showed that genetic variation is highly structured at a very fine spatial scale by local palaeogeographical events and historical population dynamics. Molecular clock estimates, calibrated here specifically for Tyrrhenian land snails, provided a framework of palaeogeographical events responsible for the observed geographical variations and migration routes. Finally, we showed for the first time well‐documented lines of evidence of selection in the past, which explains divergence of land snail shell shapes. We suggest that time and palaeogeographical history acted as constraints in the progress along the ecological speciation continuum. Our study shows that testing for correlation among palaeogeography, morphology and genetic data on a fine geographical scale provides information fundamental for a detailed understanding of ecological speciation processes.     

Singular value decomposition analysis of the torsional angles of dopamine reuptake inhibitor GBR 12909 analogs: effect of force field and charges

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis of large, flexible molecules, such as the dopamine reuptake inhibitor GBR 12909 (1), is complicated by the fact that they can take on a wide range of closely-related conformations. The first step in the analysis is to classify the conformers into groups. Over 600 conformers each of a piperazine (2) and piperidine (3) analog of 1 were generated by random search conformational analysis using the Merck Molecular Force Field (MMFF94). Singular value decomposition (SVD) was used to group the conformers of 2 and 3 by the similarity of their non-ring torsional angles. SVD uncovered subtle differences in their conformer populations due to that fact that the conformers separate along different principal components, and ultimately to the fact that different torsional angles are the chief contributors to these components. The results were compared to our previous SVD analysis (Fiorentino, et al., Journal of Computational Chemistry, 2006, 27, 609-620) of conformer populations of 2 and 3 generated by the Tripos force field and Gasteiger-Hückel charges. Except for the dominant contribution of angle B3 to principal component 8 seen with both force fields, the angles which are chiefly responsible for the grouping of the conformers of 2 and 3 are different with both force fields. This illustrates that SVD is useful in identifying unique groupings of conformers in large data sets of flexible molecules—a first step in selecting representative conformers for 3D-QSAR modeling studies.     

Musculotendon variability influences tissue strains experienced by the biceps femoris long head muscle during high-speed running

The hamstring muscles frequently suffer injury during high-speed running, though the factors that make an individual more susceptible to injury remain poorly understood. The goals of this study were to measure the musculotendon dimensions of the biceps femoris long head (BFlh) muscle, the hamstring muscle injured most often, and to use computational models to assess the influence of variability in the BFlh’s dimensions on internal tissue strains during high-speed running. High-resolution magnetic resonance (MR) images were acquired over the thigh in 12 collegiate athletes, and musculotendon dimensions were measured in the proximal free tendon/aponeurosis, muscle and distal free tendon/aponeurosis. Finite element meshes were generated based on the average, standard deviation and range of BFlh dimensions. Simulation boundary conditions were defined to match muscle activation and musculotendon length change in the BFlh during high-speed running. Muscle and connective tissue dimensions were found to vary between subjects, with a coefficient of variation (CV) of 17±6% across all dimensions. For all simulations peak local strain was highest along the proximal myotendinous junction, which is where injury typically occurs. Model variations showed that peak local tissue strain increased as the proximal aponeurosis width narrowed and the muscle width widened. The aponeurosis width and muscle width variation models showed that the relative dimensions of these structures influence internal muscle tissue strains. The results of this study indicate that a musculotendon unit’s architecture influences its strain injury susceptibility during high-speed running.     

Continuous measurement of galactolipid hydrolysis by pancreatic lipolytic enzymes using the pH-stat technique and a medium chain monogalactosyl diglyceride as substrate

Galactolipids are the main lipids from plants and galactolipases play a major role in their metabolism. These enzymes were however poorly studied so far and only few assays have been developed. A specific and continuous galactolipase assay using synthetic medium chain monogalactosyl diacylglycerol (MGDG) as substrate was developed using the pH-stat technique and recombinant human (rHPLRP2) and guinea pig (rGPLRP2) pancreatic lipase-related protein 2 as model enzymes. PLRP2s are the main enzymes involved in the digestion of galactolipids in the gastrointestinal tract. Monogalactosyl di-octanoylglycerol was mixed with bile salt solutions by sonication to form a micellar substrate before launching the assay. The nature of the bile salt and the bile salt to MGDG ratio were found to significantly affect the rate of MGDG hydrolysis by rHPLRP2 and rGPLRP2. The maximum galactolipase activity of both enzymes was recorded with sodium deoxycholate (NaDC) and at a NaDC to MGDG ratio of 1.33 and at basic pH values (8.0–9.0). The maximum rates of hydrolysis were obtained using a MGDG concentration of 10^− 2 M and calcium chloride was found to be not necessary to obtain the maximum of activity. Under these conditions, the maximum turnovers of rGPLRP2 and rHPLRP2 on mixed NaDC/MGDG micelles were found to be 8000 ± 500 and 2800 ± 60 μmol/min/mg (U/mg), respectively. These activities are in the same order of magnitude as the activities on triglycerides of lipases and they are the highest specific activities ever reported for galactolipases. For the sake of comparison, the hydrolysis of mixed bile salt/MGDG micelles was also tested using other pancreatic lipolytic enzymes and only native and recombinant human carboxyl ester hydrolase were found to display significant but lower activities (240 ± 17 and 432 ± 62 U/mg, respectively) on MGDG.