Enzymatic synthesis of butyl hydroxycinnamates and their inhibitory effects on LDL-oxidation

The potential of the Aspergillus niger type A feruloyl esterase (AnFaeA) for the synthesis of various phenolic acid esters was examined using a ternary-organic reaction system consisting of a mixture of n-hexane, 1- or 2-butanol and water. Reaction parameters including the type of methyl hydroxycinnamate, the composition of the reaction media, the temperature, and the substrate concentration were investigated to evaluate their effect on initial rate and conversion to butyl esters of sinapic acids. Optimisation of the reaction parameters lead to 78% and 9% yield for the synthesis of 1-butyl and 2-butyl sinapate, respectively. For the first time, a feruloyl esterase was introduced in the reaction system as cross-linked enzyme aggregates (CLEAs), after optimisation of the immobilisation procedure, allowing the recycling and reuse of the biocatalyst. The inhibition of copper-induced LDL oxidation by hydroxycinnamic acids and their corresponding butyl esters was investigated in vitro. Kinetic analysis of the antioxidation process demonstrates that sinapate derivatives are effective antioxidants indicating that esterification increases the free acid's antioxidant activity especially on dimethoxylated compounds such as sinapic acid compared to methoxy-hydroxy-compounds such as ferulic acid.     

Genetic structure of Pinus brutia stands exposed to wild fires

This study investigates the genetic structure of brutia pine (Pinus brutia Ten.) stands that were exposed to wild fires. A systematic investigation within the species distribution in Greece first identified areas of frequent wild forest fires and then located stands that had experienced ground fires about 20 years ago. In these stands it was possible to sample the pre-fire population that had survived the ground fire event (“mature” population) as well as the post-fire population that has reached reproductive capacity (“young” population). Gel electrophoresis was used in order to study isoenzyme variability in four such populations present in two sites (Kourteri, Lesvos island and Mytilineoi, Samos island). Results indicated absence of notable differences in genetic diversity among the remnant mature populations and the young populations after regeneration. No significant inbreeding was detected and genetic identity among young and mature stands was high. However, some differences were observed in the frequencies of rare alleles and in the presence of interspecific (P. brutia x halepensis) hybrids in the young populations. This paper discusses the above results in the context of the evolution of brutia pine and the maintenance of its genetic variation.     

Sub-arctic Populations of European Lobster, Homarus gammarus, in Northern Norway

The European lobster is distributed throughout the south and western regions of the Norwegian coast. A previous lobster allozyme investigation (1993) in the Tysfjord region, north of the Arctic Circle demonstrated that the lobster population from this region was genetically different from lobster samples collected in other parts of Norway. More detailed investigation including supplementary extensive sampling and additional allozyme, microsatellite and mtDNA analyses are reported here. This investigation supports the genetic distinctness of the Tysfjord population and shows that this is mainly due to a reduction (60–70%) in gene diversity (observed heterozygosities and number of alleles) compared with lobsters from more southern regions. In addition to the Tysfjord region, the comprehensive sampling also included lobsters found in the adjacent Nordfolda fjord system. Genetic analyses provided evidence for significant differences between the lobster populations of Tysfjord and Nordfolda, even though they are separated by a coastal distance of only 142 km. The two populations were also different with regards to several biological characteristics such as body size. The genetic difference between these two geographically close populations is likely to be due to the local hydrological conditions, preventing larval dispersal between the fjord systems. Assessment of lobster abundance in the north-west region suggests that the sub-arctic lobster populations are geographically isolated.     

Clostridium carboxidivorans Strain P7T Recombinant Formate Dehydrogenase Catalyzes Reduction of CO2 to Formate

Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7^T, expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions.     

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus

The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-beta-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.     

High apparent dielectric constant inside a protein reflects structural reorganization coupled to the ionization of an internal Asp

The dielectric properties of proteins are poorly understood and difficult to describe quantitatively. This limits the accuracy of methods for structure-based calculation of electrostatic energies and pK(a) values. The pK(a) values of many internal groups report apparent protein dielectric constants of 10 or higher. These values are substantially higher than the dielectric constants of 2-4 measured experimentally with dry proteins. The structural origins of these high apparent dielectric constants are not well understood. Here we report on structural and equilibrium thermodynamic studies of the effects of pH on the V66D variant of staphylococcal nuclease. In a crystal structure of this protein the neutral side chain of Asp-66 is buried in the hydrophobic core of the protein and hydrated by internal water molecules. Asp-66 titrates with a pK(a) value near 9. A decrease in the far UV-CD signal was observed, concomitant with ionization of this aspartic acid, and consistent with the loss of 1.5 turns of alpha-helix. These data suggest that the protein dielectric constant needed to reproduce the pK(a) value of Asp-66 with continuum electrostatics calculations is high because the dielectric constant has to capture, implicitly, the energetic consequences of the structural reorganization that are not treated explicitly in continuum calculations with static structures.