Synthesis of 3-tert-butylcatechol by an engineered monooxygenase

Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol. The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1. The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme. The biotransformation was performed in a 3-L bioreactor for 24 h. To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed. Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth. In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution. The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated. Down-stream processing consisted of two steps. First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol. Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate. The final purity of 3-tert-butylcatechol was greater than 98%.     

Mechanisms and rates of bacterial colonization of sinking aggregates

Quantifying the rate at which bacteria colonize aggregates is a key to understanding microbial turnover of aggregates. We used encounter models based on random walk and advection-diffusion considerations to predict colonization rates from the bacteria's motility patterns (swimming speed, tumbling frequency, and turn angles) and the hydrodynamic environment (stationary versus sinking aggregates). We then experimentally tested the models with 10 strains of bacteria isolated from marine particles: two strains were nonmotile; the rest were swimming at 20 to 60 microm s(-1) with different tumbling frequency (0 to 2 s(-1)). The rates at which these bacteria colonized artificial aggregates (stationary and sinking) largely agreed with model predictions. We report several findings. (i) Motile bacteria rapidly colonize aggregates, whereas nonmotile bacteria do not. (ii) Flow enhances colonization rates. (iii) Tumbling strains colonize aggregates enriched with organic substrates faster than unenriched aggregates, while a nontumbling strain did not. (iv) Once on the aggregates, the bacteria may detach and typical residence time is about 3 h. Thus, there is a rapid exchange between attached and free bacteria. (v) With the motility patterns observed, freely swimming bacteria will encounter an aggregate in <1 day at typical upper-ocean aggregate concentrations. This is faster than even starving bacteria burn up their reserves, and bacteria may therefore rely solely on aggregates for food. (vi) The net result of colonization and detachment leads to a predicted equilibrium abundance of attached bacteria as a function of aggregate size, which is markedly different from field observations. This discrepancy suggests that inter- and intraspecific interactions among bacteria and between bacteria and their predators may be more important than colonization in governing the population dynamics of bacteria on natural aggregates.     

ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha vbeta 3 and induces blood vessel formation in vivo

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.     

Multiple sequence alignment with arbitrary gap costs: computing an optimal solution using polyhedral combinatorics

Multiple sequence alignment is one of the dominant problems in computational molecular biology. Numerous scoring functions and methods have been proposed, most of which result in NP-hard problems. In this paper we propose for the first time a general formulation for multiple alignment with arbitrary gap-costs based on an integer linear program (ILP). In addition we describe a branch-and-cut algorithm to effectively solve the ILP to optimality. We evaluate the performances of our approach in terms of running time and quality of the alignments using the BAliBase database of reference alignments. The results show that our implementation ranks amongst the best programs developed so far.     

A hydroponic culture system for growing<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plantlets under sterile conditions

We developed a hydroponic cultivation system for growingArabidopsis plantlets under sterile, controlled environmental conditions. The system consists of a piece of stainless-steel wire cloth (125 μm mesh size) that is fixed between 2 flat rings and held in place by 3 legs, placed in a commercially-available glass jar, and covered by the original glass lid or a sheet of sterilized cellophane. Sterilized seeds were distributed evenly across the mesh piece, the size of which allowed root growth and kept the seeds in place. After 3 weeks of cultivation, shoot and root tissues were easily harvested without mechanical damage. Proteome and metabolite analyses were performed on root and shoot tissues and demonstrated excellent reproducibility, indicating that the system is advantageous when biological variation is minimized. Induction experiments can be performed by transferring the apparatus (with plants) to a new jar containing a different nutrient solution. The apparatus is reusable and can easily be sterilized by autoclaving or dry heat. The system can be adapted to other small-seed plants by varying the mesh size.     

Structure of the bc1 complex from Seculamonas ecuadoriensis,a jakobid flagellate with an ancestral mitochondrial genome

In eubacteria, the respiratory bc(1) complex (complex III) consists of three or four different subunits, whereas that of mitochondria, which have descended from an alpha-proteobacterial endosymbiont, contains about seven additional subunits. To understand better how mitochondrial protein complexes evolved from their simpler bacterial predecessors, we purified complex III of Seculamonas ecuadoriensis, a member of the jakobid protists, which possess the most bacteria-like mitochondrial genomes known. The S. ecuadoriensis complex III has an apparent molecular mass of 460 kDa and exhibits antimycin-sensitive quinol:cytochrome c oxidoreductase activity. It is composed of at least eight subunits between 6 and 46 kDa in size, including two large "core" subunits and the three "respiratory" subunits. The molecular mass of the S. ecuadoriensis bc(1) complex is slightly lower than that reported for other eukaryotes, but about 2x as large as complex III in bacteria. This indicates that the departure from the small bacteria-like complex III took place at an early stage in mitochondrial evolution, prior to the divergence of jakobids. We posit that the recruitment of additional subunits in mitochondrial respiratory complexes is a consequence of the migration of originally alpha-proteobacterial genes to the nucleus.     

Potential role for ADAM15 in pathological neovascularization in mice

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15(-/-) mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15(-/-) mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15(-/-) mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15(-/-) mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.