Increase of anti-metastatic efficacy by selectivity- but not affinity-optimization of synthetic serine protease inhibitors

Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model. This in vivo screening revealed efficacy of urokinase inhibitors but no correlation between urokinase selectivity or affinity and anti-metastatic effect. In contrast, factor Xa-selective inhibitors were more potent, demonstrating factor Xa or a factor Xa-like serine protease likely to be more determinant in this model. Factor Xa selectivity, but not affinity, significantly improved anti-metastatic efficacy. For example, factor Xa inhibitors CJ-504 and CJ-510 exert similar affinity for factor Xa (K(i)=14 nM versus 8.8 nM) but CJ-504 was 70-fold more selective for factor Xa. This correlated with higher anti-metastatic efficacy (58.8% with CJ-504; 28.2% with CJ-510). Our results show that among the protease inhibitors employed that have affinities in the nanomolar range, the strategy of selectivity-optimization is superior to further improvement of affinity to significantly enhance anti-metastatic efficacy. This appreciation may be important for the future rational design of new anti-proteolytic agents for cancer therapy.     

High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines

A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.     

Impact fact-or fiction?

The Journal Impact Factor dominates research assessment in many disciplines and in many countries. While research assessment will always have to rely to some extent on quantitative, standardized metrics, the focus on this single measure has gone so far as to hamper and distort scientific research. The Declaration on Research Assessment (DORA), signed by influential journals, funders, academic institutions and individuals across the natural sciences, aims to raise awareness and to redress the use of non‐objective research assessment practices.     

Subunit composition and functional properties of G-protein heterotrimers on rat chromaffin granules

Heterotrimeric G-proteins at the plasma membrane serve as switches between heptahelical receptors and intracellular signal cascades. Likewise endomembrane associated G-proteins may transduce signals from intracellular compartments provided they consist of a functional trimer. Using quantitative immunoelectron microscopy we found heterotrimeric G-protein subunits Galpha2, Galpha(q/11), Gbeta2 and Gbeta5 to reside on secretory granules in chromaffin cells of rat adrenal glands.Thus rat chromaffin granules are equipped with functional G-proteins that consist of a specific alpha-, beta- and probably gamma-subunit combination. Serotonin uptake into a crude rat chromaffin granule preparation was inhibited by activated Galphao2 (10 nM) to nearly the same extent as by GMppNp (50 microM) whereas GDPbetaS was ineffective. The data support the idea that vesicular G-proteins directly regulate the transmitter content of secretory vesicles. In this respect Galphao2 appears to be the main regulator of vesicular momoamine transporter activity.     

Lipid Formation by Aqueous Fischer-Tropsch-Type Synthesis over a Temperature Range of 100 to 400 °C

The formation of lipid compounds during anaqueous Fischer-Tropsch-type reaction was studied withsolutions of oxalic acid as the carbon and hydrogensource. The reactions were conducted in stainlesssteel vessels by heating the oxalic acid solution atdiscrete temperatures from 100 to 400 °C, atintervals of 50 °C for two days each. Themaximum lipid yield, especially for oxygenatedcompounds, is in the window of 150–250 °C. At atemperature of 100 °C only a trace amount oflipids was detected. At temperatures above150 °C the lipid components ranged from C₁₂to >C₃₃ and included n-alkanols, n-alkanoic acids, n-alkyl formates, n-alkanals, n-alkanones, n-alkanes, andn-alkenes, all with essentially no carbon numberpreference. The n-alkanes increased inconcentration over the oxygenated compounds attemperatures of 200 °C and above, with a slightreduction in their carbon number ranges due tocracking. It was also noted that the n-alkanoicacids increased while n-alkanols decreased withincreasing temperature above 200 °C. Attemperatures above 300 °C synthesis competeswith cracking and reforming reactions. At 400 °Csignificant cracking was observed and polynucleararomatic hydrocarbons and their alkylated homologswere detected. The results of this work suggest thatthe formation of lipid compounds by aqueous FTTreactions proceeds by insertion of a CO group at theterminal end of a carboxylic acid functionality toform n-oxoalkanoic acids, followed by reductionto n-alkanoic acids, to n-alkanals, thento n-alkanols. The n-alkenes areintermediate homologs for n-alkan-2-ones andn-alkanes. This proposed mechanism for aqueousFTT synthesis differs from the surface-catalyzedstepwise FT process (i.e., gaseous) of polymerization of methylene reported in the literature.     

The intracellular tyrosine residues of the ATP-gated P2X(1) ion channel are essential for its function

In order to gain a first insight into the effects of reactive oxygen species (ROS) on plant mitochondria, we studied the effect of the ROS producing system consisting of xanthine plus xanthine oxidase on the rate of membrane potential (DeltaPsi) generation due to either succinate or NADH addition to durum wheat mitochondria as monitored by safranin fluorescence. We show that the early ROS production inhibits the succinate-dependent, but not the NADH-dependent, DeltaPsi generation and oxygen uptake. This inhibition appears to depend on the impairment of mitochondrial permeability to succinate. It does not involve mitochondrial thiol groups sensitive to either mersalyl or N-ethylmaleimide and might involve both protein residues and/or membrane lipids, as suggested by the mixed nature. We propose that, during oxidative stress, early generation of ROS can affect plant mitochondria by impairing metabolite transport, thus preventing further substrate oxidation, DeltaPsi generation and consequent large-scale ROS production.     

Cathepsins B, K, and L are regulated by a defined collagen type II peptide via activation of classical protein kinase C and p38 MAP kinase in articular chondrocytes

Degradation of the extracellular matrix (ECM) is a prominent feature in osteoarthritis (OA), which is mainly because of the imbalance between anabolic and catabolic processes in chondrocytes resulting in cartilage and bone destruction. Various proteases act in concert to degrade matrix components, e.g. type II collagen, MMPs, ADAMTS, and cathepsins. Protease-generated collagen fragments may foster the destructive process. However, the signaling pathways associated with the action of collagen fragments on chondrocytes have not been clearly defined. The present data demonstrate that the N-terminal telopeptide of collagen type II enhances expression of cathepsins B, K, and L in articular chondrocytes at mRNA, protein, and activity levels, mediated at least in part through extracellular calcium. We also demonstrate that the induction is associated with the activation of protein kinase C and p38 MAP kinase.