Single-site chemical modification at C10 of the baccatin III core of paclitaxel and Taxol C reduces P-glycoprotein interactions in bovine brain microvessel endothelial cells

A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core that reduces interaction with P-glycoprotein in bovine brain microvessel endothelial cells is described. Modification and derivatization of the C10 position were carried out using a substrate controlled hydride addition to a key C9 and C10 diketone intermediate. The analogs were tested for tubulin assembly and cytotoxicity, and were shown to retain potency similar to paclitaxel. P-glycoprotein interaction was examined using a rhodamine assay and it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel and Taxol C can reduce interaction with the P-glycoprotein transporter that may allow for increased permeation of taxanes into the brain.     

A weighted least squares method for inverse dynamic analysis

Internal forces in the human body can be estimated from measured movements and external forces using inverse dynamic analysis. Here we present a general method of analysis which makes optimal use of all available data, and allows the use of inverse dynamic analysis in cases where external force data is incomplete. The method was evaluated for the analysis of running on a partially instrumented treadmill. It was found that results correlate well with those of a conventional analysis where all external forces are known.     

Inducible defences prevent strong population fluctuations in bi- and tritrophic food chains

Recent theoretical work ( Vos et al. 2004 ) predicts that inducible defences prevent strong population fluctuations under high levels of nutrient enrichment. Here we evaluate this model prediction and show that inducible defences in algae stabilize the dynamics of experimentally assembled bi‐ and tritrophic planktonic food chains. At high phosphorus levels, we observed strong population fluctuations in all food chains with undefended algae. These fluctuations did not occur when algae had inducible defences. At low phosphorus levels, we observed deterministic consumer extinctions, as predicted by stoichiometric theory. Our study thus shows that both biotically and abiotically induced changes in algal food quality affect the stability and persistence of planktonic food chains.     

The phosducin-like protein PhLP1 is essential for G{beta}{gamma} dimer formation in Dictyostelium discoideum

Phosducin proteins are known to inhibit G protein-mediated signaling by sequestering Gbetagamma subunits. However, Dictyostelium discoideum cells lacking the phosducin-like protein PhLP1 display defective rather than enhanced G protein signaling. Here we show that green fluorescent protein (GFP)-tagged Gbeta (GFP-Gbeta) and GFP-Ggamma subunits exhibit drastically reduced steady-state levels and are absent from the plasma membrane in phlp1(-) cells. Triton X-114 partitioning suggests that lipid attachment to GFP-Ggamma occurs in wild-type cells but not in phlp1(-) and gbeta(-) cells. Moreover, Gbetagamma dimers could not be detected in vitro in coimmunoprecipitation assays with phlp1(-) cell lysates. Accordingly, in vivo diffusion measurements using fluorescence correlation spectroscopy showed that while GFP-Ggamma proteins are present in a complex in wild-type cells, they are free in phlp1(-) and gbeta(-) cells. Collectively, our data strongly suggest the absence of Gbetagamma dimer formation in Dictyostelium cells lacking PhLP1. We propose that PhLP1 serves as a cochaperone assisting the assembly of Gbeta and Ggamma into a functional Gbetagamma complex. Thus, phosducin family proteins may fulfill hitherto unsuspected biosynthetic functions.     

Reversible self-association of ovalbumin at air-water interfaces and the consequences for the exerted surface pressure

In this study the relation between the ability of protein self-association and the surface properties at air-water interfaces is investigated using a combination of spectroscopic techniques. Three forms of chicken egg ovalbumin were obtained with different self-associating behavior: native ovalbumin, heat-treated ov-albumin-being a cluster of 12-16 predominantly noncovalently bound proteins, and succinylated ovalbumin, as a form with diminished aggregation properties due to increased electrostatic repulsion. While the bulk diffusion of aggregated protein is clearly slower compared to monomeric protein, the efficiency of transport to the interface is increased, just like the efficiency of sticking to rather than bouncing from the interface. On a timescale of hours, the aggregated protein dissociates and adopts a conformation comparable to that of native protein adsorbed to the interface. The exerted surface pressure is higher for aggregated material, most probably because the deformability of the particle is smaller. Aggregated protein has a lower ability to desorb from the interface upon compression of the surface layer, resulting in a steadily increasing surface pressure upon reducing the available area for the surface layer. This observation is opposite to what is observed for succinylated protein that may desorb more easily and thereby suppresses the buildup of a surface pressure. Generally, this work demonstrates that modulating the ability of proteins to self-associate offers a tool to control the rheological properties of interfaces.     

Inhibition of protein kinase CbetaII increases glucose uptake in 3T3-L1 adipocytes through elevated expression of glucose transporter 1 at the plasma membrane

The mechanism via which diacylglycerol-sensitive protein kinase Cs (PKCs) stimulate glucose transport in insulin-sensitive tissues is poorly defined. Phorbol esters, such as phorbol-12-myristate-13-acetate (PMA), are potent activators of conventional and novel PKCs. Addition of PMA increases the rate of glucose uptake in many different cell systems. We attempted to investigate the mechanism via which PMA stimulates glucose transport in 3T3-L1 adipocytes in more detail. We observed a good correlation between the rate of disappearance of PKCbetaII during prolonged PMA treatment and the increase in glucose uptake. Moreover, inhibition of PKCbetaII with a specific myristoylated PKCbetaC2-4 peptide inhibitor significantly increased the rate of glucose transport. Western blot analysis demonstrated that both PMA treatment and incubation with the myristoylated PKCbetaC2-4 pseudosubstrate resulted in more glucose transporter (GLUT)-1 but not GLUT-4 at the plasma membrane. To our knowledge, we are the first to demonstrate that inactivation of PKC, most likely PKCbetaII, elevates glucose uptake in 3T3-L1 adipocytes. The observation that PKCbetaII influences the rate of glucose uptake through manipulation of GLUT-1 expression levels at the plasma membrane might reveal a yet unidentified regulatory mechanism involved in glucose homeostasis.     

Ligand-induced changes in the conformational dynamics of a bacterial cytotoxic endonuclease

Knowledge about the conformational dynamics of a protein is key to understanding its biochemical and biophysical properties. In the present work we investigated the dynamic properties of the enzymatic domain of DNase colicins via time-resolved fluorescence and anisotropy decay analysis in combination with steady-state acrylamide quenching experiments. The dynamic properties of the apoenzyme were compared to those of the E9 DNase ligated to the transition metal ion Zn(2+) and the natural inhibitor Im9. We further investigated the contributions of each of the two tryptophans within the E9 DNase (Trp22 and Trp58) using two single-tryptophan mutants (E9 W22F and E9 W58F). Wild-type E9 DNase, E9 W22F, and E9 W58F, as well as Im9, showed multiple lifetime decays. The time-resolved and steady-state fluorescence results indicated that complexation of E9 DNase with Zn(2+) induces compaction of the E9 DNase structure, accompanied by immobilization of Trp22 along with a reduced solvent accessibility for both tryptophans. Im9 binding resulted in immobilization of Trp22 along with a decrease in the longest lifetime component. In contrast, Trp58 experienced less restriction on complexation of E9 DNase with Im9 and showed an increase in the longest lifetime component. Furthermore, the results point out that the Im9-induced changes in the conformational dynamics of E9 DNase are predominant and occur independently of the Zn(2+)-induced conformational effects.     

NIPBL mutational analysis in 120 individuals with Cornelia de Lange syndrome and evaluation of genotype-phenotype correlations

The Cornelia de Lange syndrome (CdLS) is a multisystem developmental disorder characterized by facial dysmorphia, upper-extremity malformations, hirsutism, cardiac defects, growth and cognitive retardation, and gastrointestinal abnormalities. Both missense and protein-truncating mutations in NIPBL, the human homolog of the Drosophila melanogaster Nipped-B gene, have recently been reported to cause CdLS. The function of NIPBL in mammals is unknown. The Drosophila Nipped-B protein facilitates long-range enhancer-promoter interactions and plays a role in Notch signaling and other developmental pathways, as well as being involved in mitotic sister-chromatid cohesion. We report the spectrum and distribution of NIPBL mutations in a large well-characterized cohort of individuals with CdLS. Mutations were found in 56 (47%) of 120 unrelated individuals with sporadic or familial CdLS. Statistically significant phenotypic differences between mutation-positive and mutation-negative individuals were identified. Analysis also suggested a trend toward a milder phenotype in individuals with missense mutations than in those with other types of mutations.     

Blue light reduces photosynthetic efficiency of cyanobacteria through an imbalance between photosystems I and II

Several studies have described that cyanobacteria use blue light less efficiently for photosynthesis than most eukaryotic phototrophs, but comprehensive studies of this phenomenon are lacking. Here, we study the effect of blue (450 nm), orange (625 nm), and red (660 nm) light on growth of the model cyanobacterium Synechocystis sp. PCC 6803, the green alga Chlorella sorokiniana and other cyanobacteria containing phycocyanin or phycoerythrin. Our results demonstrate that specific growth rates of the cyanobacteria were similar in orange and red light, but much lower in blue light. Conversely, specific growth rates of the green alga C. sorokiniana were similar in blue and red light, but lower in orange light. Oxygen production rates of Synechocystis sp. PCC 6803 were five-fold lower in blue than in orange and red light at low light intensities but approached the same saturation level in all three colors at high light intensities. Measurements of 77 K fluorescence emission demonstrated a lower ratio of photosystem I to photosystem II (PSI:PSII ratio) and relatively more phycobilisomes associated with PSII (state 1) in blue light than in orange and red light. These results support the hypothesis that blue light, which is not absorbed by phycobilisomes, creates an imbalance between the two photosystems of cyanobacteria with an energy excess at PSI and a deficiency at the PSII-side of the photosynthetic electron transfer chain. Our results help to explain why phycobilisome-containing cyanobacteria use blue light less efficiently than species with chlorophyll-based light-harvesting antennae such as Prochlorococcus, green algae and terrestrial plants.     

Production of a de-novo designed antimicrobial peptide in Nicotiana benthamiana

Antimicrobial peptides are important defense compounds of higher organisms that can be used as therapeutic agents against bacterial and/or viral infections. We designed several antimicrobial peptides containing hydrophobic and positively charged clusters that are active against plant and human pathogens. Especially peptide SP1-1 is highly active with a MIC value of 0.1 μg/ml against Xanthomonas vesicatoria, Pseudomonas corrugata and Pseudomonas syringae pv syringae. However, for commercial applications high amounts of peptide are necessary. The synthetic production of peptides is still quite expensive and, depending on the physico-chemical features, difficult. Therefore we developed a plant/tobacco mosaic virus-based production system following the ‘full virus vector strategy’ with the viral coat protein as fusion partner for the designed antimicrobial peptide. Infection of Nicotiana benthamiana plants with such recombinant virus resulted in production of huge amounts of virus particles presenting the peptides all over their surface. After extraction of recombinant virions, peptides were released from the coat protein by chemical cleavage. A protocol for purification of the antimicrobial peptides using high resolution chromatographic methods has been established. Finally, we yielded up to 0.025 mg of peptide per g of infected leaf biomass. Mass spectrometric and NMR analysis revealed that the in planta produced peptide differs from the synthetic version only in missing of N-terminal amidation. But its antimicrobial activity was in the range of the synthetic one. Taken together, we developed a protocol for plant-based production and purification of biologically active, hydrophobic and positively charged antimicrobial peptide.