Cells on the move: Modulation of guidance cues during germ cell migration

In Drosophila melanogaster the progenitors of the germ-line stem cells, the primordial germ cells (PGCs) are formed on the outside surface of the early embryo, while the somatic gonadal precursor cells (SGPs) are specified during mid-embryogenesis. To form the primitive embryonic gonad, the PGCs travel from outside of the embryo, across the mid-gut and then migrate through the mesoderm to the SGPs. The migratory path of PGCs is dictated by a series of attractive and repulsive cues. Studies in our laboratory have shown that one of the key chemoattractants is the Hedgehog (Hh) ligand. Although, Hh is expressed in other cell types, the long-distance transmission of this ligand is specifically potentiated in the SGPs by the hmgcr isoprenoid biosynthetic pathway. The distant transmission of the Hh ligand is gated by restricting expression of hmgcr to the SGPs. This is particularly relevant in light of the recent findings that an ABC transporter, mdr49 also acts in a mesoderm specific manner to release the germ cell attractant. Our studies have demonstrated that mdr49 functions in hh signaling likely via its role in the transport of cholesterol. Given the importance of cholesterol in the processing and long distance transmission of the Hh ligand, this observation has opened up an exciting avenue concerning the possible role of components of the sterol transport machinery in PGC migration.     

Early experimental obesity is associated with coronary endothelial dysfunction and oxidative stress

Obesity is independently associated with increased cardiovascular risk. However, since established obesity clusters with various cardiovascular risk factors, configuring the metabolic syndrome, the early effects of obesity on vascular function are still poorly understood. The current study was designed to evaluate the effect of early obesity on coronary endothelial function in a new animal model of swine obesity. As to method, juvenile domestic crossbred pigs were randomized to either high-fat/high-calorie diet (HF) or normal chow diet for 12 wk. Coronary microvascular permeability and abdominal wall fat were determined by using electron beam computerized tomography. Epicardial endothelial function and oxidative stress were measured in vitro. Systemic oxidative stress, renin-angiotensin activity, leptin levels, and parameters of insulin sensitivity were evaluated. As a result, HF pigs were characterized by abdominal obesity, hypertension, and elevated plasma lysophosphatidylcholine and leptin in the presence of increased insulin sensitivity. Coronary endothelium-dependent vasorelaxation was reduced in HF pigs and myocardial microvascular permeability increased compared with those values in normal pigs. Systemic redox status in HF pigs was similar to that in normal pigs, whereas the coronary endothelium demonstrated higher content of superoxide anions, nitrotyrosine, and NADPH-oxidase subunits, indicating increased tissue oxidative stress. In conclusion, the current study shows that early obesity is characterized by increased vascular oxidative stress and endothelial dysfunction in association with increased levels of leptin and before the development of insulin resistance and systemic oxidative stress. Vascular dysfunction is therefore an early manifestation of obesity and might contribute to the increased cardiovascular risk, independently of insulin resistance.     

A screen for genes expressed in Drosophila imaginal discs

The development of Drosophila imaginal discs serves as a model system to understand how genes determine the shape and size of an organ. The identification of genes involved in this process is an important step towards this goal. Here we describe a P-element based enhancer trap screen for genes expressed in the larval imaginal discs. Our aim was to establish a large collection of enhancer trap lines each showing expression of Gal4 in imaginal discs. To this end, we improved the well established P-element vector pGawB in order to obtain higher in vivo transposition frequencies. In addition we chose an F1-screening approach using UAS-GFP as a reporter gene. This system permits the efficient screening of larval and pupal stages of living animals and the detection of imaginal gene expression patterns through the transparent cuticle. The procedure has been optimized for high-throughput. 2'000 P-element insertions have been established which exhibit expression in imaginal discs.     

Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin

Human protein C (HPC) is an antithrombotic serine protease that circulates in the plasma as several glycoforms. To examine the role of glycosylation in the function of this protein, we singly eliminated each of the four potential N-linked glycosylation sites by site-directed mutagenesis of Asn to Gln at amino acid positions 97, 248, and 313 (HPC derivatives Q097, Q248, and Q313) or at the unusual consensus sequence Asn-X-Cys at 329 (HPC derivative Q329). The cDNAs for wild type and each derivative were inserted into expression vectors and expressed both transiently and stably in human 293 and hamster AV12-664 cells. We demonstrate that N-linked glycosylation at position 97 in the light chain of HPC is critical for efficient secretion and affects the degree of core glycosylation at Asn-329. Glycosylation at position 248 affects the intracellular processing of the internal Lys-Arg (KR) KR cleavage site, and partial glycosylation at the sequence Asn-329-X-Cys is responsible for the natural alpha-glycoform. Altering the glycosylation pattern of the protein had no significant effect on the level of fully gamma-carboxylated HPC secreted from the 293 cell line. However, elimination of glycosylation sites in the heavy chain resulted in a 2- to 3-fold increase in anticoagulant activity. Utilizing synthetic substrate, both the Km and kcat were affected, depending on the specific glycosylation site eliminated. However, there were no significant differences in the inhibition kinetics by alpha-1-antitrypsin (association rate constants of 10-11 M-1s-1 and t1/2 of 27-29 min at 40 microM alpha-1-antitrypsin) or t1/2 in human plasma (17-18 min). A comparison of the rate of activation of each derivative by thrombin alone or in complex with thrombomodulin revealed that Q313 was activated approximately 2.5-fold faster than wt HPC, independent of calcium concentration. This increase in rate was due to an enhanced affinity of thrombin-thrombomodulin for Q313, as indicated by a 3-fold reduction in Km. Overall, our studies demonstrate that glycosylation at different sites in HPC affects distinct properties of this complex protein. Furthermore, we demonstrate the ability to improve the catalytic efficiency of this enzyme through carbohydrate modifications.     

Advances in Fmoc solid‐phase peptide synthesis

Today, Fmoc SPPS is the method of choice for peptide synthesis. Very‐high‐quality Fmoc building blocks are available at low cost because of the economies of scale arising from current multiton production of therapeutic peptides by Fmoc SPPS. Many modified derivatives are commercially available as Fmoc building blocks, making synthetic access to a broad range of peptide derivatives straightforward. The number of synthetic peptides entering clinical trials has grown continuously over the last decade, and recent advances in the Fmoc SPPS technology are a response to the growing demand from medicinal chemistry and pharmacology. Improvements are being continually reported for peptide quality, synthesis time and novel synthetic targets. Topical peptide research has contributed to a continuous improvement and expansion of Fmoc SPPS applications. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Axial arrangement of crossbridges in thick filaments of vertebrate skeletal muscle.

X-ray intensity data to 1.8 Å resolution were collected from native trigonal crystals of bovine trypsinogen. The orientation and position of the trypsinogen molecules within their crystal cells were determined by Patterson search techniques using the refined model of bovine trypsin (Bode &; Schwager, 1975), and by subsequent R factor refinement. The translation functions allowed discrimination between the enantiomorphic space groups P3₂21 and P3₁21. After one constrained crystallographic refinement cycle, which reduced the crystallographic reliability factor (R) from 35% to 31%, a preliminary difference Fourier map showed several interesting details. Several refinement cycles reduced the value of R to 23%. The overall chain folding is very similar to trypsin. The chain segments, including residues 184 to 193² and 217 to 223, which form the specificity pocket in trypsin, are flexible in trypsinogen. The autolysis loop is partially mobile between residues 142 and 152. There is no continuing electron density for the N terminal residues preceding Tyr20. This indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution.     

The interaction of C-protein with heavy meromyosin and subfragment-2.

C-protein has previously been shown to bind to the light-meromyosin region of the myosin tail. Examination of mixtures of C-protein with heavy meromyosin or subfragment-2 or subfragment-1 in the analytical ultracentrifuge shows that there is also a binding site for C-protein in the subfragment-2 region of the tail.     

Delineation of the protein module that anchors HMGN proteins to nucleosomes in the chromatin of living cells

Numerous nuclear proteins bind to chromatin by targeting unique DNA sequences or specific histone modifications. In contrast, HMGN proteins recognize the generic structure of the 147-bp nucleosome core particle. HMGNs alter the structure and activity of chromatin by binding to nucleosomes; however, the determinants of the specific interaction of HMGNs with chromatin are not known. Here we use systematic mutagenesis, quantitative fluorescence recovery after photobleaching, fluorescence imaging, and mobility shift assays to identify the determinants important for the specific binding of these proteins to both the chromatin of living cells and to purified nucleosomes. We find that several regions of the protein affect the affinity of HMGNs to chromatin; however, the conserved sequence RRSARLSA, is the sole determinant of the specific interaction of HMGNs with nucleosomes. Within this sequence, each of the 4 amino acids in the R-S-RL motif are the only residues absolutely essential for anchoring HMGN protein to nucleosomes, both in vivo and in vitro. Our studies identify a new chromatin-binding module that specifically recognizes nucleosome cores independently of DNA sequence or histone tail modifications.     

Photoinducible DRONPA‐s: a new tool for investigating cell–cell connectivity

The development of multicellular plants relies on the ability of their cells to exchange solutes, proteins and signalling compounds through plasmodesmata, symplasmic pores in the plant cell wall. The aperture of plasmodesmata is regulated in response to developmental cues or external factors such as pathogen attack. This regulation enables tight control of symplasmic cell‐to‐cell transport. Here we report on an elegant non‐invasive method to quantify the passive movement of protein between selected cells even in deeper tissue layers. The system is based on the fluorescent protein DRONPA‐s, which can be switched on and off repeatedly by illumination with different light qualities. Using transgenic 35S::DRONPA‐s Arabidopsis thaliana and a confocal microscope it was possible to activate DRONPA‐s fluorescence in selected cells of the root meristem. This enabled us to compare movement of DRONPA‐s from the activated cells into the respective neighbouring cells. Our analyses showed that pericycle cells display the highest efflux capacity with a good lateral connectivity. In contrast, root cap cells showed the lowest efflux of DRONPA‐s. Plasmodesmata of quiescent centre cells mediated a stronger efflux into columella cells than into stele initials. To simplify measurements of fluorescence intensity in a complex tissue we developed software that allows simultaneous analyses of fluorescence intensities of several neighbouring cells. Our DRONPA‐s system generates reproducible data and is a valuable tool for studying symplasmic connectivity.     

Shape and flexibility of the myosin molecule

Molecules of rabbit skeletal myosin have been examined in the electron microscope after drying at low temperature from solutions containing ethylene glycol or glycerol and rotary-shadowing with platinum. Analysis of the structure has been assisted by stereo-photography. While the general appearance, two heads attached to a long tail, is similar to that described by Slayter & Lowey (1967), more detail about the shape and size of the heads can be discerned and new information has been obtained about the flexibility of the tail and the head-tail junction.The heads are 190 Å long and wider at their ends than near the junction with the tail; the shape resembles that of a pear. The length is appreciably greater than the generally accepted value for subfragment 1, the proteolytic fragment of myosin. The heads are flexibly attached to the tail and can assume a wide range of tilt angles.Because the point where the two heads join the tail can be identified, the length of the tail, 1560 (±50) Å, can be measured more accurately than formerly. While all parts of the tail are somewhat flexible, sharp bends often occur at a well-defined site 430 Å from the head-tail junction. The demonstration of hinges at the head-tail junction and in the tail provides strong support for H. E. Huxley's (1969) hypothesis for the mechanism of muscle contraction.