Tie receptors and their angiopoietin ligands are context-dependent regulators of vascular remodeling

Angiopoietins are ligands of the Tie2 receptor that control angiogenic remodeling in a context-dependent manner. Tie signaling is involved in multiple steps of the angiogenic remodeling process during development, including destabilization of existing vessels, endothelial cell migration, tube formation and the subsequent stabilization of newly formed tubes by mesenchymal cells. Beyond this critical role in blood vessel development, recent studies suggest a wider role for Tie2 and angiopoietins in lymphangiogenesis and the development of the hematopoietic system, as well as a possible role in the regulation of certain non-endothelial cells. The outcome of Tie signaling depends on which vascular bed is involved, and crosstalk between different VEGFs has an important modulating effect on the properties of the angiopoietins. Signaling through the Tie1 receptor is not well understood, but Tie1 may have both angiopoietin-dependent and ligand-independent functions. Changes in the expression of Tie receptors and angiopoietins occur in many pathological conditions, and mutations in the Tie2 gene are found in familial cases of vascular disease.     

The European carbon balance. Part 2: croplands

We estimated the long‐term carbon balance [net biome production (NBP)] of European (EU‐25) croplands and its component fluxes, over the last two decades. Net primary production (NPP) estimates, from different data sources ranged between 490 and 846 gC m^?2 yr^?1, and mostly reflect uncertainties in allocation, and in cropland area when using yield statistics. Inventories of soil C change over arable lands may be the most reliable source of information on NBP, but inventories lack full and harmonized coverage of EU‐25. From a compilation of inventories we infer a mean loss of soil C amounting to 17 g m^?2 yr^?1. In addition, three process‐based models, driven by historical climate and evolving agricultural technology, estimate a small sink of 15 g C m^?2 yr^?1 or a small source of 7.6 g C m^?2 yr^?1. Neither the soil C inventory data, nor the process model results support the previous European‐scale NBP estimate by Janssens and colleagues of a large soil C loss of 90 ± 50 gC m^?2 yr^?1. Discrepancy between measured and modeled NBP is caused by erosion which is not inventoried, and the burning of harvest residues which is not modeled. When correcting the inventory NBP for the erosion flux, and the modeled NBP for agricultural fire losses, the discrepancy is reduced, and cropland NBP ranges between ?8.3 ± 13 and ?13 ± 33 g C m^?2 yr^?1 from the mean of the models and inventories, respectively. The mean nitrous oxide (N₂O) flux estimates ranges between 32 and 37 g C Eq m^?2 yr^?1, which nearly doubles the CO₂ losses. European croplands act as small CH₄ sink of 3.3 g C Eq m^?2 yr^?1. Considering ecosystem CO₂, N₂O and CH₄ fluxes provides for the net greenhouse gas balance a net source of 42–47 g C Eq m^?2 yr^?1. Intensifying agriculture in Eastern Europe to the same level Western Europe amounts is expected to result in a near doubling of the N₂O emissions in Eastern Europe. N₂O emissions will then become the main source of concern for the impact of European agriculture on climate.     

Anti-idiotypic protein domains selected from protein A-based affibody libraries

Three pairs of small protein domains showing binding behavior in analogy with anti-idiotypic antibodies have been selected using phage display technology. From an affibody protein library constructed by combinatorial variegation of the Fc binding surface of the 58 residue staphylococcal protein A (SPA)-derived domain Z, affibody variants have been selected to the parental SPA scaffold and to two earlier identified SPA-derived affibodies. One selected affibody (Z(SPA-1)) was shown to recognize each of the five domains of wild-type SPA with dissociation constants (K(D)) in the micromolar range. The binding of the Z(SPA-1) affibody to its parental structure was shown to involve the Fc binding site of SPA, while the Fab-binding site was not involved. Similarly, affibodies showing anti-idiotypic binding characteristics were also obtained when affibodies previously selected for binding to Taq DNA polymerase and human IgA, respectively, were used as targets for selections. The potential applications for these types of affinity pairs were exemplified by one-step protein recovery using affinity chromatography employing the specific interactions between the respective protein pair members. These experiments included the purification of the Z(SPA-1) affibody from a total Escherichia coli cell lysate using protein A-Sepharose, suggesting that this protein A/antiprotein A affinity pair could provide a basis for novel affinity gene fusion systems. The use of this type of small, robust, and easily expressed anti-idiotypic affibody pair for affinity technology applications, including self-assembled protein networks, is discussed.     

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

Chronic nitric oxide synthase inhibition blunts endothelium-dependent function of conduit coronary arteries, not arterioles

Current literature suggests that chronic nitric oxide synthase (NOS) inhibition has differential effects on endothelium-dependent dilation (EDD) of conduit arteries vs. arterioles. Therefore, we hypothesized that chronic inhibition of NOS would impair EDD of porcine left anterior descending (LAD) coronary arteries but not coronary arterioles. Thirty-nine female Yucatan miniature swine were included in the study. Animals drank either tap water or water with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 mg/l), resulting in control and chronic NOS inhibition (CNI) groups, respectively. Treatment was continued for 1-3 mo (8.3 +/- 0.6 mg x kg(-1) x day(-1)). In vitro EDD of coronary LADs and arterioles was assessed via responses to ADP (LADs only) and bradykinin (BK), and endothelium-independent function was assessed via responses to sodium nitroprusside (SNP). Chronic NOS inhibition diminished coronary artery EDD to ADP and BK. Incubating LAD rings with L-NAME decreased relaxation responses of LADs from control pigs but not from CNI pigs such that between-group differences were abolished. Neither indomethacin (Indo) nor sulfaphenazole incubation significantly affected relaxation responses of LAD rings to ADP or BK. Coronary arteries from CNI pigs showed enhanced relaxation responses to SNP. In contrast to coronary arteries, coronary arterioles from CNI pigs demonstrated preserved EDD to BK and no increase in dilation responses to SNP. L-NAME, Indo, and L-NAME + Indo incubation did not result in significant between-group differences in arteriole dilation responses to BK. These results suggest that although chronic NOS inhibition diminishes EDD of LAD rings, most likely via a NOS-dependent mechanism, it does not affect EDD of coronary arterioles.     

Strontium Diffusion in Magnetron Sputtered Gadolinia‐Doped Ceria Thin Film Barrier Coatings for Solid Oxide Fuel Cells

Strontium (Sr) diffusion in magnetron sputtered gadolinia‐doped ceria (CGO) thin films is investigated. For this purpose, a model system consisting of a screen printed (La,Sr)(Co,Fe)O_3?δ (LSCF) layer, and thin films of CGO and yttria‐stabilized zirconia (YSZ) is prepared to simulate a solid oxide fuel cell. This setup allows observation of Sr diffusion by observing SrZrO₃ formation using X‐ray diffraction while annealing. Subsequent electron microscopy confirms the results. This approach presents a simple method for assessing the quality of CGO barriers without the need for a complete fuel cell test setup. CGO films with thicknesses ranging from 250 nm to 1.2 μm are tested at temperatures from 850 °C to 1000 °C which yields an in‐depth understanding of Sr diffusion through CGO thin films that may be of high scientific and technical interest for implementation of novel fuel cell materials. Sr is found to diffuse along column/grain boundaries in the CGO films but by modifying the film thickness and microstructure the breaking temperature of the barrier can be increased.     

Expression of a functional proteinase inhibitor capable of accepting xylose: bikunin

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly.     

Transport of [1-14C]-indole-3-acetic acid in Abies balsamea shoots ringed with Ethrel

The terminal (1-year-old) shoot of dormant, 2-year-old Abies balsamea (L.) Mill. seedlings was ringed with 0 or 10 mg Ethrel g^-1 lanolin. After 5 weeks of culture under environmental conditions favorable for growth, some of the treated shoots were harvested to measure tracheid number by microscopy and ethylene evolution by gas chromatography-flame ionization detection. The remaining shoots were used to measure basipetal IAA transport in the cambial region by decapitating the shoot apex, applying a pulse of [1-¹⁴C]-IAA to the cut surface, and monitoring the subsequent distribution of radioactivity. Ringing with 10 mg Ethrel g^-1 lanolin, compared with lanolin alone, stimulated cambial region ethylene evolution about 26-fold at, and 3-fold above and below the ringing site, but promoted tracheid production at the ringing site only. Ethrel ringing also increased the velocity, after 26 h transport, at which the front of the [1-¹⁴C]-IAA pulse moved below the ringing site. After 72 h of [1-¹⁴C]-IAA transport, when only immobilized radioactivity was present, the amount of radioactivity recovered in shoots ringed with 10 mg Ethrel g^-1 lanolin was higher than with lanolin alone at the ringing site but lower below it. No difference was observed above the ringing site. The distribution of radioactivity was the same in shoots ringed with lanolin alone and in unringed shoots. The results support the hypothesis that an abnormally high cambial region concentration of ethylene derived from Ethrel ringing inhibits the capacity of basipetal IAA transport at the ringing site, resulting in a local accumulation of IAA that stimulates tracheid production.