A new, versatile field immunosensor for environmental pollutants: development and proof of principle with TNT, diuron, and atrazine

This paper presents a new, versatile, portable miniaturized flow-injection immunosensor which is designed for field analysis. The temperature-controlled field prototype can run for 6h without external power supply. The bio-recognition element is an analyte-specific antibody immobilized on a gold surface of pyramidal structures inside an exchangeable single-use chip, which hosts also the enzyme-tracer and the sample reservoirs. The competition between the enzyme-tracer and the analyte for the antigen-binding sites of the antibodies yields in the final step a chemiluminescence signal that is inversely proportional to the concentration of analyte in the given range of detection. A proof of principle is shown for nitroaromatics and pesticides. The detection limits (DL; IC20) reached with the field prototype in the laboratory was below 0.1 microg l(-1) for 2,4,6-trinitrotoluene (TNT), and about 0.2 microg l(-1) for diuron and atrazine, respectively. Important aspects in this development were the design of the competition between analyte and enzyme-tracer, the unspecific signal due to unspecific binding and/or luminescence background signal, and the flow pattern inside the chip.     

Purification and characterisation of a jacalin-related, coleoptile specific lectin from Hordeum vulgare

A plant lectin was isolated from barley (Hordeum vulgare) coleoptiles using acidic extraction and different chromatographic methods. Sequencing of more than 50% of the protein sequence by Edman degradation confirmed a full-length cDNA clone. The subsequently identified open reading frame encodes for a 15 kDa protein which could be found in the soluble fraction of barley coleoptiles. This protein exhibited specificity towards mannose sugar and is therefore, accordingly named as Horcolin (Hordeum vulgare coleoptile lectin). Database searches performed with the Horcolin protein sequence revealed a sequence and structure homology to the lectin family of jacalin-related lectins. Together with its affinity towards mannose, Horcolin is now identified as a new member of the mannose specific subgroup of jacalin-related lectins in monocot species. Horcolin shares a high amino acid homology to the highly light-inducible protein HL#2 and, in addition to two methyl jasmonic acid-inducible proteins of 32.6 and 32.7 kDa where the jasmonic acid-inducible proteins are examples of bitopic chimerolectins containing a dirigent and jacalin-related domain. Immunoblot analysis with a cross-reactive anti-HL#2 antibody in combination with Northern blot analysis of the Horcolin cDNA revealed tissue specific expression of Horcolin in the coleoptiles. The function of Horcolin is discussed in the context of its particular expression in coleoptiles and is then compared to other lectins, which apparently share a related response to biotic or abiotic stress factors.     

The amino acid tryptophan prevents the biosynthesis of dermatan sulfate

An important part of the biosynthesis of proteoglycans is the epimerization of glycosaminoglycan chains. As a consequence of the conversion of chondroitin sulfate (CS) to dermatan sulfate (DS), the glycosaminoglycans become more flexible and enable DS to perform more sophisticated signaling functions. In a recent study, we generated a chimera (S222A) composed of a truncated form of a DS (decorin) and CS (CSF-1) containing proteoglycan and analyzed the influence of the core protein on the extent of epimerization. C-terminal truncation constructs from S222A enabled us to identify an amino acid segment that lies within the CSF-1 part which prevents DS synthesis. Co-localization experiments using S222A-HA and DCN-Flag showed different intracellular localizations for the proteoglycans during biosynthesis. A data base search revealed a sequence motif (TNWVP) within the CSF-1 moiety that is found to be important in other proteoglycans. A single substitution of tryptophan-216 to leucine (W216L) in the chimera S222A increased the amount of l-IdoA to 12-16%. Co-localization with an ER-marker demonstrated that the biosynthesis of recombinant decorin is similar to the chimera S222A and S222A(W216L) in HEK293 cells. Co-staining of S222A-HA and S222A(W216L)-Flag showed different intracellular localizations for the proteoglycans. A more detailed analysis of the glycosaminoglycans reflects a similar total sulfate content for S222A and S222A(W216L). The 4/6 sulfation ratio was similar for decorin and S222A, but altered for S222A(W216L). However, the binding of fibroblasts growth factor-1 to CS/DS was only partially dependent on epimerization. These results are consistent with the model in which the core protein, via the amino acid tryptophan, is responsible for routing to subcellular compartments with or without sufficient access to chondroitin-glucuronate 5-epimerase.     

Effects of enzymatic degradation on the frictional response of articular cartilage in stress relaxation

It was recently shown experimentally that the friction coefficient of articular cartilage correlates with the interstitial fluid pressurization, supporting the hypothesis that interstitial water pressurization plays a fundamental role in the frictional response by supporting most of the load during the early time response. A recent study showed that enzymatic treatment with chondroitinase ABC causes a decrease in the maximum fluid load support of bovine articular cartilage in unconfined compression. The hypothesis of this study is that treatment with chondroitinase ABC will increase the friction coefficient of articular cartilage in stress relaxation. Articular cartilage samples (n = 34) harvested from the femoral condyles of five bovine knee joints (1-3 months old) were tested in unconfined compression with simultaneous continuous sliding (+/-1.5 mm at 1 mm/s) under stress relaxation. Results showed a significantly higher minimum friction coefficient in specimens treated with 0.1 micro/ml of chondroitinase ABC for 24 h (micro(min) = 0.082+/-0.024) compared to control specimens (micro(min) = 0.047+/-0.014). Treated samples also exhibited higher equilibrium friction coefficient (micro(eq) = 0.232+/-0.049) than control samples (micro(eq) = 0.184+/-0.036), which suggest that the frictional response is greatly influenced by the degree of tissue degradation. The fluid load support was predicted from theory, and the maximum value (as a percentage of the total applied load) was lower in treated specimens (77+/-12%) than in control specimens (85+/-6%). Based on earlier findings, the increase in the ratio micro(min)/micro(eq) may be attributed to the decrease in fluid load support.     

Flow-Induced β-Hairpin Folding of the Glycoprotein Ibα β-Switch

Flow-induced shear has been identified as a regulatory driving force in blood clotting. Shear induces β-hairpin folding of the glycoprotein Ibα β-switch which increases affinity for binding to the von Willebrand factor, a key step in blood clot formation and wound healing. Through 2.1-μs molecular dynamics simulations, we investigate the kinetics of flow-induced β-hairpin folding. Simulations sampling different flow velocities reveal that under flow, β-hairpin folding is initiated by hydrophobic collapse, followed by interstrand hydrogen-bond formation and turn formation. Adaptive biasing force simulations are employed to determine the free energy required for extending the unfolded β-switch from a loop to an elongated state. Lattice and freely jointed chain models illustrate how the folding rate depends on the entropic and enthalpic energy, the latter controlled by flow. The results reveal that the free energy landscape of the β-switch has two stable conformations imprinted on it, namely, loop and hairpin—with flow inducing a transition between the two.     

Ultrastructural immuno-localization of tropoelastin in the chick eye

Summary Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.     

Species‐specific TaqMan probes for simultaneous identification of (Gadus morhua L.), haddock (Melanogrammus aeglefinus L.) and whiting (Merlangius merlangus L.).

We report the development of a multiplex, single tube (TaqMan) PCR assay for the identification of three commercially important gadoid species, the cod (Gadus morhua L.), the haddock (Melanogrammus aeglefinus L.) and the whiting (Merlangius merlangus L.). The assay unambiguously identifies known adult tissue samples, and ethanol‐preserved eggs from all three species with greater than 98% accuracy, providing a powerful tool for the development of catch independent stock assessment methods, mapping of spawning sites and the detection of commercial fraud in the fishing and food production industries.     

Suppression of oxidative phosphorylation confers resistance against bevacizumab in experimental glioma

Although bevacizumab initially shows high response rates in gliomas and other tumours, therapy resistance usually develops later. Because anti‐angiogenic agents are supposed to induce hypoxia, we asked whether rendering glioma cells independent of oxidative phosphorylation modulates their sensitivity against hypoxia and bevacizumab. LNT‐229 glioma cells without functional mitochondria (rho⁰) and control (rho⁺) cells were generated. LNT‐229 rho⁰‐cells displayed reduced expression of oxidative phosphorylation‐related genes and diminished oxygen consumption. Conversely, glycolysis was up‐regulated in these cells, as shown by increased lactate production and stronger expression of glucose transporter‐1 and lactate dehydrogenase‐A. However, hypoxia‐induced cell death in vitro was nearly completely abolished in the LNT‐229 rho⁰‐cells, these cells were more sensitive towards glucose restriction and the treatment with the glycolysis inhibitor 2‐deoxy‐D‐glucose. In an orthotopic mouse xenograft experiment, bevacizumab induced hypoxia as reflected by elevated Hypoxia‐inducible factor 1‐alpha staining in both, rho⁺‐ and rho⁰‐tumours. However, it prolonged survival only in the mice bearing rho⁺‐tumours (74 days vs. 105 days, p  = 0.024 log‐rank test) and had no effect on survival in mice carrying LNT‐229 rho⁰‐tumours (75 days vs. 70 days, p  = 0.52 log‐rank test). Interestingly, inhibition of glycolysis in vivo with 2‐deoxy‐D‐glucose re‐established sensitivity of rho⁰‐tumours against bevacizumab (98 days vs. 80 days, p  = 0.0001). In summary, ablation of oxidative phosphorylation in glioma cells leads to a more glycolytic and hypoxia‐resistant phenotype and is sufficient to induce bevacizumab‐refractory tumours. These results add to increasing evidence that a switch towards glycolysis is one mechanism how tumour cells may evade anti‐angiogenic treatments and suggest anti‐glycolytic strategies as promising approaches to overcome bevacizumab resistance.