An enzyme electrode for the detection of V-type nerve agents, VX (O-ethyl-S-2-diisopropylaminoethyl methylphosphonothioate) and R-VX (O-isobutyl-S-2-diethylaminoethyl methylphosphonothioate), is proposed. The principle of the new biosensor is based on the enzyme-catalyzed hydrolysis of the nerve agents and amperometric detection of the thiol-containing hydrolysis products at carbon nanotube-modified screen-printed electrodes. Demeton-S was used as a nerve agent mimic. 2-(Diethylamino)ethanethiol (DEAET) and 2-(dimethylamino)ethanethiol (DMAET), the thiol-containing hydrolysis product and hydrolysis product mimic of R-VX and VX, respectively, were monitored by exploiting the electrocatalytic activity of carbon nanotubes (CNT). As low as 2 microM DMAET and 0.8 microM DEAET were detected selectively at a low applied potential of 0.5 V vs Ag/AgCl at a CNT-modified mediator-free amperometric electrode. Further, the large surface area and the hydrophobicity of CNT was used to immobilize organophosphorus hydrolase mutant with improved catalytic activity for the hydrolysis of the P-S bond of phosphothiolester neurotoxins including VX and R-VX nerve gases to develop a novel, mediator-free, membrane-free biosensor for V-type nerve agents. The applicability of the biosensor was demonstrated for direct, rapid, and selective detection of V-type nerve agents' mimic demeton-S. The selectivity of the sensor against interferences and application to spiked lake water samples was demonstrated.

The adaptor protein Nck is inducibly recruited through its SH3.1 domain to a proline-rich sequence (PRS) in CD3ε after TCR engagement. However, experiments with a knockin mutant bearing an 8-aa replacement of the PRS have indicated that Nck binding to the TCR is constitutive, and that it promotes the degradation of the TCR in preselection double-positive (DP) CD4+CD8+ thymocytes. To clarify these discrepancies, we have generated a new knockin mouse line (KI-PRS) bearing a conservative mutation in the PRS resulting from the replacement of the two central prolines. Thymocytes of KI-PRS mice are partly arrested at each step at which pre-TCR or TCR signaling is required. The mutation prevents the trigger-dependent inducible recruitment of endogenous Nck to the TCR but does not impair TCR degradation. However, KI-PRS preselection DP thymocytes show impaired tyrosine phosphorylation of CD3ζ, as well as impaired recruitment of ZAP70 to the TCR and impaired ZAP70 activation. Our results indicate that Nck is recruited to the TCR in an inducible manner in DP thymocytes, and that this recruitment is required for the activation of early TCR-dependent events. Differences in the extent of PRS mutation could explain the phenotypic differences in both knockin mice.

No abstract

On TCR ligation, the adaptor Nck is recruited through its src homology 3.1 domain to a proline-rich sequence (PRS) in CD3ε. We have studied the relevance of this interaction for T cell activation in vitro and in vivo by targeting the interaction sites in both partners. The first approach consisted of studying a knockin (KI) mouse line (KI-PRS) bearing a conservative mutation in the PRS that makes the TCR incompetent to recruit Nck. This deficiency prevents T cell activation by Ag in vitro and inhibited very early TCR signaling events including the tyrosine phosphorylation of CD3ζ. Most important, KI-PRS mice are partly protected against the development of neurological symptoms in an experimental autoimmune encephalitis model, and show a deficient antitumoral response after vaccination. The second approach consisted of using a high-affinity peptide that specifically binds the src homology 3.1 domain and prevents the interaction of Nck with CD3ε. This peptide inhibits T cell proliferation in vitro and in vivo. These data suggest that Nck recruitment to the TCR is fundamental to mount an efficient T cell response in vivo, and that the Nck–CD3ε interaction may represent a target for pharmacological modulation of the immune response.

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.

Two polychlorinated biphenyl‐contaminated sites in the Czech Republic, a soil at Zamberk and a sediment sludge at Milevsko, were screened for the presence of chlorobenzoate degraders. Sixteen different chlorobenzoate degraders were isolated from the soil compared with only three strains isolated from the sediment. From these strains, only four soil degraders and one strain isolated from the sediment, respectively, were shown to possess a complete chlorobenzoate (CB) pathway. Bacteria isolated from the soil have expressed more flexibility for CB degradation, namely in the case of ortho‐chlorinated benzoates. They all possessed large plasmids, the restriction patterns of which were compared. Plasmids in Pseudomonas sp. A7, A8, A18 and A19, respectively, were cured and found to encode at least part of the metabolic pathway involved in the growth on ortho‐chlorinated benzoates.

No abstract

Cell growth limitation is known to be an important condition that enhances L: -valine synthesis in Corynebacterium glutamicum recombinant strains with L: -isoleucine auxotrophy. To identify whether it is the limited availability of L: -isoleucine itself or the L: -isoleucine limitation-induced rel-dependent ppGpp-mediated stringent response that is essential for the enhancement of L: -valine synthesis in growth-limited C. glutamicum cells, we deleted the rel gene, thereby constructing a relaxed (rel (-) ) C. glutamicum DeltailvA DeltapanB Deltarel ilvNM13 (pECKAilvBNC) strain. Variations in enzyme activity and L: -valine synthesis in rel (+) and rel (-) strains under conditions of L: -isoleucine excess and limitation were investigated. A sharp increase in acetohydroxy acid synthase (AHAS) activity, a slight increase in acetohydroxyacid isomeroreductase (AHAIR) activity, and a dramatic increase in L: -valine synthesis were observed in both rel (+) and rel (-) cells exposed to L: -isoleucine limitation. Although the positive effect of induction of the stringent response on AHAS and AHAIR upregulation in cells was not confirmed, we found the stringent response to be beneficial for maintaining increased AHAS, dihydroxyacid dehydratase, and transaminase B activity and L: -valine synthesis in cells during the stationary growth phase.