Building novel binding ligands to B7.1 and B7.2 based on human antibody single variable light chain domains.

Ligands specific for B7.1 (CD80) and B7.2 (CD86) have applications in disease indications that require inhibition of T-cell activity. As we observed significant sequence and structural similarity between the B7-binding ligand, cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and antibody variable light chain domains (VLs), we have explored the possibilities of making novel B7 binding molecules based on single VL domains.We first describe the "rational" design and construction of a VL/CTLA-4 hybrid molecule in which we have grafted both the CDR1 and CDR3-like loops of CTLA-4 onto a single VL light chain, at sites determined by sequence and structure-based alignment. This molecule was secreted as a soluble product from Escherichia coli, but did not show any binding to B7.1 and B7.2. In a second approach we constructed a VL library in which human VL genes derived from B-cells were spiked with the CDR3-like loop of CTLA-4 and further diversified by DNA shuffling. This library was displayed on phage, and after selection gave B7.1 binding ligands which competed with CTLA-4. In order to evaluate the possible general utility of VL domains as binding ligands, we have constructed a non-biased VL library. From this DNA-shuffled human VL library we have selected single VL domains specific for B7.1, B7.2 or human IgG. Two B7.1-specific VL ligands and one B7.2-specific VL ligand showed competition with CTLA-4. One candidate VL domain-specific for B7.1 was affinity matured by simultaneous randomisation of all CDR loops using DNA shuffling with degenerate CDR-spiking oligonucleotides. From this library, a single VL domain with affinity of 191 nM for B7.1 was obtained, which also showed binding to B7.1 in situ. This VL had mutations in CDR1 and CDR3, indicating that antigen recognition for this single VL is most likely mediated by the same regions as in the VL domain of whole antibodies.The B7.1 and B7.2-specific VL domains described in this study may form the basis of a new family of immunomodulatory recombinant molecules. Furthermore, our studies suggest that it is feasible to create specific single VL domains to diverse targets as is the case for single VH domains.     

Soil activity and persistence of sulcotrione and mesotrione

Greenhouse bioassays were set up using a small pot test method to determine the intrinsic sensitivity of different plant species to sulcotrione and mesotrione applied in a sandy loam soil. Herbicides were applied over an appropriate concentration range. After a 2-3 week test period, foliage fresh weight was determined. Data were subjected to a non-lineair regression analysis. Using the regression equations, ED50-values (herbicide concentrations that cause 50 percent foliage fresh weight reduction) were calculated for each combination of crop species and herbicide. To determine which replacement crops might be grown in case of failure of a crop treated with one of these herbicides, field persistence experiments were conducted over the 1993-2003 period for sulcotrione and the 1998-2003 period for mesotrione at the Experimental Farm, Biocentre Agri-Vet, Ghent University at Melle. Herbicides were applied in spring (about mid-March) on a bare soil; untreated control strips were included. Replacement crops were sown or planted approximately five weeks after herbicide applications. Visual estimations of crop injury were recorded at several intervals from sowing and fresh matter yield of plant parts was determined. Based on these data, crops were ranked according to their degree of sensitivity to either sulcotrione or mesotrione. Maize is very tolerant to both herbicides, although in some years, temporary injury could be seen in the field experiments. Italian rye-grass and fibre flax are tolerant crops; in field experiments a slight, temporary injury could be noticed in some years. Winter wheat displayed a high degree of tolerance to mesotrione (in both experiment types): however this crop was less tolerant to sulcotrione especially in the bioassay experiment. Based on its ED50-value, black salsify is tolerant to sulcotrione but under field conditions, the selectivity of this herbicide is quite variable; tolerance to mesotrione is moderate. Turnip and witloof chicory are clearly sensitive to mesotrione and sulcotrione whereas sugar beet, red clover and lettuce are extremely sensitive to both herbicides in both experiment types. Bioassays and field experiments provide a detailed and complete information about soil activity and persistence of both herbicides.     

Alpha-1-antitrypsin immunoreactivity in islet cells of adult human pancreas

alpha-1-antitrypsin immunoreactivity was demonstrated by immunofluorescence technique in the peripheral islet cells of all ten normal adult human pancreata examined; normal adult human liver was negative. The specificity of the reactions was confirmed by applying various control tests including absorption of the specific antisera with purified alpha-1-antitrypsin, inhibition and blocking tests and by ensuring the monospecificity of the antisera used. The findings suggest that the pancreatic islet may be an additional source of alpha-1-antitrypsin.     

Iatrogenic left main coronary artery dissection

We present a case of iatrogenic left main coronary artery dissection, successfully treated by prompt bail-out stenting, and provide a brief discussion on its occurrence and treatment, as well as the immediate and long-term outcome of percutaneous coronary intervention, including our own single-centre experience, for this potentially catastrophic complication.     

Light Dependent Reduction of Pertechnetate (TcO(4)) by Broken Chloroplasts

Arguments are given for a ferredoxin-mediated reduction of TcO₄⁻, preponderantly into extractable Tc(V) complexes, by illuminated, broken chloroplasts. Photosynthetic O₂- and NADP-reduction competitively inhibit Tc incorporation. As for O₂, the reaction can be stimulated by the auto-oxidizable electron acceptor methyl viologen. Furthermore TcO₄⁻ can function as terminal acceptor in the diaphorase reaction, with NADPH as electron donor.     

Development and application of cytotoxic T lymphocyte-associated antigen 4 as a protein scaffold for the generation of novel binding ligands

We have explored the possibilities of using human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) as a single immunoglobulin fold-based scaffold for the generation of novel binding ligands. To obtain a suitable protein library selection system, the extracellular domain of CTLA-4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3. CTLA-4 was shown to be functionally intact by binding to its natural ligands B7-1 (CD80) and B7-2 (CD86) both in vitro and in situ. Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA-4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3-like loop of CTLA-4 with the sequence XXX-RGD-XXX (where X represents any amino acid). Using phage display we selected several CTLA-4-based variants capable of binding to human alphavbeta3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR-like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA-4 is a suitable human scaffold for engineering single-domain molecules with one or possibly more binding specificities.     

A Globin Domain in a Neuronal Transmembrane Receptor of Caenorhabditis elegans and Ascaris suum: MOLECULAR MODELING AND FUNCTIONAL PROPERTIES*

We report the structural and biochemical characterization of GLB-33, a putative neuropeptide receptor that is exclusively expressed in the nervous system of the nematode Caenorhabditis elegans. This unique chimeric protein is composed of a 7-transmembrane domain (7TM), GLB-33 7TM, typical of a G-protein-coupled receptor, and of a globin domain (GD), GLB-33 GD. Comprehensive sequence similarity searches in the genome of the parasitic nematode, Ascaris suum, revealed a chimeric protein that is similar to a Phe-Met-Arg-Phe-amide neuropeptide receptor. The three-dimensional structures of the separate domains of both species and of the full-length proteins were modeled. The 7TM domains of both proteins appeared very similar, but the globin domain of the A. suum receptor surprisingly seemed to lack several helices, suggesting a novel truncated globin fold. The globin domain of C. elegans GLB-33, however, was very similar to a genuine myoglobin-type molecule. Spectroscopic analysis of the recombinant GLB-33 GD showed that the heme is pentacoordinate when ferrous and in the hydroxide-ligated form when ferric, even at neutral pH. Flash-photolysis experiments showed overall fast biphasic CO rebinding kinetics. In its ferrous deoxy form, GLB-33 GD is capable of reversibly binding O₂ with a very high affinity and of reducing nitrite to nitric oxide faster than other globins. Collectively, these properties suggest that the globin domain of GLB-33 may serve as a highly sensitive oxygen sensor and/or as a nitrite reductase. Both properties are potentially able to modulate the neuropeptide sensitivity of the neuronal transmembrane receptor.     

Thermodynamics of Mn2+-binding to goat α-lactalbumin

By means of reaction calorimetry we measured the apparent enthalpy change, H^app, of the binding of Mn²⁺-ions to goat -lactalbumin as a function of temperature. The observed H^app can be written as the sum of contributions resulting from a conformational and a binding process. In combination with the thermal unfolding curve of goat -lactalbumin, we succeeded in separating the complete set of thermodynamic parameters (H, G, S, C_p) into the binding and conformational contributions. By circular dichroism we showed that NH ₄ ⁺ -ions, upon binding to bovine a-lactalbumin, induce the same conformational change as do Na⁺ and K⁺: the binding constant equals 98 ± 9 M^–1.Abbreviations BLA bovine -lactalbumin - GLA goat -lactalbumin - HLA human -lactalbumin - CD circular dichroism Offprint requests to: H. Van DaelDeceased