NMR experiments reveal distinct antibody-bound conformations of a synthetic disaccharide representing a general structural element of bacterial lipopolysaccharide epitopes.

The recognition reactions between a synthetic disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and two monoclonal antibodies (mAbs) were studied by NMR, yielding two distinct bound conformations of the carbohydrate ligand. One mAb, S23-24, recognizes the disaccharides alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl and alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with similar affinities, whereas mAb S25-2 binds to the disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl with an approximately 10-fold higher affinity than to the disaccharide alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl. Compared to S25-2, S23-24 binds to alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl with an approximately 50-fold increased affinity. We used NMR experiments that are based on the transferred NOE effect, specifically, trNOESY, trROESY, QUIET-trNOESY, and MINSY experiments, to show that the (2-->8)-specific mAb, S25-2, stabilizes a conformation of the alpha-(2-->4)-linked disaccharide that is not highly populated in solution. S23-24 recognizes two conformations of alpha-Kdo-(2-->4)-alpha-Kdo-(2-->O)-allyl, one that is highly populated in aqueous solution and another conformation that is similar to the one bound by S25-2. This is the first example where it is experimentally shown that a carbohydrate ligand may adopt different bioactive conformations upon interaction with mAbs with different fine specificities. Our NMR studies indicate that a careful examination of spin diffusion is critical for the analysis of bioactive conformations of carbohydrate ligands.

Conformational analysis of a Chlamydia-specific disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl in aqueous solution and bound to a monoclonal antibody: observation of intermolecular transfer NOEs.

The disaccharide alpha-Kdo-(2-->8)-alpha-Kdo (Kdo: 3-deoxy-D-manno-oct-2-ulosonic acid) represents a genus-specific epitope of the lipopolysaccharide of the obligate intracellular human pathogen Chlamydia. The conformation of the synthetically derived disaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl was studied in aqueous solution, and complexed to a monoclonal antibody S25-2. Various NMR experiments based on the detection of NOEs (or transfer NOEs) and ROEs (or transfer ROEs) were performed. A major problem was the extensive overlap of almost all 1H NMR signals of alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. To overcome this difficulty, HMQC-NOESY and HMQC-trNOESY experiments were employed. Spin diffusion effects were identified using trROESY experiments, QUIET-trNOESY experiments and MINSY experiments. It was found that protein protons contribute to the observed spin diffusion effects. At 800 MHz, intermolecular trNOEs were observed between ligand protons and aromatic protons in the antibody binding site. From NMR experiments and Metropolis Monte Carlo simulations, it was concluded that alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl in aqueous solution exists as a complex conformational mixture. Upon binding to the monoclonal antibody S25-2, only a limited range of conformations is available to alpha-Kdo-(2-->8)-alpha-Kdo-(2-->O)-allyl. These possible bound conformations were derived from a distance geometry analysis using transfer NOEs as experimental constraints. It is clear that a conformation is selected which lies within a part of the conformational space that is highly populated in solution. This conformational space also includes the conformation found in the crystal structure. Our results provide a basis for modeling studies of the antibody-disaccharide complex.

Effect of the geometry of the intraoral position-indicating device on effective dose.

OBJECTIVE: The objective of this study was to calculate and compare the effective dose and to estimate risk from the use of intraoral position-indicating devices of differing geometries. STUDY DESIGN: Thermoluminescent dosimeters were placed at selected sites in the upper portion of a tissue-equivalent human phantom to record the equivalent dose to weighted tissues and organs. The phantom was exposed to simulated complete mouth surveys with either a long (29.8 cm) or short (19.6 cm) round open-end position-indicating device, a long (35.3 cm) or short (23.3 cm) rectangular open-end position-indicating device, or a pointed (29.6 cm) closed-end position-indicating device. RESULTS: The effective dose was calculated as the sum of the equivalent doses to each organ or tissue multiplied by that organ or tissue's weighting factor. The salivary glands were included as part of the remainder. The effective dose ranged from 362 micro Sv for the pointed position-indicating device, to 63 micro Sv for both the long and the short rectangular position-indicating devices. CONCLUSIONS: These effective doses were calculated to represent a probability for stochastic effects that range in magnitude from 26 x 10(-6) to 4.6 x 10(-6).

Conformational analysis of biantennary glycans and molecular modeling of their complexes with lentil lectin.

Some mannose-binding legume lectins show higher affinity for fucosylated glycans than for glycans without fucose. These lectins possess a secondary binding site. Owing to the possibility of additional fucose binding, oligosaccharides adopt different conformations depending on whether they contain fucose or not. To study these conformational differences, complexes of fucosylated and unfucosylated glycans with Lens culinaris lectin have been modeled. Starting points were X-ray structures of lentil lectin and complexes of the homologous Lathyrus ochrus lectin. The SYBYL molecular modeling package with the TRIPOS force field was used. Two different models were built, displaying in both a network of hydrogen bonds between the saccharide and the binding site. Furthermore, to compare the free and bound ligand, conformational analysis in the free state has been performed. A complete analysis of all possible disaccharide fragments has been performed using the MM3 force field. A CICADA analysis employing the same force field was carried out to study the complete oligosaccharide. Low-energy conformers found by CICADA were clustered in conformational families and analyzed in terms of flexibility and rotational barriers. All values of glycosidic torsion angles are in the range as calculated by MM3 for the disaccharides.

Effective dose and risk assessment from detailed narrow beam radiography.

Studies of the effectiveness of commonly used imaging techniques have shown that they are less than optimal in revealing oral disease. The diagnostic accuracy of detailed narrow beam radiography (scanography) has been reported to be significantly better than intraoral periapical radiography for the observation of periodontal pathoses and at least as good as periapical radiography for detecting periapical lesions. The purpose of this study was to calculate and compare the effective doses and risk estimates from the use of detailed narrow beam radiography and intraoral radiography. With the use of a tissue equivalent human phantom and thermoluminescent dosimetry, the effective dose from detailed narrow beam radiography was found to vary from 5 to 35 microSv depending on the anatomic location of the image layer and intraoral radiography from 9 to 150 microSv depending on the type of survey. Effective doses of these magnitudes represent 0.6 to 18.8 days of equivalent natural radiation exposure and a probability for stochastic effects on the order of 0.37 to 10.95 x 10(-6).

Effective dose and risk assessment from computed tomography of the maxillofacial complex.

The effective dose from computed tomography of the maxillofacial complex has been estimated and used for an assessment of risk. For each scan sequence 64 TLDs were placed in 27 selected sites in the upper portion of a tissue-equivalent human phantom to record the equivalent dose in radiosensitive organs/tissues. Equivalent doses ranged from 0.11 mSv (bone marrow, maxillary scan) to 20 mSv (salivary glands, mandibular scan). By the use of a calculation that included the salivary glands as part of the remainder, two contiguous 1 cm axial slices of the maxilla were found to result in an effective dose of 0.1 mSv, and four contiguous 1 cm axial slices of the mandible in an effective dose of 0.76 mSv. Effective doses of this magnitude represent a probability of stochastic effects of the order of 8 X 10(-6) and 56 X 10(-6) respectively.

Effective dose and risk assessment from film tomography used for dental implant diagnostics.

There are no data relating complex film tomography with effective dose that may be used to estimate the relative risk associated with dental implant diagnostics. The purpose of this study was to calculate the effective dose and estimate risk from the use of the Scanora multimodal imaging system. With the use of a tissue equivalent human phantom and thermoluminescent dosimetry, panoramic radiography was found to result in an effective dose of 26 microSv, while complex film tomography resulted in an effective dose of < 1 microSv to 30 microSv depending on the anatomical location of the imaging plane and the collimation option. An effective dose of this magnitude for panoramic radiography was estimated to represent a probability for stochastic effects on the order of 1.9 x 10(-6). Similarly, the effective dose associated with film tomography may be estimated to be equal to a probability for stochastic effects in the range of << 1 x 10(-6) to 2.2 x 10(-6).

Human complement factor B: functional properties of a recombinant zymogen of the alternative activation pathway convertase.

The human complement factor B is a centrally important component of the alternative pathway activation of the complement system. Here we report the isolation, characterization and eukaryotic expression of the first full length cDNA transcript for human factor B. In a factor B dependent haemolysis assay, the recombinant human factor B generated by transient COS cell transfection was shown to reconstitute haemolytic activity of factor B depleted human serum. To study the biological activities assigned to factor B, the availability of recombinant polypeptides representing definite portions of the human factor B molecule is desirable.