Structural aspects of antibody-antigen interaction revealed through small random peptide libraries

Summary Two small random peptide libraries, one composed of 4550 dodecapeptides and one of 8000 tripeptides, were synthesized in newly developed credit-card format miniPEPSCAN cards (miniPEPSCAN libraries). Each peptide was synthesized in a discrete well (455 peptides/card). The two miniPEPSCAN libraries were screened with three different monoclonal antibodies (Mabs). Two other random peptide libraries, expressed on the wall of bacteria (recombinant libraries) and composed of 10⁷ hexa- and octapeptides, were screened with the same three Mabs. The aim of this study was to compare the amino acid sequence of peptides selected from small and large pools of random peptides and, in this way, investigate the potential of small random peptide libraries. The screening of the two miniPEPSCAN libraries resulted in the identification of a surprisingly large number of antibody-binding peptides, while the screening of the large recombinant libraries, using the same Mabs, resulted in the identification of only a small number of peptides. The large number of peptides derived from the small random peptide libraries allowed the determination of consensus sequences. These consensus sequences could be related to small linear and nonlinear parts of the respective epitopes. The small number of peptides derived from the large random peptide libraries could only be related to linear epitopes that were previously mapped using small libraries of overlapping peptides covering the antigenic protein. Thus, with respect to the cost and speed of identifying peptides that resemble linear and nonlinear parts of epitopes, small diversity libraries based on synthetic peptides appear to be superior to large diversity libraries based on expression systems.Abbreviations ABTS 2,2-azino-di-3-ethylbenzthiazoline sulfonate - EGF epidermal growth factor - Mab monoclonal antibody - OD_ccd optical density obtained using CCD camera - RAMPO rabbit-antimouse peroxidase - SDS sodium dodecylsulfate - TGEV transmissible gastroenteritis virus     

Ageladine A: an antiangiogenic matrixmetalloproteinase inhibitor from the marine sponge Agelas nakamurai

A novel MMP inhibitor, ageladine A (1) with antiangiogenic activity was isolated from a marine sponge Agelas nakamurai. Structure 1 was determined by a combination of spectroscopic and chemical methods to be an unprecedented structure of 4-(4,5-dibromo-1H-pyrrol-2-yl)]-1H-imidazo[4,5-c]pyridin-2-amine.     

Novel bromotyrosine derivatives that inhibit growth of the fish pathogenic bacterium Aeromonas hydrophila, from a marine sponge Hexadella sp

Three new bromotyrosine derivatives, 11-N-methylmoloka'iamine (1), 11-N-cyano-11-N-methylmoloka'iamine (2), and kuchinoenamine (3), were isolated as antibacterial constituents from a marine sponge Hexadella sp. Their structures were elucidated on the basis of spectral and chemical methods. They exhibited moderate antibacterial activity against the fish pathogenic bacterium Aeromonas hydrophila.     

Implementation and use of the method of prudent ascent in conformational analysis using molecular mechanics

In two-dimensional conformational analysis the current practice is to perform an energy minimization for all possible combinations of two dihedral angles in the molecule, in a fixed order, and apply a certain dihedral angle step-size. A newly developed method is presented in which the order of the evaluation points on the energy-surface is not fixed, but is dependent on all previous results in a way which we call “the method of prudent ascent.” In this method the most promising calculation is carried out first, thus minimizing the risk of atomic collisions. In order to be able to take care of the many additional degrees of conformational freedom present in, e.g., carbohydrate molecules, all minimizations are performed using a set of different promising starting conformations on the basis of previous calculations, and only the lowest energy result for each point is saved. An application of the method to conformational analysis of methyl-cellobiose and the artificial sweetener trichlorogalactosucrose is also presented.     

Oil core-polymer shell microcapsules prepared by internal phase separation from emulsion droplets. I. Characterization and release rates for microcapsules with polystyrene shells

Microcapsules with an oil core surrounded by a polymeric shell have been prepared by the controlled phase separation of polymer dissolved within the oil droplets of an oil-in-water emulsion. The dispersed oil phase consists of the shell polymer (polystyrene), a good solvent for the polymer (dichloromethane), and a poor solvent for the polymer (typically hexadecane). Removal of the good solvent results in phase separation of the polymer within the oil droplets. If the three interfacial tensions between the core oil, the shell-forming polymer, and the continuous phase are of the required relative magnitudes, a polymer shell forms surrounding the poor solvent. A UV-responsive organic molecule was added to the oil phase, prior to emulsification, to investigate the release of a model active ingredient from the microcapsules. This molecule should be soluble in the organic core but also have some water solubility to provide a driving force for release into the continuous aqueous phase. As the release rate of the active ingredient is a function of the thickness of the polymeric shell, for controlled release applications, it is necessary to control this parameter. For the preparative method described here, the thickness of the shell formed is directly related to the mass of polymer dissolved in the oil phase. The rate of volatile solvent removal influences the porosity of the polymer shell. Rapid evaporation leads to cracks in the shell and a relatively fast release rate of the active ingredient. If a more gentle evaporation method is employed, the porosity of the polymer shell is decreased, resulting in a reduction in release rate. Cross-linking the polymer shell after capsule formation was also found to decrease both the release rate and the yield of the active ingredient. The nature of the oil core also affected the release yield.     

A polycationic dendrimer as noncovalent support for anionic organometallic complexes

Polycationic dendrimer 1 forms a well-defined, stoichiometric assembly with eight anionic metal complexes; this assembly is successfully applied as a Lewis acidic catalyst which performs comparably to the unsupported metal complex.     

Measurement uncertainty: practical problems encountered by accredited testing laboratories

Following the introduction of ISO 17025 in 1999, all accredited testing laboratories have started to implement a procedure for estimating measurement uncertainties for the analytical methods that they employ. These procedures and uncertainties are an integral part of the quality system of the accredited laboratory and are therefore reviewed during audits by the accreditation bodies. In January/February 2004 the status of measurement uncertainties available at accredited testing laboratories was evaluated. It was found that most laboratory customers do not make use nor want to make use of the available measurement uncertainties. Furthermore, the current lack of standardisation has caused the estimated measurement uncertainties to vary widely, even for the simplest determinations.     

Infrared spectroscopy of neutral C7H7 isomers: benzyl and tropyl

The gas-phase infrared absorption spectra of neutral benzyl and tropyl, isomers of formula C7H7, have been measured in the 400-1800 cm-1 spectral region. In addition, a quantum chemical calculation has been performed to model the infrared spectra. For the benzyl radical, the theory shows satisfactory overlap with the experiment, although vibrations involving the CH2 group might be anharmonic. The tropyl radical, which is subject to the Jahn-Teller effect, seems well modeled for the out-of-plane vibrational modes, but less so for the in-plane vibrational modes.     

Biocatalytic asymmetric hydroxylation of hydrocarbons with the topsoil-microorganism Bacillus megaterium

A Bacillus megaterium strain was isolated from topsoil by a selective screening procedure with allylbenzene as a xenobiotic substrate. This strain performed the hydroxylation chemoselectively (no arene oxidation and overoxidized products) and enantioselectively (up to 99% ee) in the benzylic and nonbenzylic positions of a variety of unfunctionalized arylalkanes. Salycilate and phenobarbital, which are potent inducers of cytochrome P-450 activity, changed the regioselectivity of the microbial CH insertion, without an effect on the enantioselectivity. The biotransformation conditions were optimized in regard to product yield and enantioselectivity by variation of the oxygen-gas supply and the time of the substrate addition. The different product distributions (alpha- versus beta-hydroxylated product) that are obtained on induction of cytochrome P-450 enzyme activity demonstrate the involvement of two or more hydroxylating enzymes with distinct regioselectivities in this biotransformation. An oxygen-rebound mechanism is assumed for the cytochrome P-450-type monooxygenase activity, in which steric interactions between the substrate and the enzyme determine the preferred face of the hydroxy-group transfer to the radical intermediate.     

New Nitrogenous Bisabolene‐Type Sesquiterpenes from a Hainan Sponge Axinyssa aff. variabilis

Two new uncommon nitrogenous sesquiterpenes, 11‐ethoxy‐3‐formamidotheonellin ( 1 ) and 7‐ethoxy‐3‐formamidobisabolane‐8,10‐diene ( 2 ), together with two known related sesquiterpenes, 3‐formamidotheonellin (=theonellin formamide; 3 ) and theonellin isothiocyanate ( 4 ), were isolated from the Hainan sponge Axinyssa aff. variabilis. Their structures were determined on the basis of extensive spectroscopic analysis and by comparison of their NMR data with those of known compounds.