Rapid identification of new bacterial alcohol dehydrogenases for (R)- and (S)-enantioselective reduction of ss-ketoesters

New bacterial alcohol dehydrogenases with high and complementary enantioselectivity for the reduction of ethyl 3-keto-4,4,4-trifluorobutyrate 1 and methyl 3-keto-3-(3'-pyridyl)-propionate 3 have been rapidly identified by use of a new methodology consisting of preselection of microorganisms based on degradation ability and high-throughput screening with a miniaturized system coupled with fast analysis of enantioselectivities.     

Mapping of a discontinuous and highly conformational binding site on follicle stimulating hormone subunit-β (FSH-β) using domain Scan™ and Matrix Scan™ technology

This paper describes the application of two novel screening technologies, i.e. Domain Scan (24- and 30-mer peptides) and Matrix Scan (24-mer peptides) technology, in the mapping of a discontinuous epitope on FSH-beta for a series of 20 monoclonal antibodies. 11 out of 20 mAb's, mapping of which was not successful by conventional Pepscan technology (12-mer peptides), showed selective binding to peptide-constructs corresponding to the beta3-loop of FSH in the Domain and/or Matrix Scan. Systematic replacement analysis studies with peptide-construct 57VYETVRVPGCAC-SAc-ADSLYTYPVATQ81 revealed that for most mAb's the amino acids R62, A70, D71, and L73 form the core of the epitope. A Domain Scan performed in the C-O format showed highly selective binding for mAb's 1 and 2 with only three beta1-beta3 peptide-constructs covering the residues 60TVRVPGCAHHADSLY74 in combination with 10IAIEKEECRFAI21, while for mAb 10 binding was observed with peptide-constructs containing the C-terminal residues 97RGLGPSYCSFGEMKE114 in combination with the residues 10IAIEKEECRFAI21. A Matrix Scan of mAb 17 showed that peptides from four different regions on FSH (1st strand beta3-loop, alpha 1-loop, long alpha2-loop, det. loop) showed enhanced binding in combination with several 70ADSL73-containing peptides. BIACORE measurements with mAb's 1, 2, 13, and 17 using a set of 21 different peptide(-construct)s partially confirmed the Domain and Matrix Scan screening results. Only 24- and 33-mer peptides covering both the 1st and 2nd strand of the beta3-loop showed measurable binding. Cyclic beta3-loop peptide mimics were found to bind significantly stronger (Kd approximately 5 microM) than the lineair analogues, in agreement with the fact that the discontinuous epitope is part of a loop structure. Coupling of the lineair beta1-peptide 1oIAIEKEECRFAI21 to the linear beta3-peptide *52TFKELVYETVRVPGCAHHADSLYTYPVATQAH83# via disulfide bond formation showed a 2-3 fold increase in Kd, thus conforming participation of the beta 1-loop in antibody binding for these mAb's.     

Fluorescence lifetimes and emission patterns probe the 3D orientation of the emitting chromophore in a multichromophoric system

In this Communication, we report on the fluorescence behavior of an individual first generation multichromophoric dendrimer. The fact that each of the chromophores in time acts as a fluorescent trap is demonstrated by directly probing the dipole orientation of the emitting chromophore by means of defocused wide-field imaging and comparing experimental and calculated emission patterns. It is shown that in such cases the electromagnetic boundary condition effect results in discrete changes in the fluorescence lifetime as a function of time for individual dendrimers.     

Temporal subtraction of thorax CR images using a statistical deformation model

We propose a voxel-based nonrigid registration algorithm for temporal subtraction of two-dimensional thorax X-ray computed radiography images of the same subject. The deformation field is represented by a B-spline with a limited number of degrees of freedom, that allows global rib alignment to minimize subtraction artifacts within the lung field without obliterating interval changes of clinically relevant soft-tissue abnormalities. The spline parameters are constrained by a statistical deformation model that is learned from a training set of manually aligned image pairs using principal component analysis. Optimization proceeds along the transformation components rather then along the individual spline coefficients, using pattern intensity of the subtraction image within the automatically segmented lung field region as the criterion to be minimized and applying a simulated annealing strategy for global optimization in the presence of multiple local optima. The impact of different transformation models with varying number of deformation modes is evaluated on a training set of 26 images using a leave-one-out strategy and compared to the manual registration result in terms of criterion value and deformation error. Registration quality is assessed on a second set of validation images by a human expert rating each subtraction image on screen. In 85% of the cases, the registration is subjectively rated to be adequate for clinical use.     

The oxidation of 6- and 7-aryl-4(3h)-pteridinones by immobilized arthrobacter M-4 cells containing xanthine oxidase

The preparation of 6- and 7-(pX-phenyl)-4(3H)-pteridinones (X = H, CH₃, OCH₃) is described. The oxidation of these compounds by (immobilized) Arthrobacter M-4 cells containing xanthine oxidase has been studied. The oxidation monitored by uv spectroscopy usually goes fast, except for 7-(pX-phenyl)-4(3H)-pteridinones (X = CH₃, OCH₃), which are slowly oxidized. With bacterial cells immobilized in gelatine crosslinked with glutaraldehyde small laboratory-scale oxidations were carried out. Based on spectral data the products of the oxidation reactions are 6- and 7-aryllumazines.     

The variation of trace element concentrations in single human head hairs

The concentrations of nine trace elements in segments of individual human head hairs have been determined by neutron activation analysis. Concentration variations have been studied along individual hairs, among hairs plucked from various locations on the scalp, among hairs in different phases of the growth cycle, and before and after hair cleaning. Recent atomic absorption analysis data indicate strong concentration fluctuations in successive single hair segments of 2 to 5 mm in length. The results are discussed in a forensic context. Work supported by the National Research Council of Canada under Grant No. A2510, by the Canada Council through a bursary to D. Maes, by a grant from the President's Research Grant Fund of Simon Fraser University, and under a contract with the Department of the Solicitor-General of Canada through the Royal Canadian Mounted Police.     

Hole trapping during low gate bias, high drain bias hot-carrierinjection in n-MOSFETs at 77 K

Injection and trapping of holes in the gate oxide of n-channel MOS transistors during operation at large drain and small gate biases are investigated at liquid-nitrogen temperature. Experimental evidence is given that about three times less trapping of holes occurs in the gate oxide at 77 K as compared to 295 K. The authors show that this is due to the small hole mobility in SiO₂ at low temperature