BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

Bicyclic [b]-heteroannulated pyridazine derivatives. 4. Cyclization reactions of 4-aryltetrahydropyridazine-3,6-dione 3-hydrazones with some keto esters

Ethoxycarbonylalkylidene derivatives 2 and 6 of the title hydrazones were obtained in the reaction with ethyl pyruvate or ethyl aroylformate and ethyl acetoacetate, respectively, in methanol. Both compounds were mixtures of geometric isomers with high predominance of one of them. Nmr spectroscopy revealed an unexpected magnetic non-equivalence of the CH₂ protons in the ester ethyl group of the major isomer of 6 . On heating (?200°) in an inert medium or on refluxing in ethanolic sodium ethoxide 2 cyclized to the corresponding pyridazino[6,1-c]-triazines 4 , whereas 6 formed pyrazolylpyridazines 7 . The structure of the latter was unambigously established by X-ray analysis. Alkylation of 4a with benzyl bromide in the presence of tetrabutylammonium bromide occurred selectively on the pyridazine N atom.     

Capillary isoelectric focusing-mass spectrometry for shotgun approach in proteomics

We report on capillary isoelectric focusing-mass spectrometry (CIEF-MS) of complex peptide mixtures in the absence of carrier ampholytes. Furthermore, the use of low concentrations of carrier ampholytes as mere spacers is investigated. Carrier ampholytes are complex mixtures of amphoteric compounds with high buffering capacity. Since all peptides are amphoteric compounds by themselves, the use of carrier ampholytes may be superfluous to establish a stable pH gradient in CIEF analysis of protein digests. Our research showed that when carrier ampholytes are omitted, the analyte ions are not focused at their isoelectric point. The analytes are charged, leading to electrophoretic mobility uncharacteristic for CIEF. The method was tested for a five-protein-mixture at 0.02 mg/mL per protein and 0.05 mg/mL per protein. At the lower concentration, the analytes were stacked during the focusing process in only a limited length of the capillary. Therefore, the higher concentration led to better separation efficiency. It was found that at low concentration (0.20%) the carrier ampholytes could work as spacers. Though it led to sensitivity losses of 15-45%, this was compensated by the higher separation efficiencies seen. The method was evaluated with an eight-protein-mixture, of which all could be identified after performing MS/MS.     

Susceptibility to Effects of UVB Irradiation on Induction of Contact Sensitivity, Relevance of Number and Function of Langerhans Cells and Epidermal Macrophages

Sensitization on skin exposed to acute low-dose UVB irradiation separates normal humans into two phenotypically distinct groups: One group, following sensitization on UVB-irradiated skin, develops contact sensitivity, designated UVB resistant (UVB-R) and the second group, following sensitization on UVB-irradiated skin, fails to develop contact sensitivity, designated UVB susceptible (UVB-S). To investigate whether UVB susceptibility in humans is related to antigen-presenting activity in the skin we studied the effect of UVB irradiation on the number and function of the epidermal antigen-presenting cells in volunteers identified as UVB-R and UVB-S. Single cell suspensions of epidermal cells from control skin and skin exposed to 3 minimal erythema doses (MED) of UVB 3 days previously were stained for Langerhans cells (CD1a+HLA-DR+) and epidermal macrophages (CD1a-HLA-DR+). The UVB exposure of the skin significantly decreased the percentage of Langerhans cells (UVB-R: n = 7, P < 0.02, UVB-S: n = 6, P < 0.03) and increased the percentage of epidermal macrophages (UVB-R: n = 7, P < 0.03, UVB-S: n = 6, P < 0.03) however to the same degree in both the UVBR and the UVB-S group. To study the effect on Langerhans cell alloreactivity, epidermal cells were harvested immediately after UVB irradiation. However, in both UVB-R and UVB-S subjects the Langerhans cell alloreactivity was blocked to the same degree immediately after UVB irradiation compared to nonirradiated epidermal cells. To determine the effect of UVB irradiation on epidermal macrophages, epidermal cells were harvested 3 days after UVB irradiation. Irradiated epidermal cells from both UVB-R and UVB-S subjects demonstrated a strong antigen-presenting capacity compared to epidermal cells from control skin leading to activation of T cells that mainly secrete interferon (1FN)-γ and not interleukin (IL)-4. In conclusion we found that UVB susceptibility was not correlated with the number of Langerhans cells or epidermal macrophages in the skin at the same time of sensitization. Neither was it correlated with the capacity of Langerhans cells nor UVB-induced epidermal macrophages to activate T cells in vitro.     

On distribution of molecular weight in anionic polymerizates of caprolactam

Measurement has been made of the dependence of the molecular weight distribution on the ratio of the catalytic system components and on the polymerization time for polycaprolactam obtained by anionic polymerization. A bimodal character of the curves was observed for products made using molar ratios of activator (N-benzoylcaprolactam) to initiator (sodium dihydro-bis(methoxyethoxy)aluminate) of 1:1 and 1:3, when the polymerization times were less than 1 hr. This course is explained by the influence of fast side-reactions, mainly condensations. For a ratio of 3:1 of the components of the catalytic system, smooth integral distribution curves are obtained, accounting for a very fast decrease in the strong base concentration in the initial stages of the polymerization.     

Hollow colloidal particles obtained by nano-extrusion in the presence of phospholipids

Using nano- and microsize extrusion, a simple synthetic procedure of preparing hollow monodispersed colloidal particles dispersed in an aqueous phase was developed. Hydrophobic styrene monomer containing 2-hydroxy-2-methyl propiophenone photoinitiator was forced into desired diameter membrane channels and stabilized by the hydrophobic regions of a liposome obtained from 1,2-dilauroyl-phosphocholine phospholipid in an aqueous phase. Such moieties exposed to 254-nm UV radiation polymerize monomers in the hydrophobic zone of the liposome, thus resulting in reinforced hollow vesicles. The size of such particles is controlled by the size of the membrane channels in the extruder and may vary from a few nanometers to micrometers, thus allowing the synthesis of monodisperse hollow colloidal spheres.     

REALISIS: a medicinal chemistry-oriented reagent selection, library design, and profiling platform

REALISIS is a software system for reagent selection, library design, and profiling, developed to fit the workflow of bench chemists and medicinal chemists. Designed to be portable, the software offers a comprehensive graphical user interface and rapid, integrated functionalities required for reagent retrieval and filtering, product enumeration, and library profiling. REALISIS is component-based, consisting of four main modules: reagent searching; reagent filtering; library enumeration; and library profiling. Each module allows the chemist to access specific functionalities and diverse filtering and profiling mechanisms. By implementing the entire process of reagent selection, library design, and profiling and by integrating all the necessary functionalities for this process, REALISIS cuts the time required to design combinatorial and noncombinatorial libraries from several days to a few hours.     

Einsatz der flammenlosen Atom-Absorption (Graphitrohrküvette) zur Bestimmung von Kupferspuren in Stahl und anderen Metall-Legierungen nach Extraktion

The determination of small and smallest amounts of copper in steel and other metal alloys by atomic absorption spectroscopy was investigated. It has been found, that the interferences caused by accompanying elements in the determination in aqueous solution with the flame can be completely removed by extracting the copper with Pb-DDTC in chloroform and determining this extract with the heated graphite atomizer. Moreover, it was recognised, that working with the heated graphite atomizer in organic solutions shows great advantages compared with the flame, because any solvent may be used. The methods of continuous variation by Job and the molar ratio by Yoe and Jones for the determination of the complex stoichiometry were transferred to the system Cu-DDTC by working with the heated graphite atomizer.     

Modular synthesis of block copolymers via cycloaddition of terminal azide and alkyne functionalized polymers

Polymeric building blocks containing terminal azide and alkyne functionalities are prepared via atom transfer radical polymerization (ATRP) and used to modularly synthesize block copolymers via 1,3-dipolar cycloaddition reactions, which are quantitative according to SEC measurements.     

Organo-noble-gas hydride compounds HKrCCH, HXeCCH, HXeCC, and HXeCCXeH: formation mechanisms and effect of 13C isotope substitution on the vibrational properties

We investigate the formation mechanism of HXeCCXeH in a Xe matrix. Our experimental results show that the HXeCCXeH molecules are formed in the secondary reactions involving HXeCC radicals. The experimental data on the formation of HXeCCXeH is fully explained based on the model involving the HXeCC+Xe+H-->HXeCCXeH reaction. This reaction is the first case when a noble-gas hydride molecule is formed from another noble-gas molecule. In addition, we investigate the (12)C/(13)C isotope effect on the vibrational properties of organo-noble-gas hydrides (HKrCCH, HXeCCH, HXeCC, and HXeCCXeH) in noble-gas matrixes. The present experimental results and ab initio calculations on carbon isotope shifts of the vibrational modes support the previous assignments of these molecules. Upon (12)C to (13)C isotope substitution, we observed a pronounced effect on the H-Kr stretching mode of HKrCCH (downshift of 1.0-3.6 cm(-1), depending on the matrix site) and a small anomalous shift (+0.1 cm(-1)) of the H-Xe stretching mode of HXeCCH and HXeCCXeH.