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).     

Extraction based on the flow-injection principle: Part 3. Fluorimetric Determination of Vitamin B1 (Thiamine) by the Thiochrome Method

The thiochrome method for determination of vitamin B₁ in pharmaceutical preparations has been adapted to a continuous flow system based on the flow-injection principle. The sample volume required for an analysis is about 150 /sml. For routine purposes a concentration range of 3 × 10^-4–6 × 10^-4 mg ml^-1 is used. Results obtained with the system agree well with results obtained manually. The consumption of organic phase is 2–3 ml/sample and the sampling rate is 30/h. A sampling rate of 70/h is easily attained if necessary. The relative standard deviation is about 1%.     

Extraction based on the flow-injection principle : Part I. Description of the Extraction System

An extraction system has been developed, essentially consisting of a pump, a rotary valve and a spectrophotometer. The sample, 12–25 μl, is introduced via the rotary valve into an aqueous stream (flow injection). The aqueous stream, containing the sample plug, is divided into small segments by an organic phase and led into a Teflon coil so that a regular pattern of the two phases is obtained. No air bubbles should be present. Separation of the two phases is achieved in a specially constructed fitting and the absorbance of the organic phase is measured. The construction and performance of the system are illustrated by analysis of caffeine samples. Up to 100 samples/h can be analysed with a relative precision of better than 1%.     

Extraction based on the flow-injection principle: Part 4. Determination of Extraction Constants

A new approach to the determination of extraction constants is described. The aqueous and organic phases, the former containing the counter ion, are pumped continuously as small segments through an extraction coil. The sample ion is introduced into the aqueous phase before it enters the coil, where the ion-pair extraction takes place (during 20 s). After leaving the extraction coil, a certain fraction of the organic phase is pumped through the flow-cell of the spectrophotometer. The extraction constants are calculated by slope analysis from the experimental data (absorbance and counter ion concentration). The constants usually agreed within ±0.1 (log units) with values obtained from batch experiments.     

Self-seeded, position-controlled InAs nanowire growth on Si: A growth parameter study

In this work, the nucleation and growth of InAs nanowires on patterned SiO(2)/Si(111) substrates is studied. It is found that the nanowire yield is strongly dependent on the size of the etched holes in the SiO(2), where openings smaller than 180?nm lead to a substantial decrease in nucleation yield, while openings larger than ≈500nm promote nucleation of crystallites rather than nanowires. We propose that this is a result of indium particle formation prior to nanowire growth, where the size of the indium particles, under constant growth parameters, is strongly influenced by the size of the openings in the SiO(2) film. Nanowires overgrowing the etched holes, eventually leading to a merging of neighboring nanowires, shed light into the growth mechanism.     

Synthesis and Biological Activity of Argiotoxin 636 and Analogues: Selective Antagonists for Ionotropic Glutamate Receptors

More discerning than the parent : Analogues of the polyamine toxin argiotoxin 636 (shown docked in the ion channel of an ionotropic glutamate (iGlu) receptor; N blue, O red) distinguish subtypes of iGlu receptors. Depending on which of the two internal amine groups is replaced with a methylene group, the analogue inhibits one or other of two receptor subtypes as potently as the natural compound, which itself inhibits both subtypes nonselectively.

     

Direct Observation of the Molten State of Nanometer-sized Particles With an Atomic Force Microscope: A Feasibility Study

An atomic force microscope (AFM) was used to directly examine the physical state of nanometer-sized particles. The critical diameter of indium particles, where evidence of melting at room temperature was observed, was 7.8 ± 1.2nm. This conclusion is based on a method relying on the manipulation of particles in ambient air and at constant temperature. This method involves a simple set-up that permits a combination of both manipulation and imaging of individual particles. To determine whether a particle is molten, three criteria are used: the merging of particles to form bigger spherical particles, a tip-induced shape change, and the formation of nanofibers. All three criteria have been checked using other particle materials. An attempt at 56°C revealed oxidation of the indium particles as the major problem for melting investigation. Manipulations under high-purity nitrogen atmosphere support the validity of the findings. The use of the AFM to determine whether a nanoparticle is molten is, however, complicated by the oxidation issue.     

Automatic potentiometric two-phase titration in pharmaceutical analysis : Part 3. Titrimetric Identification of Drugs

The conditional acidity constant in a liquid-liquid two-phase system (pK^*_HA + value) can be used as an identity parameter for drug compounds. The pK^*_HA + value can be calculated if the phase ratio and the acidity and distribution constants are known. Selectivity is obtained mainly by the choice of the organic phase. An excess of hydrochloric acid is first added to the sample in the two-phase system. The hydrochloric acid and the sample are then titrated sequentially with sodium hydroxide. The proposed titration procedure makes it possible to calibrat the electrodes, determine the pK^*_HA + value(s), quantify one or more compounds, and check the performance of the electrodes in one and the same experimental run. Lidocaine was “identified” and assayed in different dosage forms such as ointment and solution for injection. The results were in agreement with those obtained by liquid chromatography.     

Experimental Study of Bacterial Penetration into Chalk Rock: Mechanisms and Effect on Permeability

Bacterial selective plugging is one of the mechanisms through which microorganisms can be applied for enhanced oil recovery, as bacteria can plug the water-swept zones of a reservoir, thus altering the flow paths and improving sweep efficiency. However, complete understanding of the penetration behavior of bacteria is lacking, especially in chalk formations where characteristic pore throat sizes are comparable with the sizes of bacterial cells. In this study, two bacterial strains, Bacillus licheniformis 421 (spore-forming) and Pseudomonas putida K12 (non-spore forming) were used to investigate the penetration of bacteria into chalk and its effect on permeability reduction. The core plugs were produced from Stevns Klint outcrop with low permeability (2–4 mD) and with pore sizes comparable to bacterial sizes. Both types of bacteria were able to penetrate and to be transported through the cores to some extent. A significantly higher number of B. licheniformis 421 was detected in the effluents as compared to P. putida K12. It was demonstrated that the spore-forming B. licheniformis 421 penetrates in the form of spores. P. putida K12 is found to penetrate the core, however, in smaller numbers compared to B. licheniformis. It was shown that both bacteria, under different injection concentrations, were capable of plugging the porous rock, as indicated by reduction of the core permeability. An incubation period of 12 days did not allow the permeability to return to initial condition. Based on the results it can be concluded that, when injected into chalk, spore forming bacteria have higher chance to survive and penetrate into deeper formation; and both types of bacteria may cause permeability reduction.     

A microprocessor-controlled flow injection analyser for the determination of terbutaline sulphate

The determination is based on the reaction with 4-aminoantipyrine and potassium hexacyanoferrate(III). The system is automatic with a microcomputer controlling the sampler and injection valve, reading the output from the spectrophotometer and evaluating the result. The system is intended for drug analysis, and the matrix viscosity can vary considerably between different formulations without affecting peak area measurements.