Detection of mutations in PCR products from clinical samples by surface plasmon resonance

Two different strategies for scanning and screening of mutations in polymerase chain reaction (PCR) products by hybridization analysis are described, employing real-time biospecific interaction analysis (BIA) for detection. Real-time BIA was used to detect differences in hybridization responses between PCR products and different 17-mer oligonucleotide probes. For the analysis using a biosensor instrument, two different experimental formats were investigated based on immobilization of either biotinylated PCR products or oligonucleotide probes onto a sensor chip. Applied on the human tumour suppressor p53 gene, differences in hybridization levels for full-match and mismatch situations employing both formats allowed the detection of point mutations in exon 6 PCR products, derived from a breast tumour biopsy sample. In addition, a mutant sample sequence could be detected in a 50/50 background of wild type exon 6 sequence. The suitability of the different formats for obtaining a regenerable system and a high throughput of samples is discussed. © 1997 John Wiley & Sons, Ltd.     

A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products

The capacity to produce large amounts of protein in mammalian cells is important in several contexts, including large-scale generation of biologically useful proteins, gene therapy, and transdominant genetics in cultured cells. For transdominant genetics, retroviral vectors are especially useful for delivery of expression libraries. However, even the potent CMV promoter is often unable to stimulate single-copy production of protein beyond the 1 microM level. We have adapted the HIV2/Tat expression system to retroviral vectors to boost expression above levels attainable with CMV promoters. We show that the system produces protein levels in four cell types tested which exceed levels attained by wild-type CMV or modified CMV promoters. In one cell line, the increase is 10-fold above CMV. Coupled with a stable expressed protein, levels of about 4 microM can be produced from presumptive single-copy retroviral transductants, and 30 microM from multicopy transductants.     

Mimicking a Honeybee Queen?Vespa affinis indosinensis Pérez 1910 Hunts Drones of Apis cerana F. 1793

Hornets (Vespa affinis) flying in a drone congregation area attracted drones of Apis cerana. The drones followed the hornet and were ‘manoeuvred’ towards a leaf or a tree. The hornet then rushed at one of the drones. Many attempts by the hornet to catch a drone were unsuccessful and all drones fled. After failing, the hornet returned to centre of the drone congregation area and repeated the behaviour. Only after successfully seizing of a drone did the hornet leave the drone congregation area carrying its prey. In a two-choice test in the centre of the drone congregation area, free-flying A. cerana drones preferred a hornet model to a live A. cerana queen. V. affinis apparently ‘exploits’ the intraspecific communication between queen and drones of A. cerana. Hunting of drones in the drone congregation area by V. affinis may be an example of predatory mimicry.     

Genotoxic effects of fumes from asphalt modified with waste plastic and tall oil pitch

As the use of recycled materials and industrial by-products in asphalt mixtures is increasing, we investigated if recycled additives modify the genotoxicity of fumes emitted from asphalt. Fumes were generated in the laboratory at paving temperature from stone-mastic asphalt (SMA) and from SMA modified with waste plastic (90% polyethylene, 10% polypropylene) and tall oil pitch (SMA-WPT). In addition, fumes from SMA, SMA-WPT, asphalt concrete (AC), and AC modified with waste plastic and tall oil pitch (AC-WPT) were collected at paving sites. The genotoxicity of the fumes was studied by analysis of DNA damage (measured in the comet assay) and micronucleus formation in human bronchial epithelial BEAS 2B cells in vitro and by counting mutations in Salmonella typhimurium strains TA98 and YG1024. DNA damage was also assessed in buccal leukocytes from road pavers before and after working with SMA, SMA-WPT, AC, and AC-WPT. The chemical composition of the emissions was analysed by gas chromatography/mass spectrometry. The SMA-WPT fume generated in the laboratory induced a clear increase in DNA damage in BEAS 2B cells without metabolic activation. The laboratory-generated SMA fume increased the frequency of micronucleated BEAS 2B cells without metabolic activation. None of the asphalt fumes collected at the paving sites produced DNA damage with or without metabolic activation. Fumes from SMA and SMA-WPT from the paving sites increased micronucleus frequency without metabolic activation. None of the asphalt fumes studied showed mutagenic activity in Salmonella. No statistically significant differences in DNA damage in buccal leukocytes were detected between the pre- and post-shift samples collected from the road pavers. However, a positive correlation was found between DNA damage and the urinary metabolites of polycyclic aromatic hydrocarbons (PAHs) after work shift, which suggested an association between occupational exposures during road paving and genotoxic effects. Our results indicate that fumes from SMA and SMA-WPT contain direct-acting genotoxic components.     

Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold

In recent years, classical antibody-based affinity reagents have been challenged by novel types of binding proteins developed by combinatorial protein engineering principles. One of these classes of binding proteins of non-Ig origin are the so-called affibody binding proteins, functionally selected from libraries of a small (6 kDa), non-cysteine three-helix bundle domain used as a scaffold. During the first 10 years since they were first described, high-affinity affibody binding proteins have been selected towards a large number of targets for use in a variety of applications, such as bioseparation, diagnostics, functional inhibition, viral targeting and in vivo tumor imaging/therapy. The small size offers the possibility to produce functional affibody binding proteins also by chemical synthesis production routes, which has been found to be advantageous for the site-specific introduction of various labels and radionuclide chelators.     

Growth on nitrate and occurrence of nitrate reductase-encoding genes in a phylogenetically diverse range of ectomycorrhizal fungi

Ectomycorrhizal (ECM) fungi are often considered to be most prevalent under conditions where organic sources of N predominate. However, ECM fungi are increasingly exposed to nitrate from anthropogenic sources. Currently, the ability of ECM fungi to metabolize this nitrate is poorly understood. Here, growth was examined among 106 isolates, representing 68 species, of ECM fungi on nitrate as the sole N source. In addition, the occurrence of genes coding for the nitrate reductase enzyme (nar gene) in a broad range of ectomycorrhizal fungi was investigated. All isolates grew on nitrate, but there was a strong taxonomic signature in the biomass production, with the Russulaceae and Amanita showing the lowest growth. Thirty-five partial nar sequences were obtained from 43 tested strains comprising 31 species and 10 genera. These taxa represent three out of the four clades of the Agaricales within which ECM fungi occur. No nar sequences were recovered from the Russulaceae and Amanita, but Southern hybridization showed that the genes were present. The results demonstrate that the ability to utilize nitrate as an N source is widespread in ECM fungi, even in those fungi from boreal forests where the supply of nitrate may be very low.     

Heterogeneity of action potential durations in isolated mouse left and right atria recorded using voltage-sensitive dye mapping

An imaging system for di-4-ANEPPS (4-[beta-[2-(di-n-butylamino)-6-naphthylvinyl]pyridinium]) voltage-sensitive dye recordings has been adapted for recording from an in vitro mouse heart preparation that consists of both atria in isolation. This approach has been used to study inter- and intra-atrial activation and conduction and to monitor action potential durations (APDs) in the left and right atrium. The findings from this study confirm some of our previous findings in isolated mouse atrial myocytes and demonstrate that many electrophysiological properties of mouse atria closely resemble those of larger mammals. Specifically, we made the following observations: 1) Activation in mouse atria originates in the sinoatrial node and spreads into the right atrium and, after a delay, into the left atrium. 2) APD in the left atrium is shorter than in the right atrium. 3) Sites in the posterior walls have longer APDs than sites in the atrial appendages. 4) Superfusion of this preparation with 4-aminopyridine and tetraethylammonium resulted in increases in APD, consistent with their inhibitory effects on the K+ currents known to be expressed in mouse atria. 5) The muscarinic agonist carbachol shortened APD in all areas of the preparation, except the left atrial appendage, in which carbachol had no statistically significant effect on APD. These results validate a new approach for monitoring activation, conduction, and repolarization in mouse atria and demonstrate that the physiological and pharmacological properties of mouse atria are sufficiently similar to those of larger animals to warrant further studies using this preparation.     

Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand

Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these Z(HER2) affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ?-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a Z(HER2) affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of Z(HER2). This novel anti-idiotypic affibody molecule Z(E01) was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of Z(HER2) affibody molecules, having a common binding surface for HER2. Eight such Z(HER2) affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope ((??m)Tc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.