Detection of 1p and 19q loss in oligodendroglioma by quantitative microsatellite analysis, a real-time quantitative polymerase chain reaction assay.

The combined loss of chromosomes 1p and 19q has recently emerged as a genetic predictor of chemosensitivity in anaplastic oligodendrogliomas. Here, we describe a strategy that uses a novel method of real-time quantitative polymerase chain reaction, quantitative microsatellite analysis (QuMA), for the molecular analysis of 1p and 19q loss in oligodendrogliomas and oligoastrocytomas in archival routinely processed paraffin material. QuMA is performed on the ABI 7700 and based on amplifications of microsatellite loci that contain (CA)n repeats where the repeat itself is the target for hybridization by the fluorescently labeled probe. This single probe can therefore be used to determine copy number of microsatellite loci spread throughout the human genome. In genomic DNA prepared from paraffin-embedded brain tumor specimens, QuMA detected combined loss of 1p and 19q in 64% (21 of 32) of oligodendrogliomas and 67% (6 of 9) of oligoastrocytomas. We validate the use of QuMA as a reliable method to detect copy number by showing concordance between QuMA and fluorescence in situ hybridization at 37 of 45 chromosomal arms tested. These results indicate that QuMA is an accurate, high-throughput assay for the detection of copy number at multiple loci; as many as 31 loci of an individual tumor can be analyzed on a 96-well plate in a single 2-hour run. In addition, it has advantages over standard allelic imbalance/loss of heterozygosity assays in that all loci are potentially informative, paired normal tissue is not required, and gain can be distinguished from loss. QuMA may therefore be a powerful molecular tool to expedite the genotypic analysis of human gliomas in a clinical setting for diagnostic/prognostic purposes.

Superoxide anion permeability of phospholipid membranes and chloroplast thylakoids.

The permeability of phospholipid membranes to the superoxide anion (O-2) was determined using soybean phospholipid vesicles containing FMN in the internal space. The efflux of O-2 generated by the illumination of FMN was so slow that more than 90% of the radicals were spontaneously disproportionated within the vesicles before they could react with cytochrome c at the membrane exterior. The amount of diffused O-2 was proportional to the intravesicular concentration of O-2 over a range from 1 to 10 microM which was deduced from its disproportionation rate. The permeability coefficient of the phospholipid bilayer for O-2 was estimated to be 2.1 X 10(-6) cm s-1 at pH 7.3 and 25 degrees C. Superoxide dismutase trapped inside vesicles was not reactive with extravesicular O-2 unless Triton X-100 was added. O-2 generated outside spinach chloroplast thylakoids did not interact with superoxide dismutase or cytochrome c which had been enclosed in the thylakoids. Thus, chloroplast thylakoids also showed little permeability to O-2.

Reduction of plastocyanin with O2- and superoxide dismutase-dependent oxidation of plastocyanin by H2O2.

Type I copper proteins, plastocyanin and rice blue protein, were reduced with O2 (-). The reduction rate constants of plastocyanins from several sources with O2 (-) are about 10(6) m(-1) sec(-1) (1.0 × 10(6) m(-1) sec(-1) for spinach and kidney bean plastocyanins and 6.7 × 10(5) m(-1)sec(-1) for Japanese radish plastocyanin) at pH 7.8 at 25°C and not significantly different from that observed for rice blue protein (7.3 × 10(5) m(-1)sec(-1)). Reduced plastocyanin was oxidized by H2O2 through the peroxidase-like reaction of Cu,Zn-superoxide dismutase.

Isolation of monomeric cytochrome f from Japanese radish and a mechanism of autoreduction.

Monomeric cytochrome f from Japanese radish (Raphanus sativus L. var acanthiformis Makino) leaves was isolated in a homogeneous state with an A420.5/A277 of 7.6. Radish cytochrome f is a single polypeptide chain with a molecular weight of about 33,000. The midpoint potential is 350 mV. The amino acid analysis indicates the existence of 3 residues of half-cystine. Radish cytochrome f contains one thiol group which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) only after denaturation by sodium dodecyl sulfate. Ferricytochrome f is reduced by the superoxide radical at the rate of 6 X 10(6) M-1 s-1 at pH 7.8. Radish ferricytochrome f is also reduced slowly without an exogenous electron donor. A kinetic study and the effect of the thiol reagent indicate that the autoreduction is an intramolecular reaction and that the thiol group is an electron donor.