Novel frameshift mutations near short simple repeats.

In patients with Alzheimer's disease or Down's syndrome, the cerebellar cortex exhibits protein deposits in neurofibrillary tangles and neuritic plaques. Recently, the deposits have been shown to contain protein fragments of ubiquitin-B and amyloid precursor protein (APP) with an aberrant carboxyl terminus resulting from frameshift mutations (dinucleotide deletions; DeltaGU or DeltaGA) in or adjacent to GAGAG motifs in their mRNAs, a process referred to as molecular misreading. We have now used a bacterial expression system with the green fluorescent protein as a reporter to screen gene transcripts from aged controls, Alzheimer's disease, and Down's syndrome for molecular misreading. Novel frameshift mutations at a number of locations in the transcripts of the ubiquitin-B and APP genes were discovered (DeltaGA, DeltaG, DeltaGU, DeltaGG, DeltaCA, DeltaAU, DeltaA, DeltaAA, DeltaC, DeltaU, and insertion of an A). Interestingly, most mutations were in close proximity of short simple repeats (GAGAG, GGUGGU, GAGACACACA, UCAUCAUCA, CAAACAAA, and GAAGAAGAA), demonstrating that the GAGAG motif does not constitute the only hot spot for transcriptional errors. Unlike the previously detected aberrant APP fragments, some of the novel ones have the potential to generate the neurotoxic peptide beta-amyloid. We conclude that during aging molecular misreading is a widespread phenomenon.

Grass pollen immunotherapy induces highly cross-reactive IgG antibodies to group V allergen from different grass species.

Sera from two groups of patients receiving grass pollen immunotherapy were tested on IgG reactivity with group V allergen from six different grass species. One group of patients was treated with a mixture of 10 grass species, and the other with a mixture of five. Only Lolium perenne, Dactylis glomerata, and Phleum pratense were present in both mixtures. Although Anthoxanthum odoratum and Secale cereale were absent from the mixture of five, IgG responses to Ant o V and Sec c V were comparable in both patient groups. This reactivity was inhibited for 92-99% with L. perenne extract, illustrating the cross-reactive nature of the IgG antibodies. The presence of A. odoratum and S. cereale in the mixture resulted in only minor amounts of species-specific anti-group V IgG. These results indicate that application of just one grass species in immunotherapy might be sufficient to induce an IgG response that covers other relevant Gramineae species as well.

Determination of some anticonvulsants, antiarrhythmics, benzodiazepines, xanthines, paracetamol and chloramphenicol by reversed phase HPLC.

In this article assays are described for caffeine, theophylline, procainamide, N-acetylprocainamide, quinidine, dihydroquinidine, paracetamol, phenobarbital, phenytoin, carbamazepine, chloramphenicol, oxazepam, temazepam, diazepam, desmethyldiazepam, chlordiazepoxide, desmethylchlordiazepoxide and demoxepam using a uniform working procedure, five (slightly) different mobile phases and one HPLC system. Changing from one eluent to another is simple and a stable base-line is achieved within half an hour. Three of the five eluents are interchangeable and recycling of eluent causes no problems. Sample pretreatment is a single step extraction. Interferences can be overcome by changing the selectivity of the eluent by adjusting the tetrahydrofuran or triethylamine content. Furthermore it is shown how triethylamine can improve peak shapes of basic components and shorten their retention times.

Comparison of equilibrium dialysis, ultrafiltration, and gel permeation chromatography for the determination of free fractions of phenobarbital and phenytoin.

In this study, three techniques for measuring the free fractions of phenobarbital and phenytoin were compared: equilibrium dialysis, ultrafiltration, and the Hummel and Dreyer method for gel permeation chromatography. In their therapeutic range (15-40 and 10-20 mg/L, respectively) the free fractions of phenobarbital and phenytoin were independent of the drug concentrations. Free fractions of phenobarbital as determined by equilibrium dialysis, ultrafiltration, and gel permeation chromatography were 58.7 +/- 1.8, 58.3 +/- 1.5, and 55.1 +/- 1.7%, respectively. Free fractions of phenytoin were 18.1 +/- 1.1, 17.0 +/- 2.1, and 19.4 +/- 1.2%, respectively. On lowering the albumin concentration, a similar increase in the free fractions of both drugs was observed with all three techniques. The results of this study show that all three techniques are suitable for the determination of free fractions of phenobarbital and phenytoin. Moreover, these techniques seem to be suitable for the investigation of physiological factors that may influence albumin drug binding.

Simultaneous determination of 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D in plasma or serum.

We describe a simultaneous assay for the principal vitamin D metabolites: 25-hydroxyvitamin D, 24-25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D. Special attention has been paid to simplification of the extensive extraction and purification procedures used in previously described simultaneous assays. All three metabolites were isolated with a single extraction step, followed by only one gradient liquid-chromatographic procedure. For final quantitation we used competitive protein binding assays, involving readily available binding proteins and commercially purchased tritiated vitamin D metabolites. Concentrations in the plasma of healthy subjects (mean age, 27 years), sampled during December were 51 (SD 17) nmol/L, 4.1 (SD 1.3) nmol/L, and 124 (SD 26) pmol/L for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D, respectively. Intra- and interassay CVs for the three metabolites were 4.4 and 3.9%, 6.7 and 8.0%, and 7.0 and 4.8%, respectively.

Analysis for 1,25-dihydroxyvitamin D in human plasma, after a liquid-chromatographic purification procedure, with a modified competitive protein-binding assay.

1,25-Dihydroxyvitamin D in plasma is measured by competitive protein-binding assay after isolation from plasma. The present method is improved, as compared with those hitherto described, with regard to receptor preparation, isolation of vitamin D metabolites from plasma, and procedure for the competitive protein binding. Receptor preparation from healthy rather than rachitic chicks, together with a fast isolation procedure, results in a high yield of active receptor protein: 12 animals provide receptor for about 4000 incubations. No loss of binding activity was observed during one year. 1,25-Dihydroxyvitamin D is isolated from plasma by a single extraction and a one-step chromatographic purification. Analytical recovery for the entire procedure averaged 78.1% (SD 4.7%). Other vitamin D metabolites (25-hydroxyvitamin D and 24,25-dihydroxyvitamin D) can also be separated with this procedure. The main features of the modified binding assay are the use of a stabilized cytosol receptor and dextran-coated charcoal instead of polyethylene glycol. The smallest detectable amount in the binding assay is 1-2 pg (2.4-4.8 fmol). Intra- and interassay CVs are 7.0% and 4.8%, respectively. 1,25-Dihydroxyvitamin D concentrations in plasma of 20 healthy subjects averaged 51.7 (SD 10.8) ng/L [124 (SD 26) pmol/L]. Three anephric patients showed values of 3, 6, and 7 ng/L (7, 14, and 17 pmol/L).