Ruptured aneurysm at the trunk of the accessory middle cerebral artery.

We present a 32-year-old woman with intracranial haemorrhage due to rupture of a saccular aneurysm arising from the trunk of an accessory middle cerebral artery. This is the first report of an aneurysm arising distally to the anomalous vessel's origin from the A1 segment of the anterior cerebral artery.

Preparation of MUC-1 oligomers using an improved convergent solid-phase peptide synthesis.

The sequentially repeating nature of the core mucin polypeptide chain MUC-1 on the surface of malignant cells makes it an excellent target for cancer immunotherapy. We describe a reliable and efficient method of synthesizing oligomers, up to five tandem repeats and oligomer heterotope derivatives with a 15-amino acid epitope from tetanus toxin using an improved convergent solid-phase peptide synthesis. The different oligomers were easily distinguishable by reverse-phase high pressure liquid chromatography, but they were poorly fixed and migrated with the same migration rate, irrespective of size, in electrophoretic studies. In contrast, the oligomer heterotopes exhibited size-dependent electrophoretic behavior but in high pressure liquid chromatography chromatograms the different heterotopes were eluted simultaneously in two peaks representing the L- and D-enantiomers of the derivatives. The oligomer heterotopes were recognized as antigens in Western blotting with a murine monoclonal antibody against the epitope APDTR. In enzyme immunoassay studies with the same antibody an increasing reactivity was observed against the larger oligomers and confirmed by inhibition assays as the MUC-1 pentamer was the most efficient inhibitor. These results support the suggestion that the pentamer attains a structure closer to the native conformation and is more immunogenic. In conclusion, large composite peptides can be reliably synthesized with the convergent solid-phase peptide strategy offering an attractive option to vaccine designing and development.

Retrieval of DNA sequences present at an extremely low frequency.

We describe a method for retrieving sequences with one or two point mutations of a given target sequence, which are present in a DNA population at a frequency of 1 in 466 x 10(3) and 1 in 28 x 10(3) molecules, respectively. By stringent hybridization to a stable, chemically immobilized probe, a large excess of unrelated fragments is removed, and the bound sequences are dissociated and amplified. By repeating the hybridization-amplification cycles twice, we achieved an estimated enrichment of 404,000-fold and 1612-fold, respectively, which was confirmed by cloning the resultant products and sequencing 35 clones. This procedure can be applied to retrieve mutated sequences that exist at an extremely low frequency in a DNA population.

Estrogen and progesterone receptors in breast cancer microsamples simultaneously quantified by enzyme-ligand immunoassay.

A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).

Development of a direct and specific enzymeimmunoassay for the measurement of oestrone sulfate in bovine milk.

A 12-step procedure is described for the synthesis of an oestrone-3-sulfate-6-hemisuccinate-BSA immunogen with oestradiol as starting material and the production of specific polyclonal antibodies. A competitive inhibition-type enzymeimmunoassay has been developed based on these specific antibodies using 3-hemisuccinate-oestrone-peroxidase as conjugate for direct measurement of the hormone in body fluids. The method has a minimum sensitivity of 0.03 ng ml-1 in bovine milk, and satisfactory specificity, recovery and reproducibility. In a small field trial with a group of 20 pregnant cows that were followed throughout gestation, it was shown that the assay is potentially an accurate pregnancy test for assessing the viability of the fetoplacental unit at approximately 100 days after insemination. The assay is well suited for routine testing, particularly as a confirmatory bovine pregnancy test.

Comparison of different antibody-conjugate derivatives for the development of a sensitive and specific progesterone assay.

Antibodies raised against progesterone with hormone-carrier protein bridges placed at four different carbon positions were used to compare the sensitivity and specificity of homologous and heterologous enzyme-hormone conjugates. All heterologous assays were at least twice as sensitive as the corresponding homologous assays. The best results were obtained by using antibodies against 7 alpha-carboxyethyl-thioether-progesterone with 6 beta-hemisuccinate-progesterone conjugate (or hemimaleate). The sensitivity with human sera was 0.25 ng ml-1 and, the highest crossreaction 10% with 5 beta-pregnane-3,20-dione, and reproducibility, recovery and accuracy were satisfactory. The correlation coefficient with radioimmunoassay in 103 human sera tested was r = 0.915. The assay was successfully applied for the diagnosis of pregnancy in dairy cattle.