A non-azole inhibitor of lanosterol 14 alpha-methyl demethylase in Candida albicans.

6-Hydroxy-N-methyl-N-(2-[4-phenylphenyl] ethyl)-1,2,3,4-tetrahydro-1- napthalene methanamine (A60586), a new non-azole inhibitor of ergosterol biosynthesis in Candida albicans ATCC62376 has been identified. In whole cells A60586 produced a dose related reduction of [14C]acetate incorporation into ergosterol and a concurrent increase in the radiolabelling of 4,4-dimethylated sterols. Similar observations were made with [14C]mevalonic acid lactone labelled cell free extracts. The IC50s for inhibition of ergosterol in the whole cell and cell free systems were 22 microM (10 mg/L) and 7.8 microM (3.5 mg/L), respectively. Analysis by gas chromatography of sterols from cells previously incubated at 37 degrees C for 24 h with A60586 (200 mg/L) confirmed the presence of lanosterol and 14 alpha-methyl fecosterol. These data indicate that A60586, inhibits the demethylation of the C-14 methyl group of lanosterol. The MIC of A60586 for several candida strains ranged from 12.5 to 50 mg/L, and against Cryptococcus albidus and Aspergillus niger ranged from 50 to 100 mg/L. The best in-vitro activity of A60586 was against Torulopsis glabrata (MIC range = 3.12 to 50 mg/L). The membrane permeabilizing effect of this compound (50% leakage of [14C]aminoisobutyric acid at 70 mg/L A60586) may have contributed to its in-vitro antifungal activity.     

Specificity of plasma HVA response to dexamethasone in psychotic depression

Earlier reports have suggested that dexamethasone significantly increases levels of plasma homovanillic acid (HVA) in normal subjects, but that this effect may be altered in some depressed patients. To investigate the specificity of such alterations, we administered dexamethasone (1 mg p.o. at 11 p.m.) to 33 normal subjects, 27 depressed patients (8 with psychotic features), and 16 schizophrenic patients. Plasma for assay of cortisol and HVA was obtained at 4 p.m. before and on the day following dexamethasone administration. Dexamethasone induced significant increases in plasma HVA in the normal subjects and in the schizophrenic patients, but not in the depressed patients. Indeed, psychotically depressed patients tended to show a dexamethasone-associated decrease in plasma levels of HVA. In contrast to cortisol "suppression" or "nonsuppression," dexamethasone-induced changes in plasma levels of HVA (i.e., increases or decreases) sensitively and specifically discriminated between patients with affective and nonaffective psychoses.     

Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.