High density DNA methylation array with single CpG site resolution

We have developed a new generation of genome-wide DNA methylation BeadChip which allows high-throughput methylation profiling of the human genome. The new high density BeadChip can assay over 480K CpG sites and analyze twelve samples in parallel. The innovative content includes coverage of 99% of RefSeq genes with multiple probes per gene, 96% of CpG islands from the UCSC database, CpG island shores and additional content selected from whole-genome bisulfite sequencing data and input from DNA methylation experts. The well-characterized Infinium® Assay is used for analysis of CpG methylation using bisulfite-converted genomic DNA. We applied this technology to analyze DNA methylation in normal and tumor DNA samples and compared results with whole-genome bisulfite sequencing (WGBS) data obtained for the same samples. Highly comparable DNA methylation profiles were generated by the array and sequencing methods (average R² of 0.95). The ability to determine genome-wide methylation patterns will rapidly advance methylation research.     

Yeasts utilizing the surface-active substance Laurox-9

Yeasts capable of utilizing the non-ionogenic surfactant Laurox-9 were identified as Cryptococcus humicolus (Daszewska) Golubev and Rhodotorula mucilaginosa (J?erg) Harrison. IR spectrometry used for the quantitative assay of Laurox-9 showed that R. mucilaginosa utilized it as a sole carbon source. The first step of Laurox-9 metabolism was its hydrolytic cleavage yielding lauric acid and polyethyleneglycol. Optimal conditions were found for the electron-cytochemical localization of hydrolase involved in the primary degradation of Laurox-9 by yeasts. Its localization of the exocellular components was established.     

Production of lincomycin by Micromonospora halophytica culture

A Micromonospara culture designated as 991/78 with activity against gram-positive cocci and bacteria was isolated from samples of silt-covered substrates from the Amu-Darya. Directed screening on a selective medium supplemented with lincomycin in an amount of 50-100 micrograms/ml was used. Identification of the antibiotic produced by the culture showed it to be lincomycin. By its taxonomic features the culture was classified as belonging to Micromonospora (subgroup II, Cinnamomea) and in particular to M. halophytica (Weinstein, Luedemann, Oden, Wagman, 1968). Up to now, it was known that lincomycin was produced only by Streptomyces cultures.     

Neural differentiation of ectodermal explants of the early gastrula from Rana temporaria under the action of various mitogens and kainic acid

The following mitogens: concanavalin A (con A), phytohemagglutinin (PHA), hydra growth factor (HGF) as well as neurotoxic agent kainic acid, caused neural differentiation (N) effects differed in value and also in character of dependence on concentration of the agent. The lowest effective concentration of con A was 75 micrograms/ml (15% neural differentiation, treatment during 3 h), and the effect reached maximum of 50-60% at 100-200 micrograms/ml. Con A concentration 50 micrograms/ml showed no effect but after 1% rabbit gamma-globulin was added, 17% neural differentiation was detected. N-effects observed after treatment of explants with con A (200 micrograms/ml, 3h) at 2 degrees and 21 degrees were similar (58 and 42% respectively). Minimum PHA concentration used (6 micrograms/ml, 18h) led to neural differentiation in 5% of explants. N-effect of PHA increased along with the concentration of the lectin and was most pronounced at 25 micrograms/ml. However, further increase in concentration (up to 200 micrograms/ml) resulted in decrease of its N-effect to 13%. At 12 micrograms/ml PHA exerted not only neural differentiation, but also lens-inducing (32%) action on the ectoderm. N-effect of HGF (2.5, 25 and 250 micrograms/ml) was lower as compared with the maximum effects of con A and PHA (30-35%). No correlation of HGF inducing action with its concentration was observed. Kainic acid showed weak N-effect (20-30%) at 1 and 10 micrograms/ml. Higher concentration (100 micrograms/ml) had no N-effect, but in 27% of explants "free" lentoids were found. Oubain (10(-3) and 10(-4) M) and HEPES (20 mM) did not affect the differentiation of explants.     

Cyclosporin biosynthesis and dynamics of bioenergetic processes in cyclosporin-producing strain

An attempt was made to show a correlation between definite bioenergetic parameters of the cells of the cyclosporine-producing culture and biosynthesis of cyclosporine. It was found that the three strains producing cyclosporine used in the study had an alternative cyanide-resistant pathway along with the classical cytochrome chain. In the strain forming only traces of the cyclopeptide during fermentation of the cyanide-resistant respiration constituted 60 to 80%. In the isogenic highly productive strains the cyanide-resistant respiration appeared to be markedly decreased beginning from the 1st day of fermentation and during the maximum biosynthesis of cyclosporine (on day 4 or 5 of fermentation) it reached zero. The ATP content in the cells of the highly productive strain, despite its decrease by the antibiotic biosynthesis peak, remained at a much higher level than that in the strain producing only traces of cyclosporine. A procedure for isolating functionally active mitochondria from the protoplasts was developed and a bioenergetic characterization of the mitochondria isolated from the strains with different antibiotic productions is presented.     

Purification and some catalytic properties of phosphoglucomutase from maize leaves

Phosphoglucomutase (EC 2.7.5.1, PGM) was purified to homogeneity from maize (Zea mays L.) leaves. The enzyme had specific activity 11. 7 U/mg protein and molecular mass (determined by gel-chromatography) of 133 +/- 4 kD. The molecular mass of PGM subunits determined by SDS-electrophoresis was 66 +/- 3 kD. The enzyme had Km for glucose-1-phosphate and glucose-1,6-diphosphate of 20.0 +/- 0.9 and 16.0 +/- 0.8 &mgr;M, respectively. Concentrations of glucose-1-phosphate and glucose-1,6-diphosphate above 3 and 0.4 mM, respectively, cause substrate inhibition. The enzyme activity was maximal at pH 8.0 and temperature 35 degreesC. Magnesium ions activate the enzyme and manganese ions inhibit it. 3-Phosphoglycerate is an uncompetitive inhibitor of the enzyme (Ki = 1.22 +/- 0.05 mM). Fructose-6-phosphate, 6-phosphogluconate, and ADP activate PGM, whereas ATP, UTP, and AMP inhibit the enzyme. Citrate was also a potent inhibitor, inhibitory effects of isocitrate and cis-aconitate being less pronounced.     

Increased T-type Ca2+ channel activity as a determinant of cellular toxicity in neuronal cell lines expressing polyglutamine-expanded human androgen receptors

We have analyzed Ca²⁺ currents in two neuroblastoma-motor neuron hybrid cell lines that expressed normal or glutamine-expanded human androgen receptors (polyGln-expanded AR) either transiently or stably. The cell lines express a unique, low-threshold, transient type of Ca²⁺ current that is not affected by L-type Ca²⁺ channel blocker (PN 200-110), N-type Ca²⁺ channel blocker (-conotoxin GVIA) or P-type Ca²⁺ channel blocker (Agatoxin IVA) but is blocked by either Cd²⁺ or Ni²⁺. This pharmacological profile most closely resembles that of T-type Ca²⁺ channels [1-3]. Exposure to androgen had no effect on control cell lines or cells transfected with normal AR but significantly changed the steady-state activation in cells transfected with expanded AR. The observed negative shift in steady-state activation results in a large increase in the T-type Ca²⁺ channel window current. We suggest that Ca²⁺ overload due to abnormal voltage-dependence of transient Ca²⁺ channel activation may contribute to motor neuron toxicity in spinobulbar muscular atrophy (SBMA). This hypothesis is supported by the additional finding that, at concentrations that selectively block T-type Ca²⁺ channel currents, Ni²⁺ significantly reduced cell death in cell lines transfected with polyGln-expanded AR.