Factors affecting homologous overexpression of the Saccharomyces cerevisiae lanosterol 14 alpha-demethylase gene.

The Saccharomyces cerevisiae Lanosterol 14 alpha-demethylase (14DM) gene was overexpressed in S. cerevisiae using promoter sequences of the highly expressed S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase (TDH3) gene. To investigate factors affecting 14DM overproduction, the levels of 14DM-specific RNAs, apoprotein, and heme protein, respectively, were determined and the 14DM-specific RNA levels compared with the RNA levels originating from the endogenous TDH gene(s). The quantitative measurements revealed that the 14DM steady-state RNA levels reached were some three- to five-fold below the theoretically expected values. With a view towards further improving expression of the 14DM gene, the spacing between the TDH3 promoter and the AUG was adjusted precisely and to rule out possible toxic effects exerted by the 14DM protein, the TDH3 promoter was placed under galactose regulation by introducing an UASG segment. Furthermore, the effects of the gene copy number on 14DM overproduction were investigated. From the analysis of the improved expression constructs five conclusions could be reached: (1) expression from the native 14DM gene is comparable to the expression driven by the TDH3 promoter-14DM fusion construct on single copy plasmid vectors; (2) expression from the TDH3 promoter-14DM construct on single-copy vectors is nearly as efficient as expression from the corresponding endogenous TDH3 gene; (3) the gene copy number has an effect on the relative expression levels of the TDH3 promoter-14DM constructs; (4) the steady-state amounts of protein produced are very nearly proportional to gene dosage; and (5) protein toxicity does not have a major impact on 14DM production. The maximum yield of 14DM was in the order of 7% of the total yeast protein and the maximum production of functional 14DM heme protein appears to be limited by the availability of heme.     

Predicted ligand interactions of 3'5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.     

Effect of forming gas anneals on the photoluminescence from nanocrystalline silicon formed by Si implantation into SiO2 matrix

The blue region of the room temperature photoluminescence spectrum from Si nanocrystallites formed in SiO₂ by Si⁺ ion implantation has been observed for the first time after annealing in a forming gas (10% H₂ + 90% N₂) ambient. Thermally grown SiO₂ on Si substrates were implanted with a dose of 2 × 10¹⁷ Si⁺ cm⁻² at energies of 200 keV and 400 keV. For reference purposes, quartz silica was implanted also with the same dose of 200 keV Si⁺ ions. The implanted samples were annealed in nitrogen and forming gas at 900°C for 3 to 180 min. Both the SiO₂ and quartz samples exhibited luminescence at about 380 nm which was weak, but detectable, before annealing. During extended anneals in forming gas, the intensity increased by a factor of about 2 above that recorded after a nitrogen anneal but the peak position was unchanged. The intensity was greater in samples annealed in forming gas which is due to the additional hydrogen. It would seem that this blue luminescence originates from new luminescent centres in the matrix caused by the Si⁺ ion implantation.     

Salt reduction in municipal sewage allocated for reuse: the outcome of a new policy in Israel.

Israel is presently reusing about 70% of its sewage and the buildup of salts in soils has been detected in some areas of the country. Prevention of sewage salt enrichment is one of the most immediately available solutions. The discharge of brines to municipal sewers is now prohibited and they are discharged to the sea. Discharge of brines to the sea has increased from 580,000 m3 in 1999 to more than 700,000 m3 in 2002. These salts are thus definitively taken out of the reuse cycle, year after year. The concentration of salts in industrial effluents is presently limited by the national water law and a unique regulation limiting the Boron and Sodium content in detergents has been implemented. Israeli industry has undergone a radical change in recent years. Many factories and all the hospitals have adopted K or Ca for softening and neutralization, while others have shifted to reverse osmosis. The concentration of Cl and Na in the sewage reaching treatment plants is declining. The addition of Cl to sewage has dropped from 120 mg/l to 70 mg/l. The average Boron concentration in sewage has dropped from 0.6 mg/l to 0.3 mg/l and should reach 0.2 mg/l by 2008.     

Sleep-disordered breathing and neuropsychological deficits. A population-based study

The relationship of sleep-disordered breathing (SOB) to neuropsychological deficits was investigated with cross-sectional data from the Wisconsin Sleep Cohort Study, a population-based study of the natural history of SDB. A sample of 841 employed men and women ages 30 to 60 yr was studied by overnight polysomnography to assess the frequency of apneas and hypopneas per hour of sleep (apnea-hypopnea index, AHI). Prior to overnight polysomnography, the participants were given a battery of neuropsychological tests for functionally important capacities including motor skills, attention, concentration, information processing, and memory. Principal factor analysis of all the neuro-psychological test data revealed a psychomotor efficiency and a memory factor. Multiple regression analysis showed a significant negative association between logarithmically transformed AHI (LogAHI) and psychomotor efficiency score independent of age, gender, and educational status (p = 0.017). The relationship was not explained by self-reported sleepiness. No significant relationship was seen between LogAHI and memory score. In assessing the clinical significance of mild SDB, we estimate that an AHI of 15 is equivalent to the decrement in psychomotor efficiency associated with 5 additional yr of age, or to 50% of the decrement associated with hypnosedative use.     

Design rules for the wallcooled hydrothermal burner (WHB)

The results of a process analysis and an experimental exploratory study of supercritical water oxidation (SCWO) show that elements of combustion technology are suitable to overcome the technical problems of SCWO. The Film Cooled Hydrothermal Burner (FCHB) which operates at 25 MPa up to 2000 K in the reaction zone keeping wall temperature under stress at 300 to 400 K shows further development potential of the Wallcooled Hydrothermal Burner (WHB) concept for SCWO, a still promising waste treatment process.