A single mutation in 16S rRNA that affects mRNA binding and translation-termination.

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.     

Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.     

Characterization of crystals of genetically engineered human manganese superoxide dismutase

The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.     

Assignment of the human gene for muscle-type phosphofructokinase (PFKM) to chromosome 1 (region cen leads to q32) using somatic cell hybrids and monoclonal anti-M antibody

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), live-type (L), and platelet-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign human PFKM locus to a specific chromosome we have analyzed human x Chinese hamster somatic cell hybrids for the expression of human M subunits, using an anti-human M subunit-specific mouse monoclonal antibody. In 18 of 19 hybrids studied, the expression of the PFKM locus segregated concordantly with the presence of chromosome 1 (discordance rate 0.05) as indicated by chromosome and isozyme marker analysis. The discordance rates for all the other chromosomes were 0.32 or greater, indicating that the PFKM locus is on chromosome 1. For the regional mapping of PFKM, eight hybrids were studied that contained one of five distinct regions of chromosome 1. These results further localize the human PFKM locus to region cen leads to q32 chromosome 1.     

High temporal resolution of ion fluxes in semi-natural ecosystems – gain of information or waste of resources?

Monitoring programs of ion concentrations and fluxes in semi-natural ecosystems are confronted with the task to gain as much information as possible with simultaneously minimizing costs and efforts. The aim of this study was (i) to assess how much of the heterogeneity of solution concentrations is lost because of temporal integration of measurements and (ii) to estimate the error in ion fluxes due to temporal integration. High resolution measurements (daily interval) of ion concentrations (sulfate, nitrate, chloride, pH and EC) in throughfall, soil solutions and runoff at the catchment Lehstenbach (Fichtelgebirge, Northeast Bavaria, Germany) were compared over a two year period with the reference monitoring program (biweekly measurement interval). Evaluation of the maximum temporal heterogeneity of ion concentrations in throughfall, soil solution and runoff (expressed as minimum, maximum, median and 25–75% percentile) did not result in an overall higher heterogeneity of the high resolution measurements compared to the reference program. The calculation of runoff fluxes from the reference data (biweekly concentration) resulted in significant errors of up to 25% for time periods < 1 year (high resolution data was considered the "true" value and set as 100%). However, errors became minor (< 10%) if longer time periods were considered. The suitability of different interpolation methods to up-scale biweekly concentration data for the calculation of runoff fluxes was evaluated in this study. We concluded for the monitoring programs at the Lehstenbach catchment that a biweekly measurement interval seemed to be suitable to capture the heterogeneity of ion concentrations and fluxes (and thus temporal trends). In comparison, high resolution measurements with a daily measurement interval were higher in cost, work and time resources and had a relatively low information gain. While the introduced methods are applicable in all monitoring programs, conclusions on temporal resolution of measurements are most likely not valid for systems where ion concentrations have a low autocorrelation length (e.g., agricultural or urban systems with nitrate or pesticide treatment; tropical systems with extreme temperature or hydrological events).     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Drug distribution studies with microdialysis. II. Caffeine and theophylline in blood, brain and other tissues in rats

Microdialysis was applied to estimate the pharmacologically active concentration of caffeine and theophylline in blood, adipose tissue, muscle, liver and brain of rats. The concentration of the drugs in the extracellular space was estimated by perfusion with varying concentrations of the drug through the microdialysis probe (difference method). Caffeine (20 mg/kg) was found to be evenly distributed with a free concentration of approximately 120 microM. Theophylline concentration in the brain was 91 microM and in other tissues approximately 120 microM. The rate of penetration into brain extracellular space was higher for caffeine than for theophylline. It is suggested that the lower levels of theophylline attained in the brain may to some extent explain the differences in clinical action profile between caffeine and theophylline.     

Phylogeography and molecular evolution of potato virus Y

Potato virus Y (PVY) is an important plant pathogen, whose host range includes economically important crops such as potato, tobacco, tomato, and pepper. PVY presents three main strains (PVY(O), PVY(N) and PVY(C)) and several recombinant forms. PVY has a worldwide distribution, yet the mechanisms that promote and maintain its population structure and genetic diversity are still unclear. In this study, we used a pool of 77 complete PVY genomes from isolates collected worldwide. After removing the effect of recombination in our data set, we used bayesian techniques to study the influence of geography and host species in both PVY population structure and dynamics. We have also performed selection and covariation analyses to identify evolutionarily relevant amino acid residues. Our results show that both geographic and host-driven adaptations explain PVY diversification. Furthermore, purifying selection is the main force driving PVY evolution, although some indications of positive selection accounted for the diversification of the different strains. Interestingly, the analysis of P3N-PIPO, a recently described gene in potyviruses, seems to show a variable length among the isolates analyzed, and this variability is explained, in part, by host-driven adaptation.     

Morphological effects of tributyltin (TBT) in vitro on the genital system of the mesogastropodaLittorina littorea (Prosobranchia)

This ultrastructural study investigates the pathological changes in the penial, the sperm groove and the glandular cushion epithelium in maleLittorina littorea (Mesogastropoda) related to TBT (tributyltin) contamination. The results are compared with those onOcinebrina aciculata (Neogastropoda), which shows a wide range of cell changes in the penis epithelia of male and imposex affected females. The investigation of the different penis epithelia ofL. littorea revealed that the cells analysed show a low sensibility towards TBT. The cells display normal metabolism. Certain atypical structures like swelling microvilli and cristae which tend to lie parallel to the long axis in the mitochondria, were detected as pathological effects.