State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

Time course of pump inhibition by ouabain in amphibian epithelia

Inhibition by ouabain of rheogenic Na⁺ transport across the basolateral membranes of frog skin is found to be manifest within 3–4 min. This rate of pump inhibition is not different from the rate of diffusion through extracellular tissue layers between the serosal bath and the actual site of action, i.e., the epithelial cell layers. It is concluded that the well-known slow time course of decrease in transepithelial current flow is due ionic redistribution and conductance changes of the epithelial membranes secondary to pump inhibition.     

Utilization of medium-chain triglycerides and octanoic acid by cultured human fibroblasts and lymphoblasts

Cultured human fibroblasts and lymphoblasts were incubated with emulsions containing 14C-trioctanoin or 14C-tripalmitin. Both cell types were able to hydrolyse the medium-chain triglyceride but not the long-chain triglyceride to the corresponding fatty acids. At the end of a 3 days incubation period, 25-30% of the initial amount of 10 nmol/ml trioctanoin were present as triglyceride. The observed hydrolysis seems to be mediated by an esterase secreted into the culture medium, as was shown by the use of cell-conditioned medium. CO2 production from octanoic acid was below 2 nmol per mg protein and day, demonstrating that these cells have a low capacity to use this substrate for their energy metabolism.     

Dissimilatory hexaheme c nitrite reductase of “Spirillum” strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO _inf2 ^sup- × (mg protein × min)^-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/M_r 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.Abbreviations DNRA dissimilatory nitrate reduction to ammonia - EPR electron paramagnetic resonance - PAGE polyacrylamide gel electrophoresis - NaP_i sodium phosphate - SDS sodium dodecylsulfate     

Diurnal variations in abscisic acid content and stomatal response to applied abscisic acid in leaves of irrigated and non-irrigated Arbutus unedo plants under naturally fluctuating environmental conditions

Summary Endogenous abscisic acid content (ABA) of Arbutus unedo leaves growing under natural conditions in a macchia near Sobreda, Portugal, was very high (0.25 to 2.3 g g¹ fresh weight). Highest concentrations were found during the very early morning hours and at midday. During the late morning hours and in the late afternoon ABA concentrations decreased to between one-third and one-fourth of peak values. The samples for ABA content were obtained from both irrigated ( between-10 and-25 bar) and non-irrigated plants experiencing natural water stress during the dry season ( of-50 bar). During the course of the measurement day, stomatal conductance was relatively constant and conductance of watered plants was 50 to 100% greater than that of unwatered plants. No clear correlations between ABA content and stomatal conductance and/or xylem water potential were observed. Despite large differences in water potential and differences in degree of stomatal opening, absolute concentrations of ABA were not found to differ.Small quantities (8–14 pmoles cm² leaf area) of ABA were applied to leaves of irrigated and non-irrigated Arbutus unedo plants by injection into the petiole. These extremely small ABA doses resulted in transient reductions in stomatal conductance. The effectiveness with which injected ABA closed stomata was highest during the morning and decreased substantially at midday. Increased sensitivity to injected ABA may again occur in the late afternoon but recent measurements suggest that this may depend on long-term drought experience of the plants. The characteristics of the response to injected ABA were similar in irrigated and non-irrigated plants although irrigated plants responded in general more strongly.     

Characterization of recombinant human antithrombin III synthesized in Chinese hamster ovary cells

Biochemical and physiochemical properties of recombinant human antithrombin III were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human plasma when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Comparison of the NH2-terminal sequences of recombinant and human plasma-derived antithrombin III showed that on synthesis and secretion of the recombinant protein from Chinese hamster ovary cells the signal peptide is correctly cleaved by the corresponding endoplasmic signal peptidase. The recombinant antithrombin III has identical properties in heparin binding and biological activities as determined in vitro by two-dimensional immunoelectrophoresis, progressive inhibitor, and heparin cofactor assays. Analysis of the carbohydrate portion of recombinant antithrombin III synthesized in Chinese hamster ovary cells revealed glycosylation of the complex type. Characterization of the oligosaccharide chains present in the recombinant protein reveals three major fractions, A (20%), B (60%), and C (20%). Fraction A contains tri- and tetraantennary complex-type oligosaccharides, fraction B contains biantennary oligosaccharides, and fraction C partially truncated biantennary structures. Pharmacokinetic studies with recombinant and plasma-derived antithrombin III in rabbits showed that the clearance behavior of both proteins is very similar and can be described by a double exponential decrease with almost identical kinetic parameters.     

Isolation and characterization of acetonitrile utilizing bacteria

Summary Bacteria utilizing high concentrations of acetonitrile as the sole carbon source were isolated and identified asChromobacterium sp. andPseudomonas aeruginosa. Maximum growth was attained after 96 h of incubation andP. aeruginosa grew slightly faster thanChromobacterium sp. The strains were able to grow and oxidize acetonitrile at concentrations as high as 600 mM. However, higher concentrations inhibited growth and oxygen uptake. Degradation studies with (¹⁴C)acetonitrile indicated 57% of acetonitrile was degraded byPseudomonas aeruginosa as compared to 43% byChromobacterium. The isolates utilized different nitrile compounds as carbon substrates.     

The xylem sap of maple (Acer platanoides) trees--sap obtained by a novel method shows changes with season and height

Xylem sap of log pieces of maple trees was collected by a novelpressure/decompression method developed recently for the mechanicaldrying of timber. Seasonal changes and spatial distributionsof the osmotic potential, the pH and the concentrations of majorsolutes and of the plant stress-hormone abscisic acid (ABA)were investigated. Sucrose and quebrachitol were the main components of the xylemsap. The sucrose concentration varied between 10 mM and 25 mMduring the winter months and declined to a minimum during theperiod of bud burst. Quebrachitol was found in concentrationsof up to 7 mM with a high variability throughout the year. Highconcentrations of ABA were measured during the summer seasonand in mid-winter. Rainfall caused an increase of ABA in somesamples. The osmotic potential of the xylem sap increased with the heightof the collection site. The pH of the sap decreased by approximatelyone unit between the base of the trunk and the crown. The increaseof the osmotic potential was mainly due to sucrose, quebrachitoland potassium. Malate contributed to the decrease of the pH.ABA of the xylem sap increased with decreasing moisture contentof the wood. Key words: Pressure/decompression method, xylem sap, abscisic acid, sucrose, quebrachitol, Acer platanoides     

Transformation of the extremely thermoacidophilic archaeon Sulfolobus solfataricus via a self-spreading vector

Abstract The comparative chromosomal locations of polymeric β-fructosidase SUC genes have been determined by Southern blot hybridization with the SUC2 probe in 91 different strains of Saccharomyces cerevisiae . Most of the strains exhibited a single SUC2 gene, but in some strains two or three SUC genes were found. All Suc⁻ strains carried a silent suc2⁰ sequence. The accumulation of SUC genes was observed in populations derived from sources containing sucrose and seems to be absent in strains from sources promoting the MEL gene.