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.     

Anaerobic degradation of hydroaromatic compounds by newly isolated fermenting bacteria

Aerobic organisms degrade hydroaromatic compounds via the hydroaromatic pathway yielding protocatechuic acid which is further metabolized by oxygenase-mediated ring fission in the 3-oxoadipate pathway. No information exists on anaerobic degradation of hydroaromatics so far. We enriched and isolated from various sources of anoxic sediments several strains of rapidly growing gram-negative bacteria fermenting quinic (1,3,4,5-tetrahydroxy-cyclohexane-1-carboxylic acid) and shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) in the absence of external electron acceptors. Quinic and shikimic acid were the only ones utilized of more than 30 substrates tested. The marine isolates formed acetate, butyrate, and H₂, whereas all freshwater strains formed acetate and propionate as typical fermentation products. Aromatic intermediates were not involved in this degradation. Characterization of the isolates, fermentation balances for both hydroaromatic compounds, and enzyme activities involved in one degradation pathway are presented.Abbreviations BV benzyl viologen (1,1-dibenzyl-4,4-bipyridinium dichloride) - CoA coenzyme A - CTAB cetyltrimethylammonium bronide - DCPIP 2,4-dichlorophenolindophenol - DTT 1,4-dithiotheriol - MV methyl viologen (1,1-dimethyl-4,4-bipyridinium dichloride) - Tricine N-[tris-(hydroxymethyl)-methyl]-glycine - Tris tris-(hydroxymethyl)-aminomethane     

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     

Noncyclic electron transport in chromatophores from photolithotrophically grown Rhodobacter sulfidophilus

Chromatophores isolated from the marine phototrophic bacterium Rhodobacter sulfidophilus were found to photoreduce NAD with sulfide as the electron donor. The apparent K _m for sulfide was 370 M and the optimal pH was 7.0. The rate of NAD photoreduction in chromatophore suspensions with sulfide as the electron donor (about 7–12 M/h·mol Bchl) was approximately onetenth the rate of sulfide oxidation in whole cell suspensions. NAD photoreduction was inhibited by rotenone, carbonyl cyanide-m-chlorophenylhydrazone, and antimycin A. Sulfide reduced ubiquinone in the dark when added to anaerobic chromatophore suspensions. These results suggest that electron transport from sulfide to NAD involves an initial dark reduction of ubiquinone followed by reverse electron transport from ubiquinol to NAD mediated by NADH dehydrogenase.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide-m-chlorophenylhydrazone - MOPS 3(N-morpholino)-propane sulfonate - Uq ubiquinone     

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.     

Fluorescence lifetimes of dimers and higher oligomers of bacteriochlorophyll c from Chlorobium limicola

Fluorescence lifetimes have been measured for bacteriochlorophyll (BChl) c isolated from Chlorobium limicola in different states of aggregation in non-polar solvents. Two different homologs of BChl c were used, one with an isobutyl group at the 4 position, the other with n-propyl. Species previously identified as dimers (Olson and Pedersen 1990, Photosynth Res, this issue) decayed with lifetimes of 0.64 ns for the isobutyl homolog, 0.71 ns for n-propyl. Decay-associated spectra indicate that the absorption spectrum of the isobutyl dimer is slightly red-shifted from that of the n-propyl dimer. Aggregates absorbing maximally at 710 nm fluoresced with a principal lifetime of 3.1 ns, independent of the homolog used. In CCl₄, only the isobutyl homolog forms a 747-nm absorbing oligomer spectrally similar to BChl c in vivo. This oligomer shows non-exponential fluorescence decay with lifetimes of 67 and 19 ps. Because the two components show different excitation spectra, the higher oligomer is probably a mixture of more than one species, both of which absorb at 747 nm.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - % MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGGipm0dc9vqaqpepu0xbbG8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeq4Xdm2aaW% baaSqabeaacaaIYaaaaaaa!3777!\[\chi ^2 \] chi-square - FWHM full-width at half-maximum