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While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-[beta]-D-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments.

Metabonomic analysis of urine utilizing high-resolution NMR spectroscopy and chemometric techniques has proven valuable in characterizing the biochemical response to an intervention. To assess the effect of magnetic field strength on information contained in NMR-based metabonomic data sets, IH NMR spectra were acquired on 250-, 400-, 500-, and 800-MHz instruments, respectively, on the same set of human urine samples collected before and after dietary interventions with milk and with meat proteins. Partial least-squares regression discriminant analyses (PLS-DA) were performed in order to elucidate the ability of the [sup.1]H spectra acquired at various field strengths to identify possible spectral differences and discriminate between pre- and postintervention samples. The loadings from PLS-DA contained the same spectral regions, implying that the same metabolites were involved in the discrimination independent of magnetic field strength. The investigation revealed a strong increase in prediction performance and thereby spectral information content when increasing the magnetic field strength from 250 to 500 MHz, while from 500 to 800 MHz the increase was less pronounced.

Frequent exposure of terrestrial insects to temperature variation has led to the evolution of protective biochemical and physiological mechanisms, such as the heat shock response, which markedly increases the tolerance to heat stress. Insight into such mechanisms has, so far, mainly relied on selective studies of specific compounds or characteristics or studies tit the genomic or proteomic levels. In the present study, we have used untargeted NMR metabolomic profiling to examine the biological response to heat stress in Drosophila melanogaster. The metabolite profile was analyzed during recovery after exposure to different thermal stress treatments and compared with untreated controls. Both moderate and severe heat stress gave clear effects on the metabolite profiles. The profiles clearly demonstrated that hardening by moderate heat stress led to a faster reestablishment of metabolite homeostasis after subsequent heat stress. Several metabolites were identified as responsive to heat stress and could be related to known physiological and biochemical responses. The time course of the recovery of metabolite homeostasis mirrored general changes in gene expression, showing that recovery follows the same temporal pattern tit these two biological levels. Finally, our data show that heat hardening permits a quicker return to homeostasis, rather than a reduction of the acute metabolic perturbation and that the reestablishment of homeostasis is important for obtaining maximal heat-hardening effect. The results display the power of NMR metabolomic profiling for characterization of the instantaneous physiological condition, enabling direct visualization of the perturbation of and return to homeostasis. metabolomics; heat shock protein; nuclear magnetic resonance spectroscopy; insect

The copepod Apocyclops royi tolerates very low salinity while being highly capable of synthesizing n‐3 fatty acids based on PUFA‐poor diets. The feasibility of culturing A. royi in freshwater fed with baker's yeast was evaluated. The copepods were adjusted for 8 days by decreasing stepwise from salinity 20 to freshwater diluting with deionized water. Initially, the cultures exhibited adequate survival and reproduction. However, after 10 days in freshwater, they stopped reproducing presumably due to the lack of vital ions in the medium. Therefore, before continuing the freshwater cultures, we tested the survival of a batch of salinity 1 pre‐acclimated adults and copepodites in various types of freshwater: deionized, bottled 'mineral' and tap water for 48 h. All copepods died in deionized water, but a significantly higher survival, 29% ± 11%, was found in mineral water versus 11% ± 8% in tap water. Hence, we continued culturing the copepods in freshwater using 'Maglekilde spring' water as diluter. The copepods regained and kept reproducing until day 71 where the experiment was terminated. After a 48 h freshwater versus salinity 20 exposure, nauplii survival was significantly less in freshwater 25 ± 12 versus 79 ± 20 (±STD) at salinity 20 but not different in mean body length. For the first time, we conducted NMR metabolomics revealing a large decrease in glycine betaine and a large increase in lactate in the copepods when challenged by freshwater versus salinity 20. There is obviously a 'price to pay' when culturing not only the copepods in freshwater, but also perspectives in developing a 'universal' A. royi live‐feed product. [ABSTRACT FROM AUTHOR]

Research was conducted to gain an insight in the role of Mg2+ in Ca2+/CaM signaling at physiological conditions. The Ca2+ affinity of the TR1C fragment of vertebrate CaM was calculated as a function of Mg2+ concentration through the use of a fluorescent chelator. The parameter values indicate that the (Mg2+)1(Ca2+)1 state has a conformation more similar to the 'closed' apo and (Mg2+)2 states than to the 'open' (Ca2+)2 state.

The effect of structural context and amino acid sequence on the backbone dynamics of helix-loop-helix calcium-binding motifs (EF-hand) was examined via 15N spin relaxation measurements performed on the calcium-free state of the calbindin D9k mutant. The findings were then analyzed relative to a previous study regarding the apo state of the wild-type-like P43G D9k mutant. The results revealed that the N-terminal EF-hand facilitates the formation of a stable structure that enables calcium binding with only minor modifications.

The solution structure of the dicerium(III) complex of the N-terminal domain of calmodulin (Ce2-TR1C) was solved through paramagnetic T1 relaxation improvements and pseudocontact shifts from the Ce3+ ions. A family of structures was computed through the torsion angle dynamics program DYANA. Longitudinal proton relaxation times aided in approximating the location of the metal ions within the protein. The solution structure allowed comparison with the solid-state structure of calcium-loaded calmodulin at 1.7 Angstroms.

The Ca2+ activation of the C-terminal domain of bovine calmodulin and the influence of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV have been examined. The results of NMR spectroscopy showed that the (Ca2+)2 form of the mutant undergoes a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin.