Determination of intermediary metabolites in yeast. Critical examination of the effect of sampling conditions and recommendations for obtaining true levels

Summary The effect of sampling conditions on the levels of adenine nucleotides, pyridine nucleotides, glycolytic intermediates and related metabolites in yeast has been studied. A systematic examination of the conditions for harvesting has shown that it can be best accomplished by rapid filtration. Delays in the handling for removal of the medium, as is usual in the process of obtaining a number of data reported in the literature, lead to important changes in some of the metabolites examined. It is also shown that when a washing is imperative it can be carried out with a methanol-water mixture (50/50, v/v) cooled at –40° without loss of intracellular concentrations of non-readily diffusible metabolites.On the basis of this experience the outline of a generally applicable procedure is presented.     

Structural and electronic properties of the liver fluke hemoglobin heme cavity by nuclear magnetic resonance: hemin isotope labeling

Reconstitution of liver fluke (Dicrocoelium dendriticum) apo-hemoglobin with hemins selectively deuterated at specific positions has permitted the assignment of several heme resonances in the proton nuclear magnetic resonance spectrum of the Met-aquo and Met-cyano forms of the holoprotein. It was established that in the Met-aquo form the meso protons resonate at positions characteristic of a six-co-ordinated in-plane iron. From this, we deduced that the Met-aquo species retains a bound water molecule at pH values as low as 4.5. The orientation of the proximal histidine imidazole ring with respect to the heme group in the cavity was determined through the identification of the heme methyl signals and the analysis of the hyperfine shift pattern in the Met-cyano hemoglobin proton nuclear magnetic resonance spectrum. Compared to sperm whale myoglobin, the heme appears to be rotated by 180 degrees about the alpha, gamma meso-axis. Protein isomers with the heme group in a reversed orientation were not detected, even shortly after reconstitution. In the Met-cyano form, the resonances most affected by the Bohr transition were shown to arise from the heme propionates.     

Isolation of high-lipid content strains of the marine microalga <Emphasis Type="Italic">Tetraselmis suecica</Emphasis> for biodiesel production by flow cytometry and single-cell sorting

Biodiesel from algae is considered an alternative for a third generation of biofuels. However, most microalgae are not lipogenic during fast growth periods, but high-lipid content occurs at resting stages. Microalgae biomass production for biodiesel needs continuous high volumetric and aerial yields and large amount of neutral lipid in the biomass. These requirements are similar to demanding a marathon runner to be obese. We show that by using cell sorting capabilities of flow cytometers, in combination with the lipid-soluble fluorescent dye Nile Red, we can isolate and select cells with a high and stable lipid content. In our study, we were able to select the equivalent of a stable “fat marathon runner” through three sorting events obtained from wild populations of Tetraselmis suecica.     

Biomarker of food intake for assessing the consumption of dairy and egg products

Dairy and egg products constitute an important part of Western diets as they represent an excellent source of high-quality proteins, vitamins, minerals and fats. Dairy and egg products are highly diverse and their associations with a range of nutritional and health outcomes are therefore heterogeneous. Such associations are also often weak or debated due to the difficulty in establishing correct assessments of dietary intake. Therefore, in order to better characterize associations between the consumption of these foods and health outcomes, it is important to identify reliable biomarkers of their intake. Biomarkers of food intake (BFIs) provide an accurate measure of intake, which is independent of the memory and sincerity of the subjects as well as of their knowledge about the consumed foods. We have, therefore, conducted a systematic search of the scientific literature to evaluate the current status of potential BFIs for dairy products and BFIs for egg products commonly consumed in Europe. Strikingly, only a limited number of compounds have been reported as markers for the intake of these products and none of them have been sufficiently validated. A series of challenges hinders the identification and validation of BFI for dairy and egg products, in particular, the heterogeneous composition of these foods and the lack of specificity of the markers identified so far. Further studies are, therefore, necessary to validate these compounds and to discover new candidate BFIs. Untargeted metabolomic strategies may allow the identification of novel biomarkers, which, when taken separately or in combination, could be used to assess the intake of dairy and egg products.     

Isolation of plant DNA: A fast,inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.     

Anti-Osteoporotic Drug Release from Ordered Mesoporous Bioceramics: Experiments and Modeling

The release of a potent bone-resorption inhibitor such as zoledronate from a versatile drug delivery system such as SBA 15 has been modeled. The initial and boundary conditions have been defined, together with the system parameters, including the determination of equilibrium and transport parameters. Additionally, the experimental model of the same system has been observed to validate the prediction here developed. This approach represents a powerful tool for the designing of mesoporous implantable drug delivery systems because their release kinetics can be predicted in advance, and this leads to a considerable time and resources saving.