Leakage-free rapid quenching technique for yeast metabolomics

Accurate determination of intracellular metabolite levels requires reliable, reproducible techniques for sampling and sample treatment. Quenching in 60% (v/v) methanol at −40°C is currently the standard method for sub-second arrest of metabolic activity in microbial metabolomics but there have been contradictory reports in the literature on whether leakage of metabolites from the cells occurs. We have re-evaluated this method in S. cerevisiae using a comprehensive, strictly quantitative approach. By determining the levels of a large range of metabolites in different sample fractions and establishing mass balances we could trace their fate during the quenching procedure and confirm that leakage of metabolites from yeast cells does occur during conventional cold methanol quenching, to such an extent that the levels of most metabolites have been previously underestimated by at least twofold. In addition, we found that the extent of leakage depends on the time of exposure, the temperature and the properties of the methanol solutions. Using the mass balance approach we could study the effect of different quenching conditions and demonstrate that leakage can be entirely prevented by quenching in pure methanol at ≤−40°C, which we propose as a new improved method. Making use of improved data on intracellular metabolite levels we also re-evaluated the need of sub-second quenching of metabolic activity and of removing the extracellular medium. Our findings have serious implications for quantitative metabolomics-based fields such as non-stationary ¹³C flux analysis, in vivo kinetic modeling and thermodynamic network analysis.

Evaluation of bioimpedance spectroscopy for measurements of body water distribution in healthy women before, during, and after pregnancy.

Bioimpedance spectroscopy (BIS) is a technique of interest in the study of human pregnancy because it can assess extracellular (ECW), intracellular (ICW), and total body water (TBW) as ECW plus ICW. The technique requires appropriate resistivity coefficients and has not been sufficiently evaluated during the reproductive cycle. Therefore, in a methodological study, we estimated ECW, ICW, and TBW, by means of BIS, and compared the results with the corresponding estimates obtained by using reference methods. Furthermore, results obtained by means of population-specific resistivity coefficients were compared with results obtained by means of general resistivity coefficients. These comparisons were made before pregnancy, in gestational weeks 14 and 32, as well as 2 wk postpartum in 21 healthy women. The reference methods were isotope and bromide dilution. Average ICW, ECW, and TBW, estimated by means of BIS, were in agreement with reference data before pregnancy, in gestational week 14, and postpartum. The corresponding comparison in gestational week 32 showed good agreement for ICW, whereas estimates by means of BIS were significantly (P < 0.001) lower than the corresponding reference values for ECW and TBW. Thus the BIS technique, which was based on a model developed for the nonpregnant body, estimated increases in ICW accurately, whereas increases in ECW and TBW tended to be underestimated. Estimates obtained by using population-specific and general resistivity coefficients were very similar. In conclusion, the results indicated that BIS is potentially useful for studies during pregnancy but that further work is needed before it can be generally applied in such studies.     

Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase

Vacuolar (H+)-ATPase (V-ATPase) is a proton pump present in several compartments of eukaryotic cells to regulate physiological processes. From biochemical studies it is known that the interaction between arginine 735 present in the seventh transmembrane (TM7) segment from subunit a and specific glutamic acid residues in the subunit c assembly plays an essential role in proton translocation. To provide more detailed structural information about this protein domain, a peptide resembling TM7 (denoted peptide MTM7) from Saccharomyces cerevisiae (yeast) V-ATPase was synthesized and dissolved in two membrane-mimicking solvents: DMSO and SDS. For the first time the secondary structure of the putative TM7 segment from subunit a is obtained by the combined use of CD and NMR spectroscopy. SDS micelles reveal an alpha-helical conformation for peptide MTM7 and in DMSO three alpha-helical regions are identified by 2D 1H-NMR. Based on these conformational findings a new structural model is proposed for the putative TM7 in its natural environment. It is composed of 32 amino acid residues that span the membrane in an alpha-helical conformation. It starts at the cytoplasmic side at residue T719 and ends at the luminal side at residue W751. Both the luminal and cytoplasmatic regions of TM7 are stabilized by the neighboring hydrophobic transmembrane segments of subunit a and the subunit c assembly from V-ATPase.     

Pharmacokinetics of poly(hydroxyethyl-l-asparagine)-coated liposomes is superior over that of PEG-coated liposomes at low lipid dose and upon repeated administration

'Stealth' liposomes with a poly(ethylene glycol) (PEG) coating are frequently studied for drug delivery and diagnostic purposes because of their prolonged blood circulation kinetics. However, several recent reports have demonstrated that PEG-liposomes are rapidly cleared at single low lipid doses (<1 micromol/kg) and upon repeated administration (time interval between the injections 5 days-4 weeks). Recently, poly(amino acid)-based stealth liposome coatings have been developed as alternative to the PEG-coating. In this study, the pharmacokinetic behavior of liposomes coated with the poly(amino acid) poly(hydroxyethyl-l-asparagine) (PHEA) was evaluated at low lipid doses and upon repeated administration in rats. Blood circulation times and hepatosplenic localization of PHEA-liposomes were assessed after intravenous injection. When administered at a dose of 0.25 micromol/kg or less, PHEA-liposomes showed significantly longer blood circulation times than PEG-liposomes. A second dose of PHEA-liposomes 1 week after the first injection was less rapidly cleared from the circulation than a second dose of PEG-liposomes. Although the mechanisms behind these observations are still not clear yet, the use of PHEA-liposomes appears beneficial when single low lipid doses and/or repeated dosing schedules are being applied.     

Metabolism and Excretion of Mood Stabilizers and New Anticonvulsants

1. The mood stabilizers lithium, carbamazepine (CBZ), and valproate (VPA), have differing pharmacokinetics, structures, mechanisms of action, efficacy spectra, and adverse effects. Lithium has a low therapeutic index and is renally excreted and hence has renally-mediated but not hepatically-mediated drug–drug interactions.2. CBZ has multiple problematic drug–drug interactions due to its low therapeutic index, metabolism primarily by a single isoform (CYP3A3/4), active epoxide metabolite, susceptibility to CYP3A3/4 or epoxide hydrolase inhibitors, and ability to induce drug metabolism (via both cytochrome P450 oxidation and conjugation). In contrast, VPA has less prominent neurotoxicity and three principal metabolic pathways, rendering it less susceptible to toxicity due to inhibition of its metabolism. However, VPA can increase plasma concentrations of some drugs by inhibiting metabolism and increase free fractions of certain medications by displacing them from plasma proteins.3. Older anticonvulsants such as phenobarbital and phenytoin induce hepatic metabolism, may produce toxicity due to inhibition of their metabolism, and have not gained general acceptance in the treatment of primary psychiatric disorders.4. The newer anticonvulsants felbamate, lamotrigine, topiramate, and tiagabine have different hepatically-mediated drug–drug interactions, while the renally excreted gabapentin lacks hepatic drug–drug interactions but may have reduced bioavailability at higher doses.5. Investigational anticonvulsants such as oxcarbazepine, vigabatrin, and zonisamide appear to have improved pharmacokinetic profiles compared to older agents.6. Thus, several of the newer anticonvulsants lack the problematic drug-drug interactions seen with older agents, and some may even (based on their mechanisms of action and preliminary preclinical and clinical data) ultimately prove to have novel psychotropic effects.     

Guanylate cyclase activity in permeabilized Dictyostelium discoideum cells

Dictyostelium discoideum cells respond to chemoattractants by transient activation of guanylate cyclase. Cyclic GMP is a second messenger that transduces the chemotactic signal. We used an electropermeabilized cell system to investigate the regulation of guanylate cyclase. Enzyme activity in permeabilized cells was dependent on the presence of a nonhydrolysable GTP analogue (e.g., GTPγS), which could not be replaced by GTP, GDP, or GMP. After the initiation of the guanylate cyclase reaction in permeabilized cells only a short burst of activity is observed, because the enzyme is inactivated with a t_1.2 of about 15 s. We show that inactivation is not due to lack of substrate, resealing of the pores in the cell membrane, product inhibition by cGMP, or intrinsic instability of the enzyme. Physiological concentrations of Ca²⁺ ions inhibited the enzyme (half-maximal effect at 0.3 μM), whereas InsP₃ had no effect. Once inactivated, the enzyme could only be reactivated after homogenization of the permeabilized cells and removal of the soluble cell fraction. This suggests that a soluble factor is involved in an autonomous process that inactivates guanylate cyclase and is triggered only after the enzyme is activated. The initial rate of guanylate cyclase activity in permeabilized cells is similar to that in intact, chemotactically activated cells. Moreover, the rate of inactivation of the enzyme in permeabilized cells and that due to adaptation in vivo are about equal. This suggests that the activation and inactivation of guanylate cyclase observed in this permeabilized cell system is related to that of chemotactic activation and adaptation in intact cells. © 1996 Wiley-Liss, Inc.     

Hosts are more important than destinations: What genetic variation in Microctonus aethiopoides (Hymenoptera: Braconidae) means for foreign exploration for natural enemies

Nucleotide sequence data were generated from the gene regions COI, 16S, and arginine kinase to assess genetic variation within the Palearctic parasitoid, Microctonus aethiopoides, reared from Sitona discoideus, S. hispidulus, and Hypera postica collected from two proximate locations in Mediterranean France. Partitioned Bayesian phylogenetic analyses of the molecular data provided strong support for the presence of at least two M. aethiopoides biotypes, one associated with Hypera species and the other with Sitona species. These new results combined with previously published data from 14 countries show that M. aethiopoides genetic variation is much more strongly correlated with host taxon than with sampling location. This contrasts with earlier perceptions that M. aethiopoides exhibits significant geographic variation, and helps to explain the widely varying biological control outcomes that have been obtained following the introductions of M. aethiopoides to Australia, New Zealand, and North America. The results strongly suggest that success rates and environmental safety in biological control would both be improved by ensuring that parasitoids collected in the native range are reared from the same host species as the one being targeted for control in the region of introduction. The results also provided insights both on the evolution of M. aethiopoides' host range, and on its evolutionary transition between solitary and gregarious larval development.     

A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum

The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium chrysogenum was studied by manipulating the steady-state flux in both pathways. A high producing industrial strain was cultivated at a growth rate mu=0.05 h(-1) in glucose-limited chemostat cultures, both under penicillin-G producing and non-producing conditions. Non-producing conditions were accomplished in two ways: (1) by cultivation without addition of the side chain precursor phenylacetic acid and (2) by cultivation of a mutant strain which lost all copies of the gene cluster coding for the penicillin biosynthesis pathway. Manipulation of the fluxes through central metabolism was obtained by cultivation on either glucose or ethanol as sole carbon source. A positive relation was observed between metabolite concentrations and carbon flux in central metabolism. Furthermore, in many cases a positive relation was found between the concentrations of free amino acids and their direct precursors in central metabolism. This corresponds with control of the biosynthesis of these amino acids via feed back inhibition by the end product. With respect to the penicillin production pathway, the flux seems not influenced by two of the three precursor amino acids, namely alphaAAA and valine but is only influenced by cysteine, which requires a large NADPH supply, and the ATP level. An interesting observation is that the absence of penicillin production seems to stimulate storage metabolism (trehalose metabolism). This leads to the final conclusion that the penicillin production flux appears to be mostly influenced by the availability of energy and redox cofactors, where ATP is supposed to exert its influence at ACV-synthetase and NADPH at the cysteine level.     

Lipase catalyzed HEMA initiated ring-opening polymerization: in situ formation of mixed polyester methacrylates by transesterification

2-Hydroxyethyl methacrylate (HEMA) was used as initiator for the enzymatic ring-opening polymerization (ROP) of omega-pentadecalactone (PDL) and epsilon-caprolactone (CL). The lipase B from Candida antarctica was found to catalyze the cleavage of the ester bond in the HEMA end group of the formed polyesters, resulting in two major transesterification processes, methacrylate transfer and polyester transfer. This resulted in a number of different polyester methacrylate structures, such as polymers without, with one, and with two methacrylate end groups. Furthermore, the 1,2-ethanediol moiety (from HEMA) was found in the polyester products as an integral part of HEMA, as an end group (with one hydroxyl group) and incorporated within the polyester (polyester chains acylated on both hydroxyl groups). After 72 h, as a result of the methacrylate transfer, 79% (48%) of the initial amount of the methacrylate moiety (from HEMA) was situated (acylated) on the end hydroxyl group of the PPDL (PCL) polyester. In order to prepare materials for polymer networks, fully dimethacrylated polymers were synthesized in a one-pot procedure by combining HEMA-initiated ROP with end-capping using vinyl methacrylate. The novel PPDL dimethacrylate (>95% incorporated methacrylate end groups) is currently in use for polymer network formation. Our results show that initiators with cleavable ester groups are of limited use to obtain well-defined monomethacrylated macromonomers due to the enzyme-based transesterification processes. On the other hand, when combined with end-capping, well-defined dimethacrylated polymers (PPDL, PCL) were prepared.