Peter Bigler

The Path from β-Carotene to Carlactone, a Strigolactone-Like Plant Hormone

Making Carlactone Germination of parasitic witchweeds depends on strigolactones, which also regulate plant branching and signal in the context of mycorrhizal symbioses. The biosynthetic pathways that lead to strigolactones are founded in carotenoid biosynthesis, but further steps have been obscure. Alder et al. (p. 1348) have now identified a biochemical pathway that generates a strigolactone-like compound, carlactone, which shows biological actions similar to those of strigolactone. Elucidation of the biosynthetic pathway of a new plant hormone variant that may be useful in agricultural settings is shown. Strigolactones, phytohormones with diverse signaling activities, have a common structure consisting of two lactones connected by an enol-ether bridge. Strigolactones derive from carotenoids via a pathway involving the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8) and the iron-binding protein D27. We show that D27 is a β-carotene isomerase that converts all-trans-β-carotene into 9-cis-β-carotene, which is cleaved by CCD7 into a 9-cis–configured aldehyde. CCD8 incorporates three oxygens into 9-cis-β-apo-10′-carotenal and performs molecular rearrangement, linking carotenoids with strigolactones and producing carlactone, a compound with strigolactone-like biological activities. Knowledge of the structure of carlactone will be crucial for understanding the biology of strigolactones and may have applications in combating parasitic weeds.

Long-term administration of L-carnitine to humans: effect on skeletal muscle carnitine content and physical performance.

BACKGROUND Long-term administration of high oral doses of L-carnitine on the skeletal muscle composition and the physical performance has not been studied in humans. METHODS Eight healthy male adults were treated with 2 x 2 g of L-carnitine per day for 3 months. Muscle biopsies and exercise tests were performed before, immediately after, and 2 months after the treatment. Exercise tests were performed using a bicycle ergometer for 10 min at 20%, 40%, and 60% of the individual maximal workload (P(max)), respectively, until exhaustion. RESULTS There were no significant differences between V(O(2)max), RER(max), and P(max) between the three time points investigated. At submaximal intensities, the only difference to the pretreatment values was a 5% increase in V(O(2)) at 20% and 40% of P(max) 2 months after the cessation of the treatment. The total carnitine content in the skeletal muscle was 4.10 +/- 0.82 micromol/g before, 4.79 +/- 1.19 micromol/g immediately after, and 4.19 +/- 0.61 micromol/g wet weight 2 months after the treatment (no significant difference). Activities of the two mitochondrial enzymes citrate synthase and cytochrome oxidase, as well as the skeletal muscle fiber composition also remained unaffected by the administration of L-carnitine. CONCLUSIONS Long-term oral treatment of healthy adults with L-carnitine is not associated with a significant increase in the muscle carnitine content, mitochondrial proliferation, or physical performance. Beneficial effects of the long-term treatment with L-carnitine on the physical performance of healthy adults cannot be explained by an increase in the carnitine muscle stores.

Observation and identification of metabolites emerging during postmortem decomposition of brain tissue by means of in situ 1H-magnetic resonance spectroscopy.

Postmortem decomposition of brain tissue was investigated by 1H‐magnetic resonance spectroscopy (MRS) in a sheep head model and selected human cases. Aiming at the eventual estimation of postmortem intervals in forensic medicine, this study focuses on the characterization and identification of newly observed metabolites. In situ single‐voxel 1H‐MRS at 1.5 T was complemented by multidimensional homo‐ and heteronuclear high‐resolution NMR spectroscopy of an extract of sheep brain tissue. The inclusion of spectra of model solutions in the program LC Model confirmed the assignments in situ. The first postmortem phase was characterized mainly by changes in the concentrations of metabolites usually observed in vivo and by the appearance of previously reported decay products. About 3 days postmortem, new metabolites, including free trimethylammonium, propionate, butyrate, and iso‐butyrate, started to appear in situ. Since the observed metabolites and the time course is comparable in sheep and human brain tissue, the model system seems to be appropriate. Magn Reson Med 48:915–920, 2002.

Gene ste20 controls amiloride sensitivity and fertility in Schizosaccharomyces pombe

Abstract It has been shown previously that amiloride, a widely used diuretic drug, inhibits growth in Schizosaccharomyces pombe. Here we show that the drug also alleviates repression by various nutrients of mating and sporulation in fission yeast. We selected spontaneous mutants that are amiloride-resistant and unable to mate and sporulate. One of them defines the gene ste20. This gene has been cloned and sequenced. It codes for a putative protein of 1309 amino acids. Its sequence does not provide any clues to its function. In contrast to the wild-type, mutants defective in this gene can grow in a medium containing 40 μm amiloride, do not arrest in G1, and do not induce ste11 expression upon nitrogen starvation and thus are sterile. In addition the ste20 mutants are methylamine-sensitive, exhibit enhanced medium acidification and are defective in the utilization of gycerol as a carbon source.

BIRD–HMBC: improved detection of heteronuclear long‐range couplings

A new 2D pulse sequence (BIRD–HMBC) designed for the detection of heteronuclear long‐range connectivities is presented. In contrast to the basic HMBC experiment and its variants, unwanted 1J(C,H) signals are sampled together with nJ(C,H) signals but are cancelled in subsequent scans, taking advantage of a BIRD pulse sequence element. As a result, sensitivities close to the HMBC with only minor losses are measured. Excellent suppression degrees for unwanted 1J(C,H) residual peaks, superior to the state‐of‐the‐art ACCORD–HMBC experiment, are achieved for the whole range of one‐bond couplings. The influence of poorly calibrated pulse angles or off‐resonance effects is marginal and options such as refocusing with additional 13C broadband decoupling or the ACCORDION element, applied for improved sampling of the whole nJ(C,H) coupling range, may easily be implemented. These attractive characteristics, together with the easy set‐up, make this new experiment a valuable alternative to the HMBC experiment and its known variants for standard routine applications. Copyright

pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy.

The pH-dependent membrane adsorption and distribution of three chlorin derivatives, chlorin e6 (CE), rhodin G7 (RG), and monoaspartyl-chlorin e6 (MACE), in the physiological pH range (pH 6-8) were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. The chlorin derivatives were characterized with respect to their aggregation behavior, the pK(a) values of individual carboxylate groups, the extent of membrane adsorption, and their flip-flop rates across the bilayer membrane for pH 6-8. External membrane adsorption was found to be lower for RG than for CE and MACE. Both electrostatic interactions and the extent of aggregation seemed to be the main determinants of membrane adsorption. Rate constants for chlorin transfer across the membrane were found to correlate strongly with the pH of the surrounding medium, in particular, for CE and RG. In acidic solution, CE and RG transfer across the membrane was strongly accelerated, and in basic solution, all compounds were retained, mostly in the outer monolayer. In contrast, MACE flip-flop across the membrane remained very low even at pH 6. The protonation of ionizable groups is suggested to be a major determinant of chlorin transfer rates across the bilayer. pK(a) values of CE and RG were found to be between 6 and 8, and two of the carboxylate groups in MACE had pK(a) values below 6. For CE and RG, the kinetic profiles at acidic pH indicated that the initial fast membrane distribution was followed by secondary steps that are discussed in this article.

In vitro characterization of Synechocystis CYP120A1 revealed the first nonanimal retinoic acid hydroxylase.

Retinoids are C20 apocarotenoids that have various important functions in metazoans. In addition, several findings suggest their occurrence in eubacteria, including cyanobacteria. It has been shown that the Synechocystis cytochrome P450 enzyme CYP120A1 is a retinoic acid‐binding polypeptide. In this work, we determined the reaction catalyzed by CYP120A1 and investigated its substrate specificity in vitro. CYP120A1‐containing microsomes generated in yeast converted all‐trans‐retinoic acid into a compound exhibiting higher polarity in HPLC analysis. Liquid chromatography–MS analysis suggested the introduction of a single hydroxyl group, and NMR analysis of the purified product revealed C16 or C17 as the reaction site. Incubations with cis‐retinoic acids, retinal, 3(R)‐OH‐retinal, retinol, β‐apo‐13‐carotenone (C18) and β‐apo‐14′‐carotenal (C22) resulted in the formation of the corresponding hydroxyl derivatives, as suggested by HPLC and liquid chromatography–MS analyses. Comparisons of the relative product amounts revealed the highest conversion rate for all‐trans‐retinoic acid, followed by β‐apo‐13‐carotenone (C18). As shown by real‐time RT‐PCR, CYP120A1 is expressed under normal growth conditions and is slightly induced by high‐intensity light. Our work provides the first enzymatic study of a cyanobacterial cytochrome P450, showing it to be the first nonanimal retinoic acid‐metabolizing enzyme characterized so far. Moreover, the CYP120A1‐catalyzed reaction represents a novel modification of retinoids.

Interactions between selected photosensitizers and model membranes: an NMR classification

Membrane interactions of porphyrinic photosensitizers (PSs) are known to play a crucial role for PS efficiency in photodynamic therapy (PDT). In the current paper, the interactions between 15 different porphyrinic PSs with various hydrophilic/lipophilic properties and phospholipid bilayers were probed by NMR spectroscopy. Unilamellar vesicles consisting of dioleoyl-phosphatidyl-choline (DOPC) were used as membrane models. PS-membrane interactions were deduced from analysis of the main DOPC 1H-NMR resonances (choline and lipid chain signals). Initial membrane adsorption of the PSs was indicated by induced changes to the DOPC choline signal, i.e. a split into inner and outer choline peaks. Based on this parameter, the PSs could be classified into two groups, Type-A PSs causing a split and the Type-B PSs causing no split. A further classification into two subgroups each, A1, A2 and B1, B2 was based on the observed time-dependent changes of the main DOPC NMR signals following initial PS adsorption. Four different time-correlated patterns were found indicating different levels and rates of PS penetration into the hydrophobic membrane interior. The type of interaction was mainly affected by the amphiphilicity and the overall lipophilicity of the applied PS structures. In conclusion, the NMR data provided valuable structural and dynamic insights into the PS-membrane interactions which allow deriving the structural constraints for high membrane affinity and high membrane penetration of a given PS.

Superior pulse sequence for two-dimensional heteronuclear overhauser spectroscopy

Abstract In this contribution two modifications are proposed and demonstrated to improve the inherent bad sensitivity of the heteronuclear 2D NOE experiment. The principal gain, compared to the basic sequence, results from looping several times through the evolution and mixing periods before data acquisition. A minor gain is achieved by an optimized and adequate ω1-decoupling scheme.

Phosphatidyl-O-[N-(2-Hydroxyethyl)Glycine] (PHEG), a New Glycerophospholipid from Brown Algae (Phaeophyceae)

Summary The phosphoglyceride phosphatidyl- O -[ N (2-hydroxyethyl)glycine] (PHEG) has been isolated from Fucus serratus L. (Fucales, Phaeophyceae) and its structure determined by IR, NMR, MS and chemical data of the intact lipid (formerly called PX), its lyso compound and the deacylated lipid. The fatty acid composition was about 82 mol% 20: 4, 9% 20: 5 and 6% 20: 0 acids in both the sn -1 and the sn -2 positions of the glycerol part. PHEG was present in 30 different species representing the 16 orders of brown algae in amounts of 8–25 mol% of total phospholipids, and is suggested to be a compulsory lipid constituent of brown algae.