Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction.

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.     

Induction of NADPH-Cytochrome P-450 (c) Reductase in Wounded Tissues from Helianthus tuberosus Tubers

Cytochrome P-450 is not self-sufficient for the catalysis ofmonooxygenase reaction but requires NADPH and NADPH-cytochromeP-450 (c) reductase. The activity of NADPH-cyto-chrome P-450reductase was strongly enhanced by wounding and aging in Jerusalemartichoke (Helianthus tuberosus L.) tuber tissues. This stimulationwas correlated with the synthesis of the enzyme protein basedon i) quantitation of the reductase protein by Western blotting,ii) incorporation of [³⁵S]methionine into the immunoprecipitableenzyme and iii) an increase in translatable mRNA for the reductasein a cell free system. (Received April 9, 1990; Accepted September 12, 1990)     

Organization of perinuclear actin in live tobacco cells observed by PALM with optical sectioning

Actin performs a wide variety of different tasks. This functional diversity may be accomplished either by the formation of different isotypes or by suitable protein decoration that regulates structure and dynamics of actin filaments. To probe for such a potential differential decoration, the actin-binding peptide Lifeact was fused to different photoactivatable fluorescent proteins. These fusions were stably expressed in Nicotiana tabacum L. cv. Bright Yellow 2 cells to follow dynamic reorganization of the actin cytoskeleton during the cell cycle. The Lifeact–monomeric variant of IrisFP fusion protein was observed to indiscriminately label both, central and cortical, actin filaments, whereas the tetrameric Lifeact–photoswitchable red fluorescent protein fusion construct selectively labeled only a specific perinuclear sub-population of actin. By using photoactivated localization microscopy, we acquired super-resolution images with optical sectioning to obtain a 3D model of perinuclear actin. This novel approach revealed that the perinuclear actin basket wraps around the nuclear envelope in a lamellar fashion and repartitions toward the leading edge of the migrating nucleus. Based on these data, we suggest that actin that forms the perinuclear basket differs from other actin assemblies by a reduced decoration with actin binding proteins, which is consistent with the differential decoration model.     

Plant actin controls membrane permeability

The biological effects of electric pulses with low rise time, high field strength, and durations in the nanosecond range (nsPEFs) have attracted considerable biotechnological and medical interest. However, the cellular mechanisms causing membrane permeabilization by nanosecond pulsed electric fields are still far from being understood. We investigated the role of actin filaments for membrane permeability in plant cells using cell lines where different degrees of actin bundling had been introduced by genetic engineering. We demonstrate that stabilization of actin increases the stability of the plasma membrane against electric permeabilization recorded by penetration of Trypan Blue into the cytoplasm. By use of a cell line expressing the actin bundling WLIM domain under control of an inducible promotor we can activate membrane stabilization by the glucocorticoid analog dexamethasone. By total internal reflection fluorescence microscopy we can visualize a subset of the cytoskeleton that is directly adjacent to the plasma membrane. We conclude that this submembrane cytoskeleton stabilizes the plasma membrane against permeabilization through electric pulses.     

BC-spiro-estradiols. Synthesis and estrogen receptor binding affinity of four new estradiol isomers

The synthesis of four new isomers of estradiol in which the ring A to ring C planes are perpendicular to each other as a result of a spiro BC ring junction is described. Heterocyclic analogs and carbocyclic homologs of these compounds are also reported. Estrogen receptor binding studies show that the spiro compounds with the natural stereochemistry at C9 bind almost as strongly as estradiol but with greater β to α selectivity. These studies show that the estrogen receptors can readily accommodate isomers of estrogen with substantially different fixed shapes than the native ligand.     

Induced plant volatiles allow sensitive monitoring of plant health status in greenhouses

A novel approach to support the inspection of greenhouse crops is based on the measurement of volatile organic compounds emitted by unhealthy plants.This approach has attracted some serious interest over the last decade. In pursuit of this interest, we performed several experiments at the laboratory-scale to pinpoint marker volatiles that can be used to indicate certain health problems. In addition to these laboratory experiments, pilot and model studies were performed in order to verify the validity of these marker volatiles under real-world conditions. This paper provides an overview of results and gives an outlook on the use of plant volatiles for plant health monitoring.Key words: plant health, volatiles, plant pathogens, plant infection     

Tests of the antibiotic properties of the invasive vine Vincetoxicum rossicum against bacteria, fungi and insects

We tested extracts of Vincetoxicum rossicum for inhibition of bacterial and fungal growth and for anti-insect activities that may account for the invasive characteristics of this introduced species in North America. Bioassay-guided fractionation was used to identify (−)-antofine as the principle inhibitor of bacteria and fungi in root extracts. This compound had especially pronounced antifungal activity, inhibiting the growth of diverse taxa that include yeast-like and filamentous fungi and, notably, broad-host-range plant pathogens. A second compound(s), that is as yet uncharacterized but distinct from (−)-antofine, was detected as having antifeedant activity against a larval hymenopteran, rose sawfly (Allantus cinctus), and toxicity to two larval lepidopterans, the masked birch caterpillar (Drepana arcuata) and the European corn borer (Ostrinia nubilalis). That V. rossicum contains potent inhibitors of plant pathogenic fungi, diverse bacteria, and herbivorous insects likely contributes to its success as an invasive species.     

Recent origin and spread of a common Lithuanian mutation, G197del LDLR, causing familial hypercholesterolemia: positive selection is not always necessary to account for disease incidence among Ashkenazi Jews

G197del is the most prevalent LDL receptor (LDLR) mutation causing familial hypercholesterolemia (FH) in Ashkenazi Jew (AJ) individuals. The purpose of this study was to determine the origin, age, and population distribution of G197del, as well as to explore environmental and genetic effects on disease expression. Index cases from Israel (n=46), South Africa (n=24), Russia (n=7), The Netherlands (n=1), and the United States (n=1) were enlisted. All trace their ancestry to Lithuania. A highly conserved haplotype (D19S221:104-D19S865:208-D19S413:74) was identified in G197del chromosomes, suggesting the occurrence of a common founder. When two methods were used for analysis of linkage disequilibrium (LD) between flanking polymorphic markers and the disease locus and for the study of the decay of LD over time, the estimated age of the deletion was found to be 20 +/- 7 generations (the 95% confidence interval is 15-26 generations), so that the most recent common ancestor of the mutation-bearing chromosomes would date to the 14th century. This corresponds with the founding of the Jewish community of Lithuania (1338 a.d.), as well as with the great demographic expansion of AJ individuals in eastern Europe, which followed this settlement. The penetrance of mutation-linked severe hypercholesterolemia is high (94% of heterozygotes have a baseline concentration of LDL cholesterol (LDL-C) that is >160 mg/dl), and no significant differences in the mean baseline lipid level of G197del carriers from different countries were found. Polymorphisms of apolipoprotein E and of scavenger-receptor class B type I were observed to have minor effects on the plasma lipid profile. With respect to determinative genetic influences on the biochemical phenotype, there is no evidence that could support the possibility of a selective evolutionary metabolic advantage. Therefore, the founder effect in a rapidly expanding population from a limited number of families remains a simple, parsimonious hypothesis explaining the spread of G197del-LDLR-linked FH in AJ individuals.