Determination of 24S- and 27-hydroxycholesterol in plasma by high-performance liquid chromatography-mass spectrometry

24S-Hydroxycholesterol (24S-OH-Chol) and 27-hydroxycholesterol (27-OH-Chol) are oxidized derivatives of cholesterol and of potential diagnostic interest because their circulating levels may reflect the cholesterol metabolism of the brain and macrophages, respectively. We developed a sensitive and specific HPLC-MS method for the quantification of 24S-OH-Chol and 27-OH-Chol in human plasma. In contrast to currently available procedures based on gas chromatography-mass spectrometry, this methodology offers the advantage that no time-consuming derivatization is needed. After saponification, solid-phase extraction, and HPLC separation under reversed-phase column conditions, detection by MS was performed using atmospheric pressure chemical ionization and selected ion monitoring mode. The standard curves were linear throughout the calibration range for both oxysterols. Within-day and between-day coefficients of variation were less than 9%, and the recoveries ranged between 98% and 103%. The quantification limits were 40 and 25 microg/l for 24S-OH-Chol and 27-OH-Chol, respectively. Mean values for both oxysterols were determined in plasma from 22 healthy volunteers. The sensitive and selective HPLC-MS method described here combined with the appropriate workup procedure allow the quantification of 24S-OH-Chol and 27-OH-Chol in plasma samples, for example in clinical studies to elaborate the clinical usefulness of these two oxysterols.     

Physiological effects of photosystem II-herbicides on the development of the photosynthetic apparatus

Photosystem II-herbicides (bentazone*, diuron) not only block photosynthetic electron transport, but have additional effects on the cell metabolism of Raphanus seedlings. They induce the formation of shade-type chloroplasts with a different ultrastructure and prenyllipid composition. This is shown by higher grana stacks as well as by higher chlorophyll b and lutein amounts with reference to chlorophyll a, and lower levels of the plastidic prenylquinones (plastoquinone, phylloquinone, -tocoquinone) and -carotene as compared to the controls.The two herbicides bentazone and diuron change the labelling pattern of the chloroplast pigments from ³H-mevalonic acid and 2-¹⁴C-acetate and also reduce the accumulation of anthocyanin (pelargonidin), which is a further indication of a shade-type growth response. The level of ubiquinone, an indicator for mitochondrial activity, is, however, increased.     

Benzodiazepine Antagonist Ro 15–1788: Binding Characteristics and Interaction with Drug-Induced Changes in Dopamine Turnover and Cerebellar cGMP Levels

Abstract: The recently discovered benzodiazepine antagonist Ro 15-1788 was characterized in binding studies, and its potency and selectivity were determined in vivo by interaction with drug-induced changes in dopamine turnover and cerebellar cGMP level. Ro 15-1788 reduced [³H]flunitrazepam binding in the brain in vivo with a potency similar to that of diazepam and effectively inhibited [³H]diazepam binding in vitro (IC₅₀= 2.3 ± 0.6 nmol/liter). [³H]Ro 15-1788 bound to tissue fractions of rat cerebral cortex with an apparent dissociation constant ( K _D) of 1.0 ± 0.1 nmol/liter. The in vitro potency of various benzodiazepines in displacing [³H]Ro 15-1788 from its binding site was of the same rank order as found previously in [³H]diazepam binding. Autoradiograms of [³H]Ro 15-1788 binding in sections of rat cerebellum showed the same distribution of radioactivity as with [³H]flunitrazepam. The attenuating effect of diazepam on the chlorpromazine- or stress-induced elevation of homovanillic acid in rat brain was antagonized by Ro 15-1788. Among a series of compounds which either decreased or increased the rat cerebellar cGMP level, only the effect of benzodiazepine receptor ligands (diazepam, zopiclone, CL 218 872) was antagonized by Ro 15-1788. Thus, Ro 15-1788 is a selective benzodiazepine antagonist acting at the level of the benzodiazepine receptor in the central nervous system. Peripheral benzodiazepine binding sites in kidney and schistosomes were not affected by Ro 15-1788.     

Predicting oligonucleotide affinity to nucleic acid targets

A computer program, OligoWalk, is reported that predicts the equilibrium affinity of complementary DNA or RNA oligonucleotides to an RNA target. This program considers the predicted stability of the oligonucleotide-target helix and the competition with predicted secondary structure of both the target and the oligonucleotide. Both unimolecular and bimolecular oligonucleotide self structure are considered with a user-defined concentration. The application of OligoWalk is illustrated with three comparisons to experimental results drawn from the literature.     

An improved method for the isolation of total RNA from spurce tissues

After many unsuccessful attempts to obtain biologically active mRNAs from spruce (Picea abies) tissues using available protocols, we have adapted a procedure for the isolation of RNAs from needles, shoots, and callus ofPicea species. Our modifications permit the recovery of and an average of 300 μg RNA per g of needles that is suitable for translationin vitro, northern hybridizations, and the construction of cDNA libraries.     

Structure of an early intermediate in the M-state phase of the bacteriorhodopsin photocycle.

The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.     

A nuclear 3'-5' exonuclease involved in mRNA degradation interacts with Poly(A) polymerase and the hnRNA protein Npl3p

Inactivation of poly(A) polymerase (encoded by PAP1) in Saccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)(+) mRNAs. Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6. These suppressors, rrp6-1 and rrp6-2, as well as a deletion of RRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)(+) mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay. Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus. Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3'-5' exonuclease core domain within Rrp6p. As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3'-5' exonucleolytic mechanism. Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)(+) mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export. These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.     

Enabling and disabling polo-like kinase 1 inhibition through chemical genetics

Polo-like kinase 1 (Plk1) is a core regulator of cell division and an emerging target for cancer therapy. Pharmacologic inhibitors of Plk1 exist but affect other kinases, complicating their in vivo validation. To address this, we examined effects of two structurally unrelated Plk1 inhibitors (BI-2536 and TAL) against isogenic human cell lines that solely express wildtype (wt) or analogue-sensitive (as) Plk1 alleles. Unexpectedly, Plk1(as) cells displayed profound biochemical and functional resistance to both inhibitors. Cells that co-express Plk1(wt) and Plk1(as) exhibit loss-of-function phenotypes only when both kinase alleles are inhibited. Resistance to BI-2536 is linked to an intragenic suppressor mutation (C67V) that restores an otherwise invariant valine to the kinase active site. Structural modeling demonstrates that this mutation not only enables Plk1(as) to function in vivo but also occludes BI-2536 from the ATP-binding pocket. Our results reveal the molecular basis of Plk inhibitor selectivity and a potential mechanism for tumor cell resistance.