Dielectrophoresis of chloroplasts

Dielectrophoresis is the migration of neutral particles in a nonuniform electric field (a.c. or d.c.) toward the region of highest field intensity. Dielectrophoresis should be distinguished from electrophoresis which is the migration of charged particles in electric fields. Chloroplasts, isolated from spinach leaves, can be collected on platinum electrodes by dielectrophoresis. Stripped chloroplasts lacking outer envelopes and stroma were prepared from fresh spinach leaves in a 0.5 M sucrose-0.05 M Tris buffer (pH 7.4). The chloroplast preparation was desalted with a mixed anion-cation resin to a resistivity of 3 · 10⁴–5 · 10⁴ ohm · cm. Dielectrophoresis was conducted in a pin-pin type leucite cell 3.2 mm in diameter and 1.5 mm deep. The 0.425-mm diameter electrodes were 0.85 mm apart and 0.05 mm below the surface of the cell. The collection of chloroplasts with ac current is a function of the frequency. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU)-stabilized chloroplasts had collection maxima at 300, 1 · 10⁶, and 3 · 10⁷ Hz when run at 50 V. The rate of collection is a function of the square root of the time. Both DCMU and darkness tend to stabilize collections. It is suggested that dielectrophoresis may be a useful tool for the study of chloroplast physiology and perhaps, for the preparation and purification of chloroplasts.     

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity.     

Comparison of effects of exercise and diuretic on left ventricular geometry, mass, and insulin resistance in older hypertensive adults

To compare the effects of exercise training and hydrochlorothiazide on left ventricular (LV) geometry and mass, blood pressure (BP), and hyperinsulinemia in older hypertensive adults, we studied 28 patients randomized either to a group (age 66.4 +/- 1.3 yr; n = 16) that exercised or to a group (age 65.3 +/- 1.2 yr; n = 12) that received hydrochlorothiazide for 6 mo. Endurance exercise training induced a 15% increase in peak aerobic power. The reduction in systolic BP was twofold greater with thiazide than with exercise (26.6 +/- 12.2 vs. 11.5 +/- 10.9 mmHg). Exercise and thiazide reduced LV wall thickness, LV mass index (14% in each group), and the LV wall thickness-to-radius ratio (h/r) similarly (exercise: before 0.48 +/- 0.2, after 0.42 +/- 0.01; thiazide: before 0.47 +/- 0.04, after 0.40 +/- 0.04; P = 0.017). The reductions in systolic BP and h/r were correlated in the exercise group (r = 0.70, P = 0.005) but not in the thiazide group. Exercise training reduced glucose-stimulated hyperinsulinemia (before: 13.65 +/- 2.6 vs. 9.84 +/- 1.5 mU.ml(-1).min; P = 0.04) and insulin resistance. Thiazide did not affect plasma insulin levels. The results suggest that although exercise is less effective in reducing systolic BP than thiazide, it can induce regression of LV hypertrophy similar in magnitude to thiazide. Unlike hydrochlorothiazide, exercise training can improve insulin resistance and aerobic capacity in older hypertensive people.     

Antioxidant mechanisms of polyphenolic caffeic acid oligomers, constituents of Salvia officinalis

Caffeic acid, rosmarinic acid and oligomers of caffeic acid with multiple catechol groups are all constituents of Salvia officinalis. Their antioxidant potential was investigated with regard to their radical scavenging activity and the stability and structure of the intermediate radicals. Pulse-radiolytic studies revealed very high rate constants with hydroxyl radicals. Evidence from kinetic modeling calculations suggested an unusual complex behavior due to the presence of both O4- and O3-semiquinones and formation and decay of a hydroxyl radical adduct at the vinyl side chain. The radical structures observed by EPR spectroscopy after autoxidation in slightly alkaline solutions were only partially identified due to their instability and generally represented dissociated O4-semiquinones. Hybrid density-functional calculations of the potential radical structures showed distinct differences between the resonance stabilization of the O4- and O3-semiquinones of caffeic and dihydrocaffeic acids, reflected also in the considerably faster decay of the O3-semiquinone observed by pulse radiolysis. No evidence was found for dimerization reactions via Cbeta radicals typical for lignin biosynthesis.     

Structural organization of DMPC lipid layers on chemically micropatterned self-assembled monolayers as biomimetic systems

The growth structure of DMPC lipid layers on hydrophobic and hydrophilic alkylsilane-based self-assembled monolayers adsorbed on silicon has been investigated by means of X-ray reflectometry and atomic force microscopy. Hydrophilic modification of hydrophobically terminated ODS-SAMs has been achieved by dose-controlled irradiation with DUV light. While island formation of small DMPC bilayer islands is observed on hydrophobic SAM surfaces, closed layers of DMPC monolayers are formed on hydrophilic SAM surfaces. Furthermore, DMPC adsorption on chemically micropatterned substrates with alternating hydrophobic/hydrophilic surface properties has been studied by imaging ellipsometry and photoemission microscopy. Indication for at least partial bridging of hydrophobic areas by an adsorbed DMPC monolayer has been found.     

Primer design and marker clustering for multiplex SNP-IT primer extension genotyping assay using statistical modeling

MOTIVATION: The optimization of the primer design is critical for the development of high-throughput SNP genotyping methods. Recently developed statistical models of the SNP-IT primer extension genotyping reaction allow further improvement of primer quality for the assay. RESULTS: Here we describe how the statistical models can be used to improve primer design for the assay. We also show how to optimize clustering of the SNP markers into multiplex panels using statistical model for multiplex SNP-IT. The primer set failure probability calculated by a model is used as a minimization function for both primer selection and primers clustering. Three clustering algorithms for the multiplex genotyping SNP-IT assay are described and their relative performance is evaluated. We also describe the approaches to improve the speed of primer design and clustering calculations when using the statistical models. Our clustering decreases the average failure probability of the marker set by 7-25%. The experimental marker failure rate in the multiplex reaction was reduced dramatically and success rate can be achieved as high as 96%. AVAILABILITY: The primer design using statistical models is freely available from www.autoprimer.com.     

Auto-validation of fluorescent primer extension genotyping assay using signal clustering and neural networks

Background

SNP genotyping typically incorporates a review step to ensure that the genotype calls for a particular SNP are correct. For high-throughput genotyping, such as that provided by the GenomeLab SNPstream^® instrument from Beckman Coulter, Inc., the manual review used for low-volume genotyping becomes a major bottleneck. The work reported here describes the application of a neural network to automate the review of results.

Results

We describe an approach to reviewing the quality of primer extension 2-color fluorescent reactions by clustering optical signals obtained from multiple samples and a single reaction set-up. The method evaluates the quality of the signal clusters from the genotyping results. We developed 64 scores to measure the geometry and position of the signal clusters. The expected signal distribution was represented by a distribution of a 64-component parametric vector obtained by training the two-layer neural network onto a set of 10,968 manually reviewed 2D plots containing the signal clusters.

Conclusion

The neural network approach described in this paper may be used with results from the GenomeLab SNPstream instrument for high-throughput SNP genotyping. The overall correlation with manual revision was 0.844. The approach can be applied to a quality review of results from other high-throughput fluorescent-based biochemical assays in a high-throughput mode.     

Helifulvanolsäkure—ein neues diterpen mit anomalem kohlenstoffgerüst aus Helichrysum fulvum

The aerial parts of Helichrysum fulvum afforded, in addition to beyerenic acid and ent-kaurenic acid, two new diterpenic acids with the hitherto unknown carbon skeleton of an isotrachylobane type. The structures of these acids, isolated as their methyl esters, were elucidated by extensive NMR studies, some chemical transformations and by X-ray structural analysis of the corresponding acetate. The related alcohol on reaction with pyridinochlorochromate afforded a homoconjugated diene probably formed by fragmentation of a cyclopropyl carbinol. The possible biogenesis of the new carbon skeleton is discussed briefly.     

2-Phenylimidazo[2,1-i]purin-5-ones: structure-activity relationships and characterization of potent and selective inverse agonists at Human A3 adenosine receptors

Structure-activity relationships of 2-phenyl-imidazo[2,1-i]purin-5-ones as ligands for human A(3) adenosine receptors (ARs) were investigated. An ethyl group in the 8-position of the imidazoline ring of 4-methyl-2-phenyl-imidazopurinone leading to chiral compounds was found to increase affinity for human A(3) ARs by several thousand-fold. Propyl substitution instead of methyl at N4 decreased A(3) affinity but increased A(1) affinity leading to potent A(1)-selective AR antagonists. The most potent A(1) antagonist of the present series was (S)-8-ethyl-2-phenyl-4-propyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-3) exhibiting a K(i) value of 7.4 nM at rat A(1) ARs and greater than 100-fold selectivity versus rat A(2A) and human A(3) ARs. At human A(1) ARs 2-phenylimidazo[2,1-i]purin-5-ones were generally less potent and therefore less A(1)-selective (S-3: K(i)=98 nM). 2-, 3-, or 4-Mono-chlorination of the 2-phenyl ring reduced A(3) affinity but led to an increase in affinity for A(1) ARs, whereas di- (3,4-dichloro) or polychlorination (2,3,5-trichloro) increased A(3) affinity. The most potent and selective A(3) antagonist of the present series was the trichlorophenyl derivative (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8) exhibiting a subnanomolar K(i) value at human A(3) ARs and greater than 800-fold selectivity versus the other AR subtypes. Methylation of 4-alkyl-2-phenyl-substituted imidazo[2,1-i]purin-5-ones led exclusively to the N9-methyl derivatives, which exhibited largely reduced AR affinities as compared to the unmethylated compounds. [35S]GTP gamma S binding studies of the most potent 2-phenyl-imidazo[2,1-i]purin-5-ones at membranes of Chinese hamster ovary cells expressing the human A(3) AR revealed that the compounds were inverse agonists at A(3) receptors under standard test conditions. Due to their high A(3) affinity, selectivity, and relatively high water-solubility, 2-phenyl-imidazo[2,1-i]purin-5-ones may become useful research tools.