In vitro dynamics of chromatin organization and migration

The organization of eukaryotic chromatin is not static but changes as a function of cell status during processes such as proliferation, differentiation, and migration. DNA quantification has not been used extensively to investigate chromatin dynamics in combination with cellular migration. In this context, an optimized DNA-specific, nonperturbant method has been developed for studying chromatin organization, using the fluorescent vital bisbenzimidazole probe Hoechst 33342: this property has been described by Hamori et al. (1980). Computer-assisted image analysis was used to follow migratory activity and chromatin organization of L929 fibroblasts during in vitro wound healing. Cell movements were analyzed using an optical flow technique, which consists in the calculation of the velocity field of cells and nuclear movements in the frame. This system allows the correlation of cell migration and position in the cell cycle. It makes it possible to study chromatin dynamics using a quantitative analysis of nuclear differentiation reorganization (nuclear texture) and to correlate this with migration characteristics. The present system would be of interest for studying cell-extracellular matrix interactions using differing substrates, and also the migratory response to chemotactic factors. Such a model is a prerequisite for gaining better understanding of drug action.     

Turnover of Brain Monoamine Oxidase Measured In Vivo by Positron Emission Tomography Using l-[11C]Deprenyl

The distribution of carbon-11-labeled L-deprenyl, an irreversible inhibitor of monoamine oxidase type B (MAO-B), was determined in the baboon brain by positron emission tomography. The irreversible blood-to-brain transfer constant (influx constant, K_i) was measured using a complete metabolite-corrected arterial plasma concentration curve. This influx constant was used as a measure of functional enzyme activity for sequential determinations of MAO-B recovery following a single high dose of unlabeled l -deprenyl. The half-life for turnover of MAO-B was thus determined to be 30 days. Using appropriate irreversible inhibitors, this procedure should be generally useful for determining enzyme turnover rates in any organ in vivo and can be applied to some human studies as well.     

4',6-Dichloroflavan (BW683C): Enantiomeric separation by hplc/dad and antirhinovirus activity

Racemic 4',6-dichloroflavan (BW683C), a highly effective inhibitor of rhinovirus serotype 1B in vitro, was resolved by high-performance liquid chromatography on a chiral stationary phase. The enantiomers were separately collected and circular dichroism curves were obtained, in order to determine the absolute configuration of the two enantiomers. The activity of the isomers was studied on human rhinovirus serotype 1B multiplication in HeLa cell cultures, by means of the plaque reduction assay. Both enantiomers were potent inhibitors of virus replication; by comparing the IC₅₀ values, the S form was 3.5 times more effective than the R form.     

Removal of zero-quantum interference in NOESY spectra of proteins by utilizing the natural inhomogeneity of the radiofrequency field

Summary A single scan method for the suppression of signals arising from zero-quantum coherences (ZQC) is analysed with respect to its application to NMR experiments on proteins. The ZQC are dephased during a spinlock period due to the natural RF inhomogeneity of a commercial probe. A quantitative analysis of a ZQC-compensated NOESY experiment is given. Although the build-up curve for the cross peaks in ZQC-compensated NOESY experiments differ from those in uncompensated experiments, interproton distances in medium-sized proteins can be evaluated with high accuracy. The proposed method is compared with other techniques for ZQC suppression.     

Computed tomography and automated image analysis of prehistoric femora

Non-invasive characterization of limb bone cross-sectional geometry would be useful for biomechanical analyses of skeletal collections. Computed tomography (CT) is potentially the method of choice. Additionally, CT images are suitable for automated analysis. CT is here shown to be both accurate and precise in the analysis of cross-sectional geometry of prehistoric femora. Beam hardening artifacts can be reduced by using a water bath. As the availability of CT for research increases, both bone density and geometry could be determined simultaneously with this method.     

Molecular motion underlying activation and inactivation of sodium channels in squid giant axons

Summary Measurements of the changes in birefringence associated with changes in membrane potential were made with internally perfused squid giant axons in low sodium solutions at 0–8°C. The time course of the birefringence changes share many properties of the gating (polarization) currents previously studied in this nerve. Both can be demonstrated as an asymmetry in the response to voltage pulses symmetrical about the resting potential which is not present about a hyperpolarized holding potential. Both have a rapid relaxation, which precedes the sodium permeability change. Both exhibit an initial delay or rising phase. Both are reversibly blocked by perfusion with 30mm colchicine; neither are altered by changes on sodium concentrations or 300nm tetrodotoxin. The birefringence response has a decrease in the amplitude of the rapid relaxation associated with the appearance of a slow relaxation. This is similar to the immobilization of fast gating charges which parallels sodium current inactivation.The amplitude of the birefringence and the gating current responses is consistent with a change in the alignment of several hundred peptide bonds per sodium channel.     

Stereoisomeric flavour compounds XXXV: Chiral aroma compounds of sherry I. Optically pure stereoisomers of solerone and solerole

The enantioseparation of the sherry aroma components 5-oxo-4-hydroxyhexanoic acid γ-lactone (solerone) and 4,5-dihydroxyhexanoic acid γ-lactone (solerole) is achieved, using Chiraspher (Merck) as the chiral HPLC phase and the optical purity ascertained directly by HRGC with heptakis(3-O-acetyl-2,6-di-O-pentyl)-β-cyclodextrin (Lipodex D) as the chiral stationary phase. The absolute configurations of 4,5-dihydroxyhexanoic acid γ-lactones are assigned by ¹H-NMR spectral data of diastereomeric α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) esters, according to Mosher's model. Sensory qualities of the isomers are given.     

Chiral stationary phase high-performance liquid chromatographic resolution and absolute configuration of enantiomeric benzo[a]pyrene diol-epoxides and tetrols

Enantiomers of diastereomeric benzo[a]pyrene (BP) diol-epoxides, r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-anti-9,10-epoxide), r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-BP (BP 7,8-diol-syn-9,10-epoxide), r-9,t-10-dihydroxy-t-7,8-epoxy-7,8,9,10-tetrahydro-BP (BP 9,10-diol-anti-7,8-epoxide), and several 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes (BP tetrols) were resolved by high-performance liquid chromatography (HPLC) using columns packed with either (R)-N-(3,5-dinitrobenzoyl)phenylglycine[(R)-DNBPG] or (S)-N-(3,5-dinitrobenzoyl)leucine [(S)-DNBL], which is either ionically or covalently bonded to gamma-aminopropylsilanized silica. Resolution of enantiomers was confirmed by ultraviolet-visible absorption and circular dichroism spectral analyses. Resolved enantiomers of BP diol-epoxides were each hydrolyzed in acidic solution to a pair of diastereomeric tetrols which were separated by reversed-phase HPLC. Absolute stereochemistries of enantiomeric diol-epoxides were deduced by the absolute configuration of their hydrolysis products.     

Temporally incoherent magnetic fields mitigate the response of biological systems to temporally coherent magnetic fields

We have previously demonstrated that a weak, extremely-low-frequency magnetic field must be coherent for some minimum length of time (≈? 10 s) in order to affect the specific activity of ornithine decarboxylase (ODC) in L929 mouse cells. In this study we explore whether or not the superposition of an incoherent (noise) magnetic field can block the bioeffect of a coherent 60 Hz magnetic field, since the sum of the two fields is incoherent. An experimental test of this idea was conducted using as a biological marker the twofold enhancement of ODC activity found in L929 murine cells after exposure to a 60 Hz, 10 μT_rms magnetic field. We superimposed an incoherent magnetic noise field, containing frequencies from 30 to 90 Hz, whose rms amplitude was comparable to that of the 60 Hz field. Under these conditions the ODC activity observed after exposure was equal to control levels. It is concluded that the superposition of incoherent magnetic fields can block the enhancement of ODC activity by a coherent magnetic field if the strength of the incoherent field is equal to or greater than that of the coherent field. When the superimposed, incoherent noise field was reduced in strength, the enhancement of ODC activity by the coherent field increased. Full ODC enhancement was obtained when the rms value of the applied EM noise was less than one-tenth that of the coherent field. These results are discussed in relation to the question of cellular detection of weak EM fields in the presence of endogenous thermal noise fields. © 1994 Wiley-Liss, Inc.     

Developments in tomographic methods for biological trace element research

Neutron-induced γ-ray emission tomography for quantitative determination of the concentration and distribution of elements in a selected plane through a biological specimen is briefly explained and applied by way of illustration to the analysis of gallstones. A system capable of carrying out studies of the binding site of⁷⁵Se in different matrices using time differential perturbed angular correlation spectroscopy is also briefly described. Developments in the detector technology of positron emission tomography have allowed small-diameter imaging devices to be built for in vivo preclinical evaluation of new tracers in small animals and are discussed in the context of a proposed experiment combining the techniques mentioned above.