Positive contrast in the detection of magnetically labeled cells by MRI--in vitro experiments]

PURPOSE: Magnetically labeled cells (MLC) cause local field inhomogenities within the single voxels as well as on a macroscopic scale. The related Larmor frequency shift near MLC was exploited to obtain bright visualization applying spectral selective saturation (SSS). METHODS: SK-Mel28 cells were labeled with the superparamagnetic iron oxide contrast agent SHU 555A. Low cell concentrations (0, 5, 20, 30, 50, 75, and 150 MLC/microl) and high cell concentrations (10 x 10(3), 30 x 10(3), 60 x 10(3), and 100 x 10(3) MLC/10 microl) were examined at 3 Tesla. Shimming and frequency adjustment to spectrometer reference frequency v0 was performed with the built in routine of the scanner. A 2D spin echo sequence with broadband excitation and refocusing pulses was used (BWex = 1.000 Hz). Prior to each TR, a non-selective saturation sinc pulse centred at v0 was applied. Bandwidth (BWsat) of this pulse was varied from 100 Hz to 800 Hz in logarithmic steps. RESULTS: Without SSS the highest value of Crel (i.e. relative MR contrast between labeled to unlabeled samples) was found for 150 MLC/microl and was given by 10%. Applying SSS led to positive contrast of the complete labeled volumes and to remarkable improvements in Crel. With increasing cell concentrations Crel raised to maximum, that was given by 52% (BWsat=100 Hz) and 28% (BWsat = 200 Hz) found for 75 MLC/microl. For 150 MLC/microl Crel decreased. A contrast clarification could also be detected near cell aggregations despite saturation. CONCLUSION: Using SSS positive contrast can be achieved for voxels containing MLC and voxels close to cell clusters. Under in vitro conditions positive contrast improved the sensitivity to detect MLC as compared to negative contrast imaging techniques. It seems reasonable, that positive contrast approaches can be applied in vivo as the underlying physical mechanism are comparable.     

Glucose-Raising Polymorphisms in the Human Clock Gene Cryptochrome 2 (CRY2) Affect Hepatic Lipid Content

Circadian rhythms govern vital functions. Their disruption provokes metabolic imbalance favouring obesity and type-2 diabetes. The aim of the study was to assess the role of clock genes in human prediabetes. To this end, genotype-phenotype associations of 121 common single nucleotide polymorphisms (SNPs) tagging ARNTL, ARNTL2, CLOCK, CRY1, CRY2, PER1, PER2, PER3, and TIMELESS were assessed in a study population of 1,715 non-diabetic individuals metabolically phenotyped by 5-point oral glucose tolerance tests. In subgroups, hyperinsulinaemic-euglycaemic clamps, intravenous glucose tolerance tests, and magnetic resonance imaging/spectroscopy were performed. None of the tested SNPs was associated with body fat content, insulin sensitivity, or insulin secretion. Four CRY2 SNPs were associated with fasting glycaemia, as reported earlier. Importantly, carriers of these SNPs’ minor alleles revealed elevated fasting glycaemia and, concomitantly, reduced liver fat content. In human liver tissue samples, CRY2 mRNA expression was directly associated with hepatic triglyceride content. Our data may point to CRY2 as a novel switch in hepatic fuel metabolism promoting triglyceride storage and, concomitantly, limiting glucose production. The anti-steatotic effects of the glucose-raising CRY2 alleles may explain why these alleles do not increase type-2 diabetes risk.     

Dynamic light scattering measurements of the diffusion of probes in filamentous actin solutions

The diffusion coefficients of monodisperse polystyrene latex spheres in solutions of polymerized actin were measured using dynamic light scattering. Four different probes with radii R, ranging from 50 to 500 nm, were separately used in actin solutions with concentrations c, ranging from 1.5 to 21 microM, which had been polymerized with either 1 mM MgCl2, 1 mM CaCl2, or 100 mM KCl. Under all conditions, and at four different scattering angles in the range of 30 degrees-90 degrees, the measured average diffusion coefficients D of the probes were systematically smaller for samples of increased actin concentration or of increased probe radius. Control experiments indicated that the probes did not bind to the actin. These data for Mg2+- and Ca2+-polymerized actin agree and were found to be quite well summarized by the scaling relation D/D0 = exp[-alpha R delta c nu], where D0 is the measured diffusion coefficient of the probes in water (and, as also measured, in the starting actin solutions prior to polymerization with added salt), with values of delta = 0.73 +/- 0.05, nu = 1.08 +/- 0.09, and alpha = (1.1 +/- 0.6) x 10(-3) (with c in microM and R in nm). Data for KCl-polymerized actin show much more restricted diffusivities of the probes at comparable actin concentrations. Inhomogeneities in the solution are reflected in the "effective polydispersity" of the probe diffusion coefficients, which depend on local microviscosity differences.     

INORGANIC PYROPHOSPHATASE OF RAT LIVER MITOCHONDRIA : Correlation of Latency with Catalytic Properties and Intramitochondrial Location

Stimulation of Mg²⁺-dependent inorganic pyrophosphatase activity several fold by disruption of mitochondrial membranes does not appreciably alter the catalytic properties of the enzyme. Stimulation is due to increased accessibility of substrate to the enzyme, which is not solublized on activation. The enzyme is attached to the inside of the inner membrane, and under physiological conditions probably hydrolyzes only intramitochondrially-produced PP_i.     

Intracerebroventricular injections of cholecystokinin octapeptide suppress feeding in rats--pharmacological characterization of this action

Cholecystokinin octapeptide (CCK-8), administered intracerebroventricularly (i.c.v.), will suppress feeding. The aim of the present study was to determine the pharmacological characteristics of this satiety inducing effect in rats. For this purpose, we employed a feeding bioassay model in 24 h fasted rats and examined the effects of CCK-8 and a variety of structurally related analogs on latency to feed after i.c.v. injection and on the amount of food and water consumed as measured after the initiation of feeding in sequential 20-min epochs for 1 h. CCK-8, given in doses of 0.1, 1 and 10 nmol, produced a dose-dependent increase in feeding latency and a reduction of food intake during the first 20 min after initiation of feeding. Food intake during the next 40 min and water consumption were not altered. Plasma levels of CCK-like immunoreactivity after an i.c.v. injection of a dose of CCK-8 which blocked feeding (10 nmol) rose insignificantly from 117 to 125 pg/ml. In contrast, at the minimally effective dose of CCK-8 after i.v. administration (10 nmol), which also produced an inhibition of feeding, the plasma level was 1430 pg/ml. This difference indicates that plasma levels of CCK after i.c.v. CCK-8 are not adequate to produce the observed feeding suppression and suggests that the effects of i.c.v. CCK-8 are not mediated by a peripheral redistribution. Systematic dose response studies revealed the following rank order of potencies: CCK-8 greater than or equal to G-17 II much greater than CCK-8 NS = G-17 I greater than or equal to CCK-4 = CCK 26-29 = 0. Only gastrin-17 II (sulfated) produced an effect comparably significant to CCK-8. I.c.v. proglumide at 2500 nmol failed to modify the effects of CCK-8 at 10 nmol after i.c.v. injection. These data demonstrate that the structural requirements for feeding suppressive activity in rat brain are the carboxyterminus with a sulfated tyrosine residue, located 6 to 7 residues from the carboxyterminus, as present in CCK-8 and gastrin-17 II.     

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