Imaging of radiocarbon-labelled tracer molecules in neural tissue using accelerator mass spectrometry

Autoradiography is widely and successfully used to image the distribution of radiolabelled tracer molecules in biological samples. The method is, however, limited in resolution and sensitivity, especially for 14C. Here we describe a new method for imaging 14C-labelled tracers in sections of biological tissue. A highly focused beam of gallium ions bombards the tissue, which is eroded (sputtered) into constituent atoms, molecules and secondary ions. The 14C ions are detected in the secondary beam by the most sensitive method available, namely accelerator mass spectrometry. The specimen is scanned pixel by pixel (1 x 2 microm), generating an image in a manner analogous to scanning electron microscopy. The method can thus be regarded as a specialized form of scanning secondary ion mass spectrometry (SIMS), referred to here as SIAMS (ref. 2). We have used SIAMS to localize the neurotransmitter gamma-aminobutyric acid (GABA) in thin sections of cerebral cortex, and show that it can generate 14C images that are much improved on 14C autoradiography. A scan takes 10-20 min and reveals individual axons, neurons and glial cells at high sensitivity. In principle, the resolution could be increased by up to tenfold, and the method could be extended to some other nuclides.     

Zinc modulates mononuclear cellular calcitriol metabolism in peritoneal dialysis patients

Zinc has long been known to play a role in maintaining immunologic function. Hypozincemia, however, is common in patients with end-stage renal disease (ESRD) treated with continuous ambulatory peritoneal dialysis (CAPD). We previously demonstrated that zinc depletion limits the ability of animals to achieve maximum circulating calcitriol levels in response to the stress of calcium or phosphorus depletion. It was unclear, however, whether changes in the circulating levels of calcitriol in these settings was associated with a direct effect on renal 1-alpha hydroxylase activity, or whether the zinc dependence of the stimulated calcitriol response involved an integrated systemic response in intact animals. In addition it was unclear whether circulating zinc levels or zinc nutritional status modified calcitriol metabolism in humans. To better understand the role zinc plays in the immune response in patients with ESRD, we studied IL-1, calcitriol and tumor necrosis factor-alpha production by mononuclear cells from blood and peritoneal effluents of 22 patients with ESRD treated with CAPD. Macrophages from peritoneal effluents and peripheral blood mononuclear cells were isolated and pulsed with phytohemagglutinin in medium to which different concentrations of zinc chloride, copper chloride, and carbonyl cyanide p-(trifluoromethoxy)-phenyl-hydrazone (FCCP), an inhibitor of mitochondrial function were added. Supernatant interleukin-1, calcitriol, and tumor necrosis factor-alpha levels were subsequently measured. We demonstrated a zinc concentration dependent increase in stimulated IL-1 alpha and -beta, and TNF-alpha release in both peripheral mononuclear cells and peritoneal macrophages from patients with ESRD treated with CAPD. The effect is zinc specific, as it is not reproduced by copper or chloride supplementation. A zinc concentration dependent increase in peritoneal macrophage calcitriol release was also noted. FCCP blocked the cellular production of IL-1 alpha, IL-1 beta, and TNF-alpha, but had little effect on zinc-induced stimulated mononuclear cell supernatant calcitriol levels. The different shape of the zinc dose response curve, and the lack of correlation between paired IL-1 and calcitriol supernatant levels suggests the effect of zinc on mononuclear cellular cytokine and calcitriol production is mediated through different pathways.     

The organization of leg movements in preterm and full-term infants after term age

In a sample of 13 full-term and 10 preterm infants, the development of kicking movements was studied at 6, 12, and 18 weeks (corrected) age. In healthy full-term infants some characteristics are strikingly stable, such as the duration of the flexion and extension phase and the within-joint organization. These parameters did not differ in preterm compared to full-term infants. For other features, however, developmental changes and differences were observed. Full-term infants tended to decrease their kick frequencies slightly with age. In preterm infants much higher initial kick rates were found, followed by a steep decrease, which resulted in kick frequencies comparable to the full-term levels after the (corrected) age of 12 weeks. There is a tight coupling between the movements in the different joints of the leg in full-term newborns. Preterm infants, in contrast, initially show much lower cross-correlations between hip and ankle and between knee and ankle. This is particularly the case for those preterm infants who were born before 32 weeks gestation. Again, the differences resolved after the age of 12 weeks, which might be related to a transformation in neural functions reported previously around this age. The initial differences in the characteristics of kicking appeared to be more readily explainable by differences in neurological condition than by contrasts in leg volume or postural control.     

Signal transduction in vertebrate growth cones navigating in vivo

Navigating growth cones need signal transduction machinery to amplify and transmit the effects of extracellular signals throughout the growth cone. In culture, many drugs that affect second messengers are known to modulate neurite extension (with different effects on different neurons), and gradients of calcium influx and cyclic nucleotide analogs can cause growth cones to turn. However, it is not clear which of these responses are physiologically relevant, as axons grow through much more complex environments in vivo. The "exposed brain" preparation in Xenopus embryos provides an experimentally tractable system in which it is possible to study growth, pathfinding, and target recognition of retinal growth cones in vivo, while pharmacologically manipulating their signal transduction systems. These growth cones can also be easily studied in explant culture. We describe preliminary results of parallel in vivo and in vitro experiments using an array of drugs that perturb transduction molecules. Surprisingly, calcium ionophores and cyclic nucleotide analogs have no significant effect on retinal axon growth or pathfinding. Several agents including herbimycin A, ML-7, mastoparan, and RHC80267 inhibit retinal axon growth, both in vivo and in vitro, suggesting that tyrosine kinases, myosin, heterotrimeric G-proteins, and diacylglycerol lipase are important for retinal growth cones navigating in the optic pathway.     

Metal ion separations in polyethylene glycol-based aqueous biphasic systems: correlation of partitioning behavior with available thermodynamic hydration data

Solvent extraction utilizing an oil-water mixture (e.g., chloroform-water) and a suitable complexant, is a proven technology for the selective removal and recovery of metal ions from aqueous solutions. Aqueous biphasic systems (ABS), formed by mixing certain inorganic salts and water-soluble polymers, or by mixing two dissimilar water-soluble polymers, have been studied for more than 40 years for the gentle, non-denaturing separation of fragile biomolecules, yet ABS have been virtually ignored as a possible extraction technology for metal ions. In this report we review our metal ion partitioning work and discuss the three major types of partitioning: (1) those rare instances that the metal ion species present in a given solution partitions to the PEG-rich phase without an extractant; (2) the use of halide salts which produce a metal anion complex that partitions to the PEG-rich phase; and (3) the use of a water-soluble extractant which distributes to the PEG-rich phase. In addition, we correlate the partitioning behavior we observed with available thermodynamic data for metal ions and their complexes.     

Overview of randomized trials of intravenous heparin in patients with acute myocardial infarction treated with thrombolytic therapy

Intravenous heparin is routinely given after thrombolytic therapy for patients with acute myocardial infarction in the United States and in some, but by no means all, other countries. Several trials have documented improved infarct-artery patency in patients treated with heparin; however, none was large enough individually to assess the effect of heparin on clinical outcomes. We performed a systematic overview of the 6 randomized controlled trials (1,735 patients) to summarize the available data concerning the risks and benefits of intravenous heparin versus no heparin after thrombolytic therapy. Mortality before hospital discharge was 5.1% for patients allocated to intravenous heparin compared with 5.6% for controls (relative risk reduction of 9%, odds ratio 0.91, 95% confidence interval 0.59 to 1.39). Similar rates of recurrent ischemia and reinfarction were observed among those allocated to heparin therapy or control. The rates of total stroke, intracranial hemorrhage, and severe bleeding were similar in patients allocated to heparin; however, the risk of any severity of bleeding was significantly higher (22.7% vs 16.2%; odds ratio 1.55, 95% confidence interval 1.21 to 1.98). There was no significant difference in the observed effects of heparin between patients receiving tissue-type plasminogen activator and those receiving streptokinase or anisoylated plasminogen streptokinase activator complex, or between patients who did and did not receive aspirin. The findings of this overview demonstrate that insufficient clinical outcome data are available to support or to refute the routine use of intravenous heparin therapy after thrombolysis. It is not known if these findings are due to lack of statistical power, inappropriate levels of anticoagulation, or lack of benefit of intravenous heparin. Large randomized studies of heparin (and of new antithrombotic regimens) are needed to establish the role of such therapy.     

Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus

Leptin, the protein product of the adipose tissue-specific ob (obese) gene (1), reduces the body weight, adiposity and food intake of obese ob/ob mice on peripheral or central injection (2, 3, 4). [125I]leptin binding has been detected in mouse choroid plexus (5), from which a leptin receptor gene was expression cloned (5). The gene has at least 6 splice variants (6, 7). Leptin receptor mRNA was localized in the hypothalamus by in situ hybridization being particularly abundantly expressed in the arcuate nucleus (8). There is evidence linking the physiological effects of injected leptin with hypothalamic neuropeptide Y (9, 10) (NPY), which has potent central effects on food intake and energy balance (11), and is also expressed in the arcuate nucleus. Here we report dual in situ hybridization studies for leptin receptor and NPY gene expression in the mouse arcuate nucleus, where the majority of cells examined expressed both genes. This provides the first direct evidence that leptin acts on cells that express NPY mRNA.     

Effect of seminal plasma on implantation rates

Of 2774 consecutive, prospectively documented fractures of the distal radius, 225 (8 per cent) occurred as sports injuries, mainly in young men. Soccer produced the greatest number of wrist fractures with 112 cases (50 per cent). Skiing, dancing and rugby caused 12 per cent, 9 per cent and 7 per cent of all sporting wrist fractures respectively. Skiing, horseriding and dancing consistently resulted in more complex fractures. In soccer, synthetic pitches increased the likelihood of a fracture following a fall by a factor of five. Twelve per cent of fractures required further treatment because of instability leading to redisplacement. The complication rate was 14 per cent with the majority being cases of malunion (12 per cent). Of the 131 patients who returned a questionnaire, 72.5 per cent had returned to their original sport. This was influenced mainly by the patient's age and pre-injury standard of competition.     

Assessment of popliteal arterial occlusive disease with 2D time-of-flight MRA

OBJECTIVE: The purpose of this study was to evaluate both morphology and blood flow in peripheral arteries with occlusive lesions using MR angiography (MRA) and velocity-encoded cine MRI. MATERIALS AND METHODS: Two-dimensional time-of-flight MRA and velocity-encoded cine MRI were performed in nine patients with peripheral arterial occlusive disease. Findings on MR angiograms were verified by conventional angiography. RESULTS: All the stenotic lesions in the popliteal arteries were depicted by MRA. The degree of the stenoses in the artery was overestimated by MRA. Major collateral circulations were demonstrated. Velocity-encoded cine MRI provided flow velocity information on the arteries above and below the stenoses. The flow velocity waveform was monophasic above and below the stenosis. The peak systolic velocity in the artery below the stenosis was reduced compared with that above the stenosis (p < 0.05). CONCLUSION: The combination of MRA and velocity-encoded cine MRI has clinical potential for the evaluation of peripheral arterial occlusive disease.     

The effectiveness and safety of low dose pravastatin in elderly hypertensive hypercholesterolemic subjects on antihypertensive therapy

To evaluate the efficacy and safety of low dose (10 mg) pravastatin in hypercholesterolemic, hypertensive elderly subjects undergoing antihypertensive treatment, a randomized, double-blind, placebo-controlled 6-month trial was conducted. The subjects had a total plasma cholesterol of at least 250 mg/dL and had been, for at least 3 months, consuming a standard lipid-lowering diet (American Heart Association Step 1 Diet). Sixty elderly hypertensive patients randomly received placebo (n = 30) or pravastatin (n = 30) treatment. The dosage consisted of 10 mg of pravastatin daily during the 6-month trial. Over that period, in the pravastatin group, plasma levels of total cholesterol and LDL-cholesterol significantly (P < .01) dropped (-20% and -25%, respectively) compared to the placebo group. The plasma level of HDL-cholesterol increased (+5%) while triglycerides slightly decreased (-8%) (P < .05). No serious side effects occurred, and pravastatin was generally tolerated. Fasting hyperinsulinemia (11.0 +/- 0.8 v 9.3 +/- 0.7 microU/mL; P = .06) also improved, although not significantly, after 6 months of pravastatin therapy. Results from this study confirmed that a low dose (10 mg) of pravastatin daily is a safe and effective method of reducing plasma total and LDL-cholesterol in hypercholesterolemic, hypertensive elderly patients who are on concurrent antihypertensive drug therapy.