L-Homocysteine and L-homocystine stereospecifically induce endothelial nitric oxide synthase-dependent lipid peroxidation in endothelial cells

Atherothrombotic cardiovascular disease associated with hyperhomocysteinemia has been proposed to result, at least in part, from increased vascular oxidative stress. Here we characterize one mechanism by which homocyteine may induce a vascular cell type-specific oxidative stress. Our results show that L-homocysteine at micromolar levels stereospecifically increases lipid peroxidation in cultured endothelial cells, but not in vascular smooth muscle cells or when medium is incubated in the absence of cells. Consistent with these observations, homocysteine also increases the formation of intracellular reactive oxygen species. The pro-oxidant effect of homocysteine can be fully replicated by an equivalent concentration of homocystine (i.e., an oxidized form of homocysteine), but not with cysteine or glutathione. Homocyst(e)ine-dependent lipid peroxidation is independent of H(2)O(2) and alterations in glutathione peroxidase activity, but dependent on superoxide. Mechanistically, the pro-oxidant effect of homocysteine appears to involve endothelial nitric oxide synthase (eNOS), as it is blocked by the eNOS inhibitor L-N(G)-nitroarginine methyl ester. Thus, homocyst(e)ine actively promotes oxidative stress in endothelial cells via an eNOS-dependent mechanism.     

Epithelial trafficking: new routes to familiar places

Research carried out in mammalian epithelial cell systems over the past 25 years has delineated pathways and sorting signals involved in polarized delivery of plasma membrane proteins. Recently some progress has been made in the identification of mechanisms underlying this polarized trafficking and in the visualization of trafficking routes in live cells. A promising area of research is the study of trafficking functions of novel polarity genes identified in Drosophila and Caenorhabditis elegans.     

Phenotypical evidence for a gender difference in cardiac norepinephrine transporter function

Norepinephrine transporter (NET) function has a central role in the regulation of synaptic norepinephrine concentrations. Clinical observations in orthostatic intolerance patients suggest a gender difference in NET function. We compared the cardiovascular response to selective NET inhibition with reboxetine between 12 healthy men and 12 age-matched women. Finger blood pressure, brachial blood pressure, and heart rate were measured. The subjects underwent cardiovascular autonomic reflex testing and a graded head-up tilt test. In a separate study, we applied incremental concentrations of tyramine and isoproterenol through subcutaneous microdialysis catheters in eight men and in eight women. NET inhibition elicited a threefold greater increase in supine blood pressure in men than women (P < 0.05). The pressor response was driven by an increased cardiac output. The orthostatic heart rate increase during NET inhibition was greater in men than women (56 +/- 5 beats/min in men, 42 +/- 4 beats/min in women, P < 0.001). In contrast, NET inhibition resulted in a similar suppression in the cold pressor and handgrip response, low-frequency blood pressure oscillations, and venous norepinephrine in the supine position. Men and women were similarly sensitive to the lipolytic effect of isoproterenol and tyramine. We conclude that NET inhibition results in more pronounced changes in cardiac regulation in men than women. Our observations suggest that the NET contribution to cardiac norepinephrine turnover may be decreased in women. The gender difference in NET function may not be expressed in tissues that are less NET dependent than the heart.     

Indicators of hippocampal neurogenesis are altered by 56Fe-particle irradiation in a dose-dependent manner

The health risks to astronauts exposed to high-LET radiation include possible cognitive deficits. The pathogenesis of radiation-induced cognitive injury is unknown but may involve loss of neural precursor cells from the subgranular zone (SGZ) of the hippocampal dentate gyrus. To address this hypothesis, adult female C57BL/6 mice received whole-body irradiation with a 1 GeV/nucleon iron-particle beam in a single fraction of 0, 1, 2 and 3 Gy. Two months later mice were given BrdU injections to label proliferating cells. Subsequently, hippocampal tissue was assessed using immunohistochemistry for detection of proliferating cells and immature neurons. Routine histopathological methods were used to qualitatively assess tissue/cell morphology in the hippocampal formation and adjacent areas. When compared to controls, irradiated mice showed progressively fewer BrdU-positive cells as a function of dose. This observation was confirmed by Ki-67 immunostaining in the SGZ showing reductions in a dose-dependent fashion. The progeny of the proliferating SGZ cells, i.e. immature neurons, were visualized by doublecortin staining and were significantly reduced by irradiation, with the decreases ranging from 34% after 1 Gy to 71% after 3 Gy. Histopathology showed that in addition to cell changes in the SGZ, (56)Fe particles induced a chronic and diffuse astrocytosis and changes in pyramidal neurons in and around the hippocampal formation. The present data provide the first evidence that high-LET radiation has deleterious effects on cells associated with hippocampal neurogenesis.     

Brain natriuretic peptide and left ventricular dysfunction in chagasic cardiomyopathy

Global left ventricular (LV) systolic dysfunction is the strongest predictor of morbidity and mortality in Chagas disease. Echocardiography is considered the gold standard for the detection of LV dysfunction, but not always available in endemic areas where chagasic cardiomyopathy is most common. Brain natriuretic peptide (BNP) is a neurohormone that has been recently described as a simple and inexpensive diagnostic and prognostic marker for patients with congestive heart failure. Chagasic patients (n = 63) and non-infected healthy individuals (n = 18) were recruited prospectively and underwent complete clinical examination, echocardiography and 24-h Holter monitoring. BNP was measured from thawed plasma samples using the Triage BNP test. We observed high levels of BNP in association with depression of LV ejection fraction, with increase of LV end-diastolic diameter and with LV premature complexes. An elevated concentration of BNP, defined as a concentration of 60 pg/ml or more, had a sensitivity of 91.7%, specificity of 82.8%, positive predictive value of 52.4%, and negative predictive value of 98% for detecting LV dysfunction (LV ejection fraction < 40%).BNP measurement using a simple, relatively inexpensive and rapid test has a promising role in identifying LV dysfunction associated with chagasic cardiomyopathy. Equally important, patients with Trypanosoma cruzi infection who have low levels of BNP level in plasma have a very low likelihood of severe cardiac involvement, and echocardiography is probably not necessary.     

Correction of image instability in confocal microscopy using image realignment. Effects on the analysis of intracellular calcium

Using confocal microscopy, we have examined the increases in [Ca(2+)](i) evoked by sodium channel toxins in cells labelled with the fluorescent dye INDO-1. We describe a new image analysis method that improves the detection of region-specific, toxin-induced patterns of change of intracellular calcium. This method is based on correction of global image motion followed by calculation of the strength of correlation between calcium changes in "seed" or reference pixels chosen to represent different regions of cells and those in other regions of the image. When the selected "seed" pixel was chosen to be in either varicosities or neurites, correlations were detected in the same regions of other cells as well as in the soma, indicating specific but spatially distinct patterns of behaviour. Control images (without changes in [Ca(2+)](i)) did not reveal significant interpixel correlations. The ability to recognize correlated patterns of calcium change in different regions of cells was greatly improved by correction for global motion.     

Glycosyltransferase activity can be selectively modulated by chemical modifications of acceptor substrates

A range of N-acetyllactosamine derivatives, which are modified by a wide range of functionalities at C-2(') and C-6, have been synthesised and the kinetic parameters of transfer catalysed by recombinant alpha-2,6-sialyltransferase and alpha-1,3-fucoyltransferase VI determined. Several of the chemical modifications led to selective modulate the activity the enzymes and offer promising lead compounds for the development of oligosaccharide primers for selective metabolic inhibition of oligosaccharide biosynthesis.