Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells

Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.     

Some ways to improve QTL mapping accuracy

In this paper, we review some approaches for QTL mapping developed by our research group in collaboration with, following the recommendation of, or under inspiration of Moshe Soller. Specifically, we explain at a simple intuitive level the main principles and ideas of: (a) QTL mapping by fractioned DNA pooling, (b) increasing the detection power of QTL mapping (in the case of individual genotyping) by multiple-trait analysis, and (c) the role of variance-covariance effects in QTL mapping. On each of these themes we had long and deep discussions with Soller on the statistical aspects of the proposed procedures. We hope that together we made important contributions towards making QTL mapping procedures easier and more effective.     

Quantitation of Hydroxyl Radicals Produced by Radiation and Copper-Linked Oxidation of Ascorbate by 2-Deoxy-d-Ribose Method

We have established controlled conditions for studying the reaction of chemically and radiolytically produced hydroxyl radical (OH) with 2-deoxy-D-ribose (2-DR). Ascorbate (ASC) or dithiothreitol (DTT) and cuprous or cupric ions were used to generate the OH-radical. The OH-radical was detected using the classical method of measuring the amount of thiobarbituric acid reactive products (TBARP) formed by OH-mediated 2-DR degradation, but using sensitive fluorescent detection of the TBARP production to quantify the OH-radical. All experiments were performed with adequate O₂ concentrations. The copper reaction with ASC consumes O₂ in a manner that is strongly dependent on copper concentration, and less dependent on ascorbate concentration. For an independent check of the Cu²⁺ catalyzed ASC oxidation kinetics, the decay of ASC absorbency at 265 nm, as well as the increase of H₂O₂ absorbency at 240 nm, were also monitored. These spectral changes agree well with the O₂ consumption data. TBARP production from 2-DR incubated with a Cu²⁺–ASC mixture or γ-irradiated were also compared. γ-Irradiation of 2-DR solutions shows a dose and 2-DR concentration dependent increase of TBARP generation. Other electron donors, such as DTT, are more complicated in their mechanism of OH-radical production. Incubation of 2-DR with Cu²⁺-DTT mixtures shows a delay (50 min) before OH-radical generation is detected. Our results suggest that the Cu²⁺-ASC reaction can be used to mimic the effects of ionizing radiation with respect to OH-radical generation. The good reproducibility and relative simplicity of the 2-DR method with fluorescence detection indicates its usefulness for the quantitation of the OH-radical generated radiolytically or chemically in carefully controlled model systems. © 1997 Elsevier Science Inc.     

Interaction of prostaglandin E1 with human high density lipoproteins

The assembly of cytochrome c oxidase subunits I-III was studied in vitro in isolated rat liver mitochondria pre-labeled with [35S]methionine. Individual subunits were immunoabsorbed with monospecific antibodies. Isolated heme a from rat liver mitochondria, when added to radiolabeled mitochondria, induced assembly of subunit I with subunits II and III. Assembly of these subunits was not observed in mitochondria incubated in the presence of heme b(hemin) or in the absence of heme. Quantitative analysis of immunoabsorbed, radiolabeled subunits suggests that the predominant effect of heme a is on the assembly of subunit I with subunit III.     

Interaction of prostaglandins with low-density lipoproteins in human blood

Interaction of prostaglandins (PG) with human plasma low density lipoproteins (LDL) was studied, using fluorescent spectroscopy and photoreactive labeling. It was demonstrated that PGE1 at low concentrations (less than 10(-9) M) induces specific lipid rearrangements on the surface of LDL globules. It was assumed that these rearrangements are brought about by the interaction of PG with apolipoprotein B to form short-living complexes. A possible mechanism and biological significance of the observed phenomenon are discussed.     

Parallel in vivo analysis of large-effect autism genes implicates cortical neurogenesis and estrogen in risk and resilience

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DNA-phospholipid interactions. A study using lipid-specific fluorescent and photoreactive probes

The interaction of phospholipids with phage T7 DNA was investigated using anthryl-vinyl-labeled and photoactivable phosphatidylcholine and sphingomyelin. Fluorescence polarization studies demonstrated that, in the presence of DNA, the fluorophore mobility is diminished as its distance from the polar head-group is increased. Immobilization of lipid chains is enhanced by Ca2+ ions, the effect being more pronounced for sphingomyelin than for phosphatidylcholine derivatives. On the other hand, phospholipids with a photoactivable group could not be crosslinked to DNA in the DNA-phospholipid complexes, evidencing against the presence of contacts between lipids and DNA.