Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis

This report describes a simple, rapid, automated microassay for measuring in vitro changes of oxidative burst of phagocytes following challenge with metals for orthopedic devices. The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) was measured using 2',7'-dichlorofluorescin-diacetate (DCFH-DA) as fluorescent probe. DCFH-DA enters the cells and is oxidized by ROS to fluorescent DCF. The DCF generated was directly proportional to ROS produced intracellularly: The fluorescence intensity was read and converted to an index of ROS production by cells. In our experimental system, granulocytes (PMNs) were isolated from normal human blood and seeded in microplates. To verify if metals could influence ROS production, chromium, cobalt, nickel, molybdenum, titanium, aluminum, and vanadium prepared as aqueous extracts in phosphate-buffered saline were tested onto PMNs using phorbolmyristate acetate (PMA) as positive control. Molybdenum, aluminum, and vanadium increased ROS generation by PMNs, while signals not different from unstimulated PMNs were recorded for chromium, cobalt, nickel, and titanium. The DCFH-DA microplate-based assay provides an in vitro tool for the detection of oxygen-reactive species generated by PMNs as a response to metals.     

Differential time course of angiotensin-induced AP-1 and Krox proteins in the rat lamina terminalis and hypothalamus

Binding to olfactory receptors is the first step in odorant and pheromonal recognition and discrimination. These receptors constitute one of the most important, although poorly known, families of neuronal receptors. In this study we used degenerated oligonucleotides and a RT-PCR approach to selectively amplify olfactory receptors in the nasal epithelium of the domestic pig Sus scrofa. Several combinations of oligonucleotide were tested and allowed the isolation of eleven different partial sequences belonging to the seven transmembrane olfactory receptor family. These receptors formed a separate family within the seven transmembrane receptor superfamily in pigs. Using the criteria of Ben Arie et al. [Ben-Arie N., Lancet D., Taylor C., Khen M., Walker N., Ledbetter DH., Carrozzo R., Patel K., Sheer D., Lehrah H. and North M., Hum. Mol. Genet., 3 (1994) 229-235], the 11 receptors described here can be classified into three known families and seven subfamilies (one known and six new).     

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na+]i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na+]i. In dopamine neurons voltage-clamped at a holding potential of -60 mV elevations of [Na+]i were induced by long-lasting (45-60 s) voltage jumps to more negative membrane potentials (-90 to -120 mV) but not by corresponding voltage jumps to -30 mV. These hyperpolarization-induced elevations of [Na+]i were depressed during inhibition of I(h), a hyperpolarization-activated inward current, by Cs+. Hyperpolarization-induced elevations in [Na+]i might occur also in other cell types which express a powerful I(h) and might signal lack of postsynaptic activity.     

Morphologic image processing operators: reduction of partial volume effects for improved 3D visualization of CT data

AIM: The quality of segmentation and three-dimensional reconstruction of anatomical structures in tomographic slices is often impaired by disturbances due to partial volume effects (PVE). The potential for artefact reduction by use of the morphological image processing operators (MO) erosion and dilation is investigated. DESIGN: The CT examinations of 31 patients with pathological alterations in lung or brain were segmented using automatic region growing and the MO were applied in a different number of iterations. The processed regions were 3D-reconstructed (shaded surface display, MIP, volume rendering) and the occurrence of PVE-related artefacts using the signal-to-background ratio (SBR) prior to and after MO application was compared. RESULTS: For all patients under review, the artefacts caused by PVE were significantly reduced by erosion (lung: mean SBRpre = 1.67, SBRpost = 4.83; brain: SBRpre = 1.06, SBRpost = 1.29) even with only a small number of iterations. Region dilation was applied to integrate further structures (e.g. at tumor borders) into a configurable neighbourhood for segmentation and quantitative analysis. CONCLUSIONS: The MO represent an efficient approach for the reduction of PVE artefacts in 3D-CT reconstructions and allow optimized visualization of individual objects.     

Application of nested PCR and mass spectrometry for DNA-based virus detection: HBV-DNA detected in the majority of isolated anti-HBc positive sera

DNA preparations from three different groups of serum samples were examined for HBV-DNA via a nested polymerase chain reaction assay (lower detection limit: 10 viral genomes in 100 microliters serum): Group I consisted of 11 uninfected control sera, group II consisted of sera obtained from 11 HBV infected patients and group III consisted of 21 isolated anti-HBc positive samples. The 21 samples from group III were HBV-DNA negative according to a conventional non-nested PCR assay and hybridization with a 32P-labelled probe. Using nested PCR and mass spectrometry, HBV-DNA was detected in none of group I and in all of group II samples. In 11 out of 21 (52%) of the isolated anti-HBc positive sera from group III, HBV-DNA was detected. No correlation was observed between HBV-DNA positivity and anti-HBc titers. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provided a fast, sensitive and non-radioactive assay for the detection of PCR products without the need for gel electrophoresis or hybridization with labelled probes.     

Immunological identification of candidate proteins involved in regulating active shape changes of outer hair cells

By employing immunological methods, it has been demonstrated that myosin, myosin light chain (MLC) and myosin light chain kinase (MLCK) proteins in outer hair cells (OHC) are immunologically different from isoforms in platelets, smooth muscle and heart muscle, and are probably more related to isoforms found in red blood cells (RBC). Moreover, proteins related to band 3 protein (b3p) and protein 4.1 (p 4.1), ankyrin as well as fodrin and spectrin, but not glycophorin, have been identified in isolated OHCs. Both OHCs and RBC differ from other motile non-muscle cells in their lack of smooth muscle isoforms of actin, their common high levels of spectrin-, ankyrin- and band 3-like proteins, as well as the expression of the 80 kDa protein 4.1 isoform. The data support the notion that motility of OHC may be based upon regulation of the b3p/p 4.1/ankyrin complex, and thus may be reminiscent to the active shape changes in RBC.     

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes

The inhibitors of the Na+/H+-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Nai) and calcium (Cai) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na+ and Ca2+ (pHi, Nai and Cai) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2, 7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pHi 7.2 to pHi 6.8) and an increase in Cai from about 50 nmol/l to about 1 micromol/l. The decrease in pHi began before the cells underwent hypoxic (rigor) contracture, whereas Cai only began to rise after rigor shortening had taken place. After reoxygenation, pHi returned to its control value and Cai oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 micromol/l Hoe 642 was present from the beginning of the experiment, pHi and Cai were not significantly different from control experiments. At reoxygenation, pHi did not recover, but Cai oscillated and returned to its resting level. To monitor Nai, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 micromol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 micromol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 micromol/l) attenuated the CN--induced rise in Nai. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell's autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Cai overload, nor CN--induced Nai and Cai overload. Hoe 642 prevented the recovery of pHi from anoxic acidification. This low pHi maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca2+ overload in mitochondria, cannot be ruled out. The increase in Nai during anoxia is possibly due to an influx of Na+ via persistent Na+ channels.     

Effect of hydrogen loading on temperature/electric-field poling ofSiO2-based thin films on Si

Waveguide stacks of GeSiO₂-based glass deposited on Si exhibit a permanent second-order nonlinearity following temperature electric-field poling. High temperature hydrogen loading increases the second harmonic signal under certain conditions