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Neutrophil labeling: problems and pitfalls.

The use of neutrophils labeled with gamma-emitting radionuclides has been shown to be acceptable for in vivo kinetic studies as well as for imaging inflammatory foci. Among the gamma-emitting radionuclides, indium-111 appears to be the agent of choice. Labeling neutrophils with 111In, however, is a relatively new technique. Although simple to perform, it involves several stages, none of which could be carried out without problems. These are discussed and the current research aimed at eliminating the problems is outlined. The knowledge of specific chemotactic receptors and surface antigens has stimulated investigations into selective neutrophil labeling that will continue to be challenging and exciting.

Animals↗

Methyl mercury interactions with phospholipid membranes as reported by fluorescence, 31P and 199Hg NMR.

Methylmercury (CH3Hg(II)) interactions with multilamellar vesicles of dimyristoyl(DM)- and dipalmitoyl(DP)-phosphatidylcholine (PC), -phosphatidic acid (PA), -phosphatidylglycerol (PG), -phosphatidylserine (PS) and -phosphatidylethanolamine (PE) have been investigated from the metal viewpoint by solution 199Hg-NMR and from the membrane side by diphenylhexatriene fluorescence polarization and solid state 31P-NMR. Results can be summarized as follows: (1) CH3Hg(II) strong binding to membranes results in a progressive decrease of the free CH3HgOH 199Hg-NMR isotropic signal and because of a slow exchange, in the NMR time scale, between free and bound methylmercury pools the lipid/water partition coefficients, K(lw), of the CH3HgOH species can be determined in the lamellar gel (fluid) phase. It is found: K(lw)(DMPC) approximately 2 +/- 2 (2 +/- 2); K(lw)(DMPE) approximately 7 +/- 3 (16 +/- 3); K(lw)(DMPG) = 170 +/- 10 (110 +/- 10); K(lw)(DMPS) = 930 +/- 50 (1250 +/- 60); K(lw)(DMPA) = 1250 +/- 60 (300 +/- 20). CH3Hg(II) interactions with membrane phospholipids are therefore electrostatic in nature and the phosphate moiety is proposed as a potential binding site. (2) The presence of CH3HgOH stabilizes the PG gel phase and destabilizes that of PS. No effect is observed on PC, PA and PE thermotropism. (3) methylmercury promotes the formation of isotropic 31P-NMR lines with PG, PA and PE systems suggesting the presence of non-bilayer phases and hence membrane reorganization. The above effects are compared to those of inorganic mercury Hg(II) and discussed in the context of cell toxicity.

Fluorescence Polarization↗

Biological ion exchanger resins. I. Quantitative electrostatic correspondence of fixed charge and mobile counter ion.

Utilizing Escherichia coli as the prototype of an ion-accumulating cell, the ion exchange isotherm is introduced as a concise method of characterizing biological ion exchange events. The ion exchange isotherm for the alkali cation exchange, K <--> Na, is described. The total charge profile of this bacterium is compiled and compared for bacteria in the Na form and in the K form. Macromolecule fixed charge was found to provide 80% of the counter ions that pair with potassium. Therefore, in its physiological state, 80% of the cell potassium in E. coli is associated with an ion exchange site on a macromolecule. The primary cation exchange sites are found to be about equally divided between carboxylate and phosphate sites indicating that E. coli is a bifunctional resin with respect to cation exchange. During substrate-dependent cation accumulation ("active transport"), phosphate esters and organic acids were shown to accumulate. One may conclude that the role of intermediate metabolism in "active transport" is to increase the ion exchange capacity of the biological resin by the production of charged metabolites that sorb to the framework of the resin.

Amines↗

Si(3)N(4)-bioglass composites stimulate the proliferation of MG63 osteoblast-like cells and support the osteogenic differentiation of human bone marrow cells.

The in vitro osteocompatibility of a novel Si(3)N(4)-bioglass composite (70-30% weight proportion) with improved mechanical properties (fracture toughness = 4.4 M Pa m(1/2); bending strength = 383 +/- 47 MPa) is reported. Immersion of the composite samples in culture medium (30 min to 7 days) resulted in rapid protein adsorption to the surface and, also, dissolution of the intergranular phase of bioglass (time-dependent process) with the formation of different size cavities. "As-received" and pre-treated material samples presented a similar behaviour concerning the proliferation of MG63 osteoblast-like cells, evaluated during a 5-day culture period. Seeded materials showed a higher cell growth rate as compared to cultures performed on the standard plastic culture plates. To assess the osteogenic potential of the composite, "as-received" material samples were seeded with human bone marrow cells and cultured for 35 days in experimental conditions that favour the development of the osteoblastic phenotype. The cell adhesion process was similar to that observed in control cultures. Cells successfully adapted to the irregularities of the surface and were able to grow towards inside the cavities; in addition, osteogenic differentiation occurred with the formation of abundant cell-mediated mineralised deposits. Results suggest that this Si(3)N(4)-bioglass composite seems to be a promising candidate for high-stress medical applications.

Adsorption↗

In vivo incorporation of cytidine-monophosphate-ciliatine into rat liver lipids.

1. In vivo this investigation was carried out in order to compare the incorporation into rat lipids of free [1,2-minus 14C]-ciliatine and CMP-[1,2-minus 14C]-ciliatine which is the precursor in phosphonolipid biosynthesis. 2. The incorporation of the radioactivity from CMP-[1,2-minus 14C]-ciliatine took place more rapidly than that from free [1,2-minus 14C]-ciliatine in both liver and kidney. The amount of radioactivity from the CMP-[1,2-minus 14C]-ciliatine incorporated into total liver lipids was about 5 times higher than that incorporated into total liver lipids of rat two hrs after injecting free-[1,2-minus 14C]-ciliatine. 3. The amount of [1,2-minus 14C]-ciliatine incorporated into total liver lipids was 15 and 21 times higher than that incorporated into total kidney lipids of rat two and four hrs after injecting free [1,2-minus 14C]-ciliatine. 4. If the main pathway for the phosphonolipid biosynthesis is via CMP-ciliatine, the rate of phosphonolipid formation from CMP-ciliatine must therefore be higher than that from free-ciliatine. The results obtained here indicate therefore that the main pathway for phosphonolipid biosynthesis is a pathway involving CMP-ciliatine. 5. An unknow compound was detected in the water soluble fraction of the acid hydrolyzate of liver phosphonolipids. This material migrated with the N-trimethyl-derivative of ciliatine on the thin-layer chromatogram. The result shows that there is therefore a possibility of methylation of exogenous ciliatine to the phosphonate analogue of choline in the mammalian body.

Aminoethylphosphonic Acid↗

Phospholipid metabolism and resistance to glucocorticoid-induced apoptosis in a human leukemic cell line: a 31P-NMR study using a phosphonium analog of choline.

In order to evaluate any connection between phospholipid metabolism and the dexamethasone resistance in CEM-C1-15 leukemic cells, a phosphonium analog of choline was used to study the phospholipid metabolism in dexamethasone-sensitive and dexamethasone-resistant leukemic cells using 31P-NMR spectroscopy. Measurements were done on the two cell lines in the presence and absence of dexamethasone, a synthetic glucocorticoid steroid. Dexamethasone was found to cause a significant reduction in the phospholipid metabolism of the dexamethasone-sensitive CEM-C7-14 cells. In contrast, dexamethasone caused a significant enhancement of the phospholipid metabolism in the dexamethasone-resistant CEM-C1-15 cells, indicating a significant difference between the two cell lines in phospholipid metabolism during apoptosis. The results of this study suggest the involvement of phospholipid metabolism in the mechanism of dexamethasone resistance in the CEM-C1-15 cells.

Apoptosis↗

The surface structure of catalysts activated with hydrogen donors as elucidated by multinuclear solid-state NMR.

1H, 27Al and 31P MAS, and 13C and 29Si CP/MAS NMR spectroscopies, were used to characterize catalysts of Pd supported on various solids including SiO2, AlPO4 and Mg3(PO4)2 that were activated with the chiral hydrogen-donor limonene. The above-mentioned techniques were used to check for the formation of an organopalladium complex between Pd2+ atoms and the olefin bonds in the limonene molecule on the catalyst surface. The results are compared with those obtained for catalysts activated in a hydrogen stream.

Aluminum↗

Struvite precipitation in anaerobic swine lagoon liquid: effect of pH and Mg:P ratio and determination of rate constant.

Because of increased concern about surface water eutrophication from nutrient-enriched agricultural runoff, many swine producers are encouraged to decrease application rates of waste-based P. Precipitation and subsequent removal of magnesium ammonium phosphate (MgNH(4)PO(4) x 6H(2)O), commonly known as struvite, is a promising mechanism for N and P removal from anaerobic swine lagoon effluent. The objectives of this research were to (i) quantify the effects of adjusting pH and Mg:P ratio on struvite precipitation and (ii) determine the rate constant pH effect for struvite precipitation in anaerobic swine lagoon liquid. Concentrations of PO(4)-P in liquid from two anaerobic swine lagoons were determined after 24 h of equilibration for a pH range of 7.5-9.5 and Mg:P ratios between 1:1 and 1.6:1. Struvite formation reduced the PO(4)-P concentration in the effluents to as low as 2 mgl(-1). Minimum concentrations of PO(4)-P occurred between pH 8.9 and 9.25 at all Mg:P ratios. Struvite precipitation decreased PO(4)-P concentrations by 85% within 20 min at pH 9.0 for an initial Mg:P ratio of 1.2:1. The rate of PO(4)-P decrease was described by a first-order kinetic model, with rate constants of 3.7, 7.9, and 12.3 h(-1) at pH 8.4, 8.7 and 9.0 respectively. Our results indicate that induced struvite formation is a technically feasible method to remove N and P from swine lagoon liquid and it may allow swine producers to recover nutrients for off-farm sale.

Anaerobiosis↗

Use of Chinese LiF:Mg,Cu,P (GR-200) TL detectors at therapy-level absorbed doses.

The objective was to find out whether or not the highly radiation-sensitive Chinese thermoluminescent (TL) LiF:Mg,Cu,P (GR-200) detector pellets can be used in therapy-level dosimetry. For this purpose the precision was investigated at an absorbed dose level of 1 Gy using a commercial TL read-out instrument. Relative standard deviations, sigma rel, of the individual calibration factors were found to be in the range between 0.2% and 0.6% from 30 randomly selected detectors, with the mean at 0.37%. Apart, linearity checks between 1 and 6 Gy resulted into a sublinear response of up to -4% which was successfully corrected for by a third-order polynomial curve fit. The residual signal turned out to be about 0.1% after one annealing cycle almost independent of the applied dose, and this figure could be reduced by two more decades after repeated annealing cycles.

Copper↗

(Aminomethyl)phosphonate derivatives of oligonucleotides.

Oligothymidylate (aminomethyl)phosphonates have been prepared, and their enzymatic and physicochemical properties have been studied. The individual isomers of the protected dimers have been separated, characterized, and incorporated into oligonucleotides in which the backbone consists of alternating (aminomethyl)phosphonate and phosphodiester linkages. One of these net neutral, single isomer oligonucleotides forms a duplex with its complementary sequence which is more stable than the corresponding natural counterpart, whereas the other isomer is considerably less stable. Specificity of hybridization is maintained, as determined by the reduction in melting temperature observed upon the introduction of mismatches into the complementary strand of the duplex. The (aminomethyl)-phosphonate linkage is stable toward enzymatic degradation but can be hydrolyzed in aqueous solution at elevated temperature.

Base Sequence↗

Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of DNA is highly specific for duplex DNA.

We show that iron(II)-EDTA-catalyzed cleavage of duplex DNA is much more rapid than cleavage of single-stranded DNA that does not form intramolecular base pairs. Comparisons of the extent of cleavage of the fully single-stranded oligonucleotides d(pT)70, d(pA)70, and d(pC)35 and the duplex DNA d(pT)70.d(pA)70 indicate that the extent of cleavage increases significantly upon formation of the duplex structure. These observations indicate that accessibility of the DNA sugars to the presumed cleaving agent, hydroxyl radical, is not the major determinant for cleavage and that most likely a direct interaction between Fe(II) and the DNA is required. As a result, the interpretation of DNA cleavage experiments performed with this reagent to obtain detailed structural information should be pursued with caution until the mechanism of the cleavage reaction is better understood.

DNA↗