Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Measurement variability in DNA flow cytometry of replicate samples

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.     

S-(2-chloroacetyl)glutathione, a reactive glutathione thiol ester and a putative metabolite of 1,1-dichloroethylene

Conversion of the toxic vinyl halide 1,1-dichloroethylene (DCE) to S-(2-S-glutathionyl-acetyl)glutathione (GSCH2COSG) involves sequential acylation and alkylation of two glutathione (GSH) molecules by the microsomal DCE metabolite ClCH2COCl. To examine its possible role in DCE biotransformation, we synthesized the putative intermediate S-(2-chloroacetyl)glutathione (ClCH2COSG). In aqueous buffer, ClCH2COSG did not hydrolyze to release GSH, but instead underwent a two-step rearrangement to yield a cyclic product. Product analyses by liquid secondary ion mass spectrometry and 1H-13C heteronuclear correlation nuclear magnetic resonance spectroscopy indicated that rearrangement involved initial transfer of the chloroacetyl moiety from the cysteinyl thiol to the gamma-glutamyl alpha-amine. The cysteinyl thiol then displaced chloride from the 2-chloroacetyl methylene carbon to yield the cyclic product. Incubation of 2 mM ClCH2COSG with 20 mM GSH yielded approximately 4.5-fold more cyclic product than GSCH2COSG. ClCH2COSG alkylated oxytocindithiol and N-acetyl-L-cysteine to yield S-[2-(alkylthio)acetyl]glutathione adducts analogous to GSCH2COSG. S-2-Chloroacetylation products were absent. In reacting with thiols by alkylation and in decomposing by rearrangement, ClCH2COSG displayed properties strikingly different from those of ClCH2COCl. Although much less reactive than its acyl halide precursor, ClCH2COSG may display greater selectivity in covalent modification of cellular targets in DCE intoxication.     

Structural and conformational features that affect the interaction of polylactosaminoglycans with immobilized wheat germ agglutinin

We examined the interaction between immobilized wheat germ agglutinin and the large, polylactosamine-containing glycans from human erythrocytes and human K-562 erythroleukemic cells. Three classes of interaction were identified. One class of glycan was merely retarded during chromatography. The other two classes were retained and could be distinguished by their ease of displacement with N-acetylglucosamine (GlcNAc); one was a moderate-affinity fraction displaced by 0.1 M GlcNAc and the other was a high-affinity fraction subsequently displaced by 1.0 M GlcNAc. A relatively small fraction of the K-562 polylactosamines were in the high-affinity class. We explored the role that fucose and sialic acid substitutions play in the strength of the lectin-glycan interaction. Although sialic acid is recognized by wheat germ agglutinin, sialylation was not required for the high-affinity interaction, and the presence of sialic acids actually prevented some glycans from binding with high affinity. In contrast, fucose is not part of the binding determinant, yet the removal of fucose resulted in decreased affinity. The possibility that some of these changes in affinity were the result of conformational changes was explored using matrices that had wheat germ agglutinin immobilized at different densities. At low wheat germ agglutinin densities, adult and fetal erythroglycans and K-562 glycophorin-like glycans were not retained by the matrix. As the density increased, the proportion of glycans that were retarded, and ultimately retained, increased. While these increases in the proportions retained occurred in parallel for the three different glycans, the apparent affinities of the glycan-lectin interactions differed. The glycophorin-like glycans were always readily displaced by 0.1 M GlcNAc, even at higher wheat germ agglutinin densities. In contrast, as the wheat germ agglutinin density increased, the proportion of erythroglycans that could be displaced by 0.1 M GlcNAc decreased; at 10 mg/ml immobilized wheat germ agglutinin, greater than 80% of the erythroglycans exhibited this tighter interaction. We suggest that this higher affinity interaction is the result of the large glycans spanning adjacent wheat germ agglutinin molecules, and is determined by the proximity of these molecules and the conformation of the glycans.