Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo

Incorporation of 8 mol percent lactosylceramide into small unilamellar vesicles consisting of cholesterol and sphingomyelin in an equimolar ratio and containing [³H]inulin as a marker resulted in an increase in total liver uptake and a drastic change in intrahepatic distribution of the liposomes after intravenous injection into rats. The control vesicles without glycolipid accumulated predominantly in the hepatocytes, but incorporation of the glycolipid resulted in a larger stimulation of Kupffer-cell uptake (3.2-fold) than of hepatocyte uptake (1.2-fold). Liposome preparations both with and without lactosylceramide in which part of the sphingomyelin was replaced by phosphatidylserine, resulting in a net negative charge of the vesicles, were cleared much more rapidly from the blood and taken up by the liver to higher extents. The negative charge had, however, no influence on the intrahepatic distributions. The fast hepatic uptake of the negatively charged liposomes allowed competition experiments with substrates for the galactose receptors on liver cells. Inhibition of blood clearance and liver uptake of lactosylceramide-containing liposomes by $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ indicated the involvement of specific recognition sites for the liposomal galactose residues. This inhibitory effect of $\text{N-}\text{acetyl-}\text{d}\text{-galactosamine}$ was shown to be mainly the result of a decreased liposome uptake by the Kupffer cells, compatible with the reported presence of a galactose specific receptor on this cell type (Kolb-Bachofen et al. (1982) Cell 29, 859–866). The difference between the results on sphingomyelin-based liposomes as described in this paper and those on phosphatidylcholine-based liposomes as published previously (Spanjer and Scherphof (1983) Biochim. Biophys. Acta 734, 40–47) are discussed.     

Immunocytochemical localization of tubulin with colloidal gold in cilia of rabbit tracheal epithelial cultures

Ciliated tracheal epithelia cell cultures were investigated immunocytochemically with anti-tubulin and colloidal gold. When rabbit tracheal cultures were fixed in paraformaldehyde, treated with acetone, anti-tubulin and a second antibody coupled to FITC, fluorescence was associated with cytoskeletal and axonemal microtubules. Cilia covering the apical surface of the ciliated tracheal cells fluoresced very brightly thus facilitating identification of this cell type. Electron microscopy of tracheal cultures fixed as above, treated with Triton-X 100 and incubated in anti-tubulin and protein A coupled to colloidal gold resulted in the highly specific localization of tubulin in ciliary axonemes and basal bodies. Omission of primary or secondary antibody resulted in extremely low levels of fluorescence while no colloidal gold particles could be detected in cultures at the electron microscopy level when rabbit anti-tubulin was omitted.     

Design and operation of a completely automated Beckman microsequencer

A unique, efficient, and inexpensive system has been designed and built for the automatic conversion of anilinothiazolinone derivatives extracted from a Beckman spinning-cup sequencer with subsequent on-line high-pressure liquid chromatography separation of the phenylthiohydantoin derivatives. The Auto Converter-Auto Sampler system is controlled by a tape programmer or microprocessor and operates by transfer of the sample from the conversion vial into an HPLC injection loop by nitrogen pressure. Incorporation of a minor programming change on the sequencer allows the introduction of nitrogen vapor into the spinning cup during phenylisothiocyanate coupling. These modifications have resulted in a completely automated subnanomole protein sequencer.     

Germplasm Modification and Its Potential for Finding New Sources of Resistance to Diseases

In vitro procedures are playing a major role in plant breeding. Embryo rescue, either through the culture of excised embryos derived from incompatible crosses or by means of ovule culture, has been a standard procedure for the introgression of genes conferring disease resistance into economically important plants. Somatic hybridization (i.e., protoplast fusion) has also been demonstrated to have some potential in obtaining hybrids that result from very wide interspecific and intergeneric crosses. Wide crosses have also been achieved by means of in vitro pollination of excised ovaries or ovules. Tissue culture-induced variability in regenerated plant (i.e., somaclonal variation) appears to be an effective way of obtaining undirected genetic change that can enhance disease resistance and yield and alter the growth habit of crops that are normally propagated vegetatively (e.g., potato) or by seed (e.g., tomato). In the near future, the isolation and sequencing of genes that confer resistance to specific plant pathogens will be possible, and transfer of this information between species will become a reality.     

Preparation of fatty acid methyl esters for gas-liquid chromatography

A convenient method using commercial aqueous concentrated HCl (conc. HCl; 35%, w/w) as an acid catalyst was developed for preparation of fatty acid methyl esters (FAMEs) from sterol esters, triacylglycerols, phospholipids, and FFAs for gas-liquid chromatography (GC). An 8% (w/v) solution of HCl in methanol/water (85:15, v/v) was prepared by diluting 9.7 ml of conc. HCl with 41.5 ml of methanol. Toluene (0.2 ml), methanol (1.5 ml), and the 8% HCl solution (0.3 ml) were added sequentially to the lipid sample. The final HCl concentration was 1.2% (w/v). This solution (2 ml) was incubated at 45°C overnight or heated at 100°C for 1–1.5 h. The amount of FFA formed in the presence of water derived from conc. HCl was estimated to be <1.4%. The yields of FAMEs were >96% for the above lipid classes and were the same as or better than those obtained by saponification/methylation or by acid-catalyzed methanolysis/methylation using commercial anhydrous HCl/methanol. The method developed here could be successfully applied to fatty acid analysis of various lipid samples, including fish oils, vegetable oils, and blood lipids by GC.     

Evaluation of acute and chronic toxicity of selected compounds in higher plants using cell culture

Summary The feasibility of using plant cell culture to measure toxicity was determined by investigating the toxicological effects of three chemical compounds, allyl alcohol, propargylglycine, and cadmium chloride, on cell cultures ofCatharanthus roseus G. Don (Madagascar periwinkle). Suspension cultures ofC. roseus were maintained in modified B5 medium and transferred every 5 d. Five-day-old cell cultures were exposed to various concentrations (10,3,1,0.3,0.1,0.03,0.01,0.003,0.001,0.0003,0.0001, 0.00003, and 0.0 mM) of the toxicants in both acute and chronic toxicity tests. In the acute test, cells were exposed to the toxicant for 24 h, washed three times with sterile medium, and plated in petri plates with an equal volume of 1.4% agar medium. Cells in the chronic test were plated with an equal volume of 1.4% agar medium containing various concentrations of the toxicant. Cells were incubated 28 d at 30°C in the dark. The colonies were counted and the results plotted as percent survival versus toxicant concentration. The results indicate, at the concentrations tested, thatC. roseus assay may be feasible in that it fulfills the criteria for a practical assay (e.g., rapid, simple, quantifiable, and reproducible). This work was submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Master of Environmental Science, Institute of Environmental Sciences.     

A bivariate radioimmunoassay for arginine vasopressin and the synthetic antidiuretic agent 1-deamino-8-D-arginine vasopressin (desmopressin)

Pairs of radioimmunoassays, each of which include a two-dimensional matrix of standards, have been previously employed to resolve specificity problems in steroid immunoassay. In this study the bivariate radioimmunoassay principle has been applied to simultaneous measurement of plasma antidiuretic hormone, arginine vasopressin, and the synthetic antidiuretic agent 1-deamino-8-D-arginine vasopressin (desmopressin), by utilizing two arginine vasopressin antisera which show significantly different cross-reactivities with the synthetic analog. Data processing consists of mathematical representation of two curved dose-response surfaces followed by solution of this pair of nonlinear simultaneous equations for the unknown arginine vasopressin and desmopressin concentrations. Details of numerical procedures are given in the Appendix. The assay appears entirely adequate in terms of sensitivity, accuracy, and precision for measurement of these antidiuretic agents in clinical samples. No evidence of significant covariance in estimated concentrations could be detected but precision of estimation is (not unexpectedly) a function of the concentration of both agents. The plasma disappearance half-time of desmopressin (probably the second of a biphasic disappearance) was estimated as 37 min in one normal subject, which is in good agreement with a previously reported value of 30 min.     

The effect of tumor promoters and antagonists on in vitro directional migration of 10T1/2 mouse embryo cells

In vitro directional migration of 10 T1/2 fibroblasts is partially inhibited by TPA but not by its non promoting analogues. Other tumor promoters, e.g., phenobarbital, saccharin, and benzoylperoxide had no measurable effect when added in concentrations known to affect in vitro two-step transformation or intercellular communication. Inhibitors of in vitro transformation do not affect migration, except for dexamethasone, which inhibited it. Hence, there is no evidence for a general correlation between tumor promoting potential and inhibition of in vitro directional migration.Abbreviations DDT 1,1,1-trichloro-2,2-bis(p-cholorophenyl) ethane - DEXA dexamethasone - DMSO dimethylsulfoxide - RA retinylacetate - SD standard deviation - SOD superoxide dismutase - TPA 12-0-tetradecanoylphorbol-13-acetate; 4-0-Me-TPA, 4-0-methyl-TPA     

Cell Cycle activation and ouabain-sensitive ion movements of 3T3 and C3H-10T½ fibroblasts

The introduction of either PGF_2α (10^?7 M) or TPA (10^?7 M) stimulated, ouabain-sensitive ⁸⁶Rb⁺ influx at 30 min in postconfluent 3T3-4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF_2α at these concentrations stimulated the incorporation of ³H-TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na⁺/K⁺ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3-4 and C3H-1 0T½ cells there were parallel increases in ³H-TdR incorporation and ouabain-sensitive ⁸⁶Rb⁺ influxes with 10^?7 M TPA, whereas PGF_2α stimulated a significant increase in ³H-TdR incorporation in 3T3-4 but not C3H-10T½ cells and only marginal increases in ouabain-sensitive ⁸⁶Rb⁺ influx in both. Therefore, although there appears to be a close correlation between Na⁺/K⁺ pump activation and subsequent S-phase entry following TPA stimulation, a similar correlation for PGF_2α cannot be confirmed.