DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography

Sodium fluoride (NaF) was assayed for the induction of DNA-repair synthesis in WI-38 human diploid fibroblasts and in primary cultures of rat hepatocytes. DNA-repair synthesis in non-replicating DNA was measured by ultracentrifugation of density-labeled DNA in CsCl gradients. When this method was used, NaF did not induce DNA-repair synthesis in either of these cell types. However, when NaF was assayed for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes by autoradiography, an increased net nuclear grain count was observed. Because the autoradiographic results were not confirmed by density-gradient ultracentrifugation of hepatocyte DNA, which is a more definitive technique, it is doubtful whether the autoradiographic results actually represent DNA-repair synthesis. Modifications of the UDS/autoradiography protocol to include more extensive washing resulted in no UDS response. Published reports (Hellung-Larsen and Klenow, 1969; Srivastava et al., 1981) describe the formation of precipitable complexes of Mg2+, F-, and [3H]thymidine triphosphate which suggests that autoradiographic measurement of UDS may lead to artifacts when testing NaF unless extensive washing of the cultures is employed.     

Advantages and limitations of density gradient ultracentrifugation in the fractionation of human serum lipoproteins: role of salts and sucrose

Two density gradient ultracentrifugation methods, Redgrave et al. (1975. Anal. Biochem. 65: 42-49) and Nilsson et al. (1981. Anal. Biochem. 110: 342-348), currently used for the separation and analysis of plasma lipoproteins were compared with respect to their resolving power and capacity to obtain pure products as a function of time of ultracentrifugation using the same rotor (Beckman SW-40), speed (150,000 g), and temperature (14 degrees C). The effects of sucrose and salts were also investigated. The Redgrave gradient insured the separation of the major classes of plasma lipoproteins after 24 hr of centrifugation; however, equilibrium conditions were only reached after 48 hr, at which time the lipoproteins were contaminated by albumin. When the effluents from each rotor tube were continuously monitored at 280 nm, each lipoprotein band gave values that were higher than those from mass analyses. This was due to a light scattering effect, the extent of which was dependent on the concentration of lipoproteins and salts. Sucrose prevented the scattering effect and was found to bind irreversibly to the apolipoproteins. In contrast, after 66 hr centrifugation, the lipoproteins obtained from the Nilsson gradient exhibited a close correspondence between protein mass and absorbance values at 280 nm, had no scattering effect, and were uncontaminated by albumin. The difference in spectroscopic behavior between the Redgrave and the Nilsson procedures was attributed to three factors: 1) the presence of sucrose in the latter gradient and incorporation of this sugar into lipoproteins as assessed by mass and radioactivity measurements; 2), the salt density to which the serum samples were exposed to at the beginning of the ultracentrifugation; and 3) the final lipoprotein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of very high density lipoproteins by differential density gradient ultracentrifugation

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.     

Spectrophotometric,potentiometric, and density gradient ultracentrifugation studies of the binding of silver ion by DNA

The equilibrium and the stoichiometry for the reversible complexing of silver ion by DNA have been studied by potentiometric titrations, proton release pH-stat titrations, and by spectrophotometry. The complexing reactions involve primarily the purine and pyrimidine residues, not the phosphate groups. There are at least three types of binding (types I, II, and III), of which the first two have been intensively studied in this work. The sum of type I and type II binding saturates at one silver atom per two nucleotide residues. In the type I and type II reactions, zero and one proton, respectively, are displaced per silver ion bound. At pH 5.6, the reactions occur stepwise, type I being first, while at pH 8.0, they occur simultaneously. The silver ion binding curve is very sharp at pH 8, indicating a cooperative reaction. The strength of the binding increases with increasing GC content. Type I binding is more important for GC-rich DNA's than for GC-poor ones. Denatured DNA binds more strongly than does native DNA. The silver ion complexing reaction is chemically and biologically reversible. We propose that type II binding essentially involves the conversion of an \documentclass{article}\pagestyle{empty}\begin{document}$ {\rm N} - {\rm H} \cdots {\rm N} $\end{document} hydrogen bond of a complementary base pair to an N—Ag—N bond. The nature of type I binding is less clear, but it may involve a π interaction with stacked bases. The buoyant density (ρ₀) of DNA in a Cs₂SO₄ density gradient increases when the DNA reacts with silver ion. The buoyant density change is about 0.15 g./ml. for 0.5 silver bound per nucleotide. The large buoyant density changes and the selective nature of the complexing reaction make it possible to perform good separations between native and denatured DNA or between GC-rich and GC-poor native DNA's by density gradient centrifugation.     

Isolation of crustacean egg yolk lipoproteins by differential density gradient ultracentrifugation

• 1.1. Egg yolk lipoproteins from four species of Crustacea were isolated by differential density gradient ultracentrifugation.
• 2.2. Egg yolk proteins from freshwater prawn, striped stone crab and mitten crab consissted of high-density lipoprotein (HDL) and lipid-free protein, while low-density lipoprotein (LDL) was present in the egg yolk protein of sand crayfish as well as HDL and lipid-free protein.
• 3.3. HDL was a major component in the egg yolk proteins from four species of Crustacea. HDL was identical to egg yolk lipovitellin.
• 4.4. Both HDL and LDL possessed phospholipid as a major lipid.
• 5.5. HDL, but not LDL, contained carotenoids. The color of HDL from mitten crab showed a reddish purple and was distinct from other Crustacea whose color was orange. The reddish purple color was characterized by an absorption flexion at 600–650 nm.

     

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.     

Comparison of gradient gel electrophoresis and zonal ultracentrifugation for quantitation of high density lipoproteins

The study was conducted to compare gradient gel electrophoresis (GGE) and zonal ultracentrifugation for quantitation of human plasma high density lipoproteins (HDL). Plasma samples were obtained from seven normal subjects consuming a high fat diet (65% total calories) followed by a high carbohydrate diet (65% total calories). HDL were fractionated into HDL2 and HDL3 by zonal ultracentrifugation and lipid and protein mass were determined. HDL were also fractionated by GGE and the results were compared to the zonal method. Zonally isolated HDL2 represented a homogeneous particle population that was equivalent to HDL2b as determined by GGE. By the zonal method, HDL2 accounted for 27 +/- 4% (mean +/- SEM) of total HDL mass in subjects on the high fat diet as compared to 16 +/- 2% in subjects fed the high carbohydrate diet; by GGE, the HDL2b values were 27 +/- 4% and 14 +/- 1%, respectively. The coefficient of correlation (n = 25) for the two methods was 0.894 (P less than 0.001).     

Isolation of DNA from fungal mycelia and sclerotia without use of density gradient ultracentrifugation

A rapid method for extracting total DNA from Aspergillus flavus and Aspergillus parasiticus has been developed. The procedure can be completed in 2 h and yields 200 to 350 micrograms of DNA from 0.5 to 1.0 g wet wt of mycelia and 150 micrograms from 0.5 g of sclerotia. DNA samples had an OD260/OD280 of 1.6 to 1.8. Most of the DNA was at least 50 kb pairs in size and showed little degradation. DNA prepared by this method was used for restriction endonuclease digestion and Southern blotting. A DNA fragment containing the repeat unit of the ribosomal RNA genes of A. flavus has been identified.     

Retinol induces platelet aggregation via activation of phospholipase A2

All-trans-retinol induced aggregation of rabbit platelets, and this effect could be inhibited by a cyclooxygenase inhibitor and a thromboxane A2 (TXA2) receptor antagonist, indicating an essential role for endogenously produced TXA2. We found a two-phase arachidonic acid release in retinol-stimulated platelets. The first phase was induced by the action of retinol alone and not inhibited by TXA2 receptor antagonist. The second phase was induced via synergistic action of retinol and initially generated small amount of TXA2, and was inhibited by the antagonist. Moreover, we discussed that the arachidonic acid release may be mediated by the action of phospholipase A2.     

Single spin density gradient ultracentrifugation method for the detection and isolation of light and heavy low density lipoprotein subfractions

A single spin density gradient ultracentrifugation method in a swinging bucket rotor has been applied for the detection and isolation of low density lipoprotein (LDL) subfractions. The visualization of the LDL heterogeneity was facilitated by prestaining the serum with Coomassie Brilliant Blue R prior to density gradient ultracentrifugation for 19.5 hr. A total of 13 human serum pools was analyzed. In each pool, two LDL subfractions, a lighter LDL1 subfraction, occasionally showing a subdivision into two bands, LDL1A and LDL1B, and a heavier LDL2 could be clearly distinguished by the banding pattern in the density gradient. Physicochemical characteristics of the isolated LDL subfractions were determined. The simple method for detection and isolation of these subfractions presented here may facilitate future studies on LDL heterogeneity.     

Conversion of plasma VLDL and IDL precursors into various LDL subpopulations using density gradient ultracentrifugation

The contribution of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) to various low density lipoprotein (LDL) subfractions was examined in three normal subjects and two with familial combined hyperlipidemia. Autologous VLDL + IDL (d less than 1.019 g/ml) or VLDL only (d less than 1.006 g/ml; one subject only) were isolated by sequential ultracentrifugation, iodinated, and injected into each subject. The appearance, distribution, and subsequent disappearance of radioactivity into LDL density subpopulations was characterized using density gradient ultracentrifugation. These techniques help determine the contribution of precursors to various LDL subpopulations defined uniquely for each subject. The results from these studies have suggested: 1) it took up to several days of intravascular processing of precursor-derived LDL before it resembled the distribution of the 'steady-state' plasma LDL protein; 2) plasma VLDL and IDL precursors contributed rapidly to a broad density range of LDL; 3) the radiolabeled plasma precursors did not always contribute to all LDL density subfractions within an individual in proportion to their relative LDL protein mass as determined by density gradient ultracentrifugation; 4) with time, the distribution of the precursor-derived LDL became more buoyant or more dense than distribution of the LDL protein mass; and 5) the kinetic characteristics of precursor-derived particles within LDL changed within a relatively narrow density range and were not always related to the LDL density heterogeneity of each subject. These studies emphasize the complexities of apoB metabolism and the need to design studies to carefully examine the production of various LDL subpopulations, the kinetic fate and interconversions among the subpopulations, and ultimately, their relationship to the development of atherosclerosis.     

Isolation of serum chylomicrons prior to density gradient ultracentrifugation of other serum lipoprotein classes

A method for the removal of serum chylomicrons before density gradient ultracentrifugation of the other serum lipoproteins using an SW 41 swinging bucket rotor is presented. In a preliminary spin, the chylomicrons with an Sf greater than 400 X 10(-13) s float to the top of the gradient, whereas the other lipoproteins are retained in the infranatant fraction. After removal of the chylomicrons, the other serum lipoproteins are subsequently fractionated by isopycnic density gradient ultracentrifugation. Analysis of the separated lipoprotein fractions suggested that this procedure permits isolation of a chylomicron fraction consisting solely of chylomicrons but that the very low density lipoprotein fraction subsequently isolated also contains chylomicrons or chylomicron remnants with an Sf less than 400 X 10(-13) s, and that there is considerable overlap in flotation rate and particle size of very low density lipoproteins and chylomicrons.