G1- and S-phase syntheses of histones H1 and H1o in mitotically selected CHO cells: utilization of high-performance liquid chromatography

We have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [3H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O4; the total HC1O4-soluble, Cl3CCO2H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns [Gurley, L. R., D'Anna, J. A., Blumenfeld, M., Valdez, J. G., Sebring, R. J., Donahue, D. K., Prentice, D. A., & Spall, W. D. (1984) J. Chromatogr. 297, 147-165]. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases (3.6 +/- 0.5)-fold as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases (3.1 +/- 0.2)-fold so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure.     

Suitability of primary monolayer cultures of adult rat hepatocytes for studies of cholesterol and bile acid metabolism

Monolayer cultures of hepatocytes isolated from cholestyramine-fed rats and incubated in serum-free medium converted exogenous [4-14C]cholesterol into bile acids at a 3-fold greater rate than did cultures of hepatocytes prepared from untreated rats. Cholic acid and beta-muricholic acid identified and quantitated by gas-liquid chromatography and thin-layer chromatography were synthesized by cultured cells for at least 96 h following plating. The calculated synthesis rate of total bile acids by hepatocytes prepared from cholestyramine-fed animals was approximately 0.058 micrograms/mg protein/h. beta-Muricholic acid was synthesized at approximately a 3-fold greater rate than cholic acid in these cultures. Cultured hepatocytes rapidly converted the following intermediates of the bile acid pathway; 7 alpha-hydroxy[7 beta-3H]cholesterol, 7 alpha-hydroxy-4-[6 beta-3H] cholesten-3-one, and 5 beta-[7 beta-3H]cholestane-3 alpha, 7 alpha, 12 alpha-triol into bile acids. [24-14C]Chenodeoxycholic acid and [3H]ursodeoxycholic acid were rapidly biotransformed to beta-muricholic acid. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity measured in microsomes of cultured hepatocytes decreased during the initial 48 h following plating, but remained relatively constant for the next 72 h. In contrast, cholesterol 7 alpha-hydroxylase activity appeared to decrease during the first 48 h, followed by an increase over the next 48 h. Despite the apparent changes in enzyme activity in vitro, the rate of bile acid synthesis by whole cells during this time period remained constant. It is concluded that primary monolayer cultures of rat hepatocytes can serve as a useful model for studying the interrelationship between cholesterol and bile acid metabolism.     

Phenytoin free fraction determination: comparison of an improved direct serum injection high-performance liquid chromatographic method to ultrafiltration coupled with fluorescence polarization immunoassay

Recent developments in restricted-access media (RAM) liquid chromatography make the simultaneous determination of total and free phenytoin concentrations possible by direct injection of drug-containing serum samples. A comparison of phenytoin free fraction determination by ultrafiltration coupled with fluorescence polarization immunoassay (TDX) to an improved direct injection RAM-HPLC method is presented. Our improved method differs from those previously reported with regard to column type, mobile-phase composition, and column temperature. Replicate samples analyzed by each method yielded similar values for serum phenytoin free fraction.     

Histone acetylation and heterochromatin content of cultured Peromyscus cells

In order to explore the relationship between unacetylated arginine-rich histones and condensed chromatin structure, the extent of histone acetylation was examined in cultured cell lines derived from three species of deer mice. These species differ considerably in their genomic content of heterochromatin but contain essentially the same euchromatin content. Cells of Peromyscus eremicus, containing 34–36% more constitutive heterochromatin than Peromyscus boylii or Peromyscus crinitus cells were found to contain 28–35% more unacetylated histone H4, 22–29% more unacetylated histone H3, and 18–22% more unacetylated histone H2B. This relationship between unacetylated histones and heterochromatin content was further explored by inducing hyperacetylation of P. eremicus and P. boylii histones through treatment of cells with 15 mM sodium butyrate for 24 h. It was found that the percentages of unacetylated histones H3 and H4 remaining after butyrate treatment were proportional to the amount of constitutive heterochromatin in the genome. These data support the concept that a small core of histones in constitutive heterochromatin is inaccessible to acetylation. It was also found that the acetylated state of isolated histones was sensitive to the method of histone extraction. Thus concern must be given to preparative procedures when studying histone acetylation in order to minimize these acetate losses.     

An HPLC procedure for the analysis of proteins in lung lavage fluid

A high-performance liquid chromatography method has been developed for fractionating the protein components of the lung's extracellular lining fluid, as sampled by bronchoalveolar lavage. With this method, 10 ml (or less) of rat bronchoalveolar lavage fluid (BALF) in phosphate-buffered saline can be quantitatively analyzed rapidly and reproducibly. This volume (25% of the lavage fluid volume from one rat using a standardized lavage technique) is made 0.2% with respect to trifluoroacetic acid (TFA) and pumped through a microBondapak C18 Radial-PAK HPLC column equilibrated with H2O/0.2% TFA. Six fractions are then eluted with a series of acetonitrile gradients and isocratic steps that progress from H2O/0.2% TFA to 65% CH3 CN/0.2% TFA. Following this, 5 additional fractions are eluted with methanol. All 11 fractions are detected by monitoring the column effluents at 206 nm and can be recovered by lyophilization since all the components of the HPLC solvent system are volatile. Nine of the 11 fractions were found to contain protein. Three of the fractions contained proteins common to the blood compartment. The largest fraction of these was albumin, followed by a fraction containing immunoglobulins. Six other protein fractions appeared to be derived from the cells of the lung inasmuch as they were not detected in plasma. Two fractions contained no protein or phospholipids, whereas the most hydrophobic protein fraction did contain phospholipids. A major phospholipid fraction containing no protein eluted early in the chromatogram and was not detectable at 206 nm. This HPLC procedure offers significant utility for identifying and quantifying alterations in several BALF constituents during the development and progression of environmentally induced lung diseases as well as other pulmonary disorders.