Philip Feigelson

Practical aspects of internal-sample liquid scintillation counting☆

Abstract The appearance of commercially available equipment has made it practicable for chemists and biochemists to consider the use of liquid-scintillation counting for the assay of radioactive samples. The method offers excellent sensitivity for weak beta-emitting isotopes, including tritium. This is because of its 4π geometry for samples as large as several grams. The disadvantages of the technique are discussed in detail. The equipment is several times as expensive as most other counting apparatus. It is more complex to understand and to maintain. The method involves certain prerequisites of the sample material. These include adequate solubility in the rather limited group of alkyl benzene and polyther solvents that suffice as liquid-scintillation solvents, absence of color, and freedom from quenching action upon the scintillation phenomenon. Toluene remains the solvent of choice, but its usefulness has been broadened through the use of 2-ethylhexanoic acid and a quarternary ammonium base which form toluene-soluble salts with many cations and anions. The applicability of liquid-scintillation counting to any counting problems should be evaluated specifically. In general, however, some of the applications in which it appears to be the method of choice include nearly all tritium counting, assaying C 14 and S 35 samples of low specific activity, any type of counting where speed and ease of sample preparation are necessary, and the counting of volatile compounds.

Analysis of the complexity and diversity of mRNA from chicken liver and oviduct

We have analyzed the sequence complexity and diversity of poly(A)-containing mRNA derived from two highly differentiated chicken tissues. Two independent approaches were used in our analyses. The first involves the annealing of cDNA copies of mRNA to a vast excess of the template RNA; the second procedure uses hybridization between highly radioactive single-copy genomic DNA and mRNA. The results obtained using these two experimental approaches are in good accord and reveal the presence of 12,000-15,000 diverse mRNA species in both chicken liver and oviduct. In both cell types, the kinetics of annealing of cDNA to its template mRNA demonstrate discrete frequency classes with most of the different mRNA species present in fewer than 10 copies per cell. 70% of oviduct mRNA, however, consists of about 10 abundant RNA species, which probably are responsible for the synthesis of the egg white proteins. The diversity of mRNA species in chicken liver and oviduct was further studied by heterologous annealing reactions between cDNA or singlecopy genomic DNA and a vast excess of mRNA. These studies demonstrate that 85% of the different mRNA sequences detected are present in both liver and oviduct, and suggest that the vast majority of the information expressed as mRNA is required for the maintenance of cellular functions common to all tissues.

Isolation of eukaryotic messenger RNA on cellulose and its translation in vitro

Abstract Chromatography on plain cellulose of RNA from rabbit reticulocyte polysomes or chicken oviduct results in the retention and enrichment of a RNA fraction with messenger properties. Addition of appropriate amounts of this RNA to a cell-free system from Krebs-II-ascites cells, supplemented with rabbit reticulocyte initiation factors, leads to 20- to 30-fold stimulation of protein synthesis. The product of the reaction directed by RNA purified from rabbit reticulocytes was shown to consist of both α- and β-globin chains, as judged by SDS-gel electrophoresis and CM-cellulose chromatography. RNA isolated from chicken oviduct directs the synthesis of ovalbumin which has been identified by SDS-electrophoresis of the released chains after immunoprecipitation with antiovalbumin.

Correlation between glucocorticoid binding to specific liver cytosol receptors and enzyme induction in vivo

Abstract The degree of saturation in , vivo of a soluble hepatic glucocorticoid binding protein as a function of time and dosage of cortisol administration to adrenalectomized rats was found to correlate closely with the extent of hormonal induction of the hepatic enzymes tryptophan dioxygenase and tyrosine amino-transferase. These findings in conjunction with the high affinity and specificity for glucocorticoids manifested by this binding protein indicate that it may be the functionally significant glucocorticoid “receptor” of rat liver cytosol.

The differential effects of glucocorticoid on tissue and plasma amino acid levels.

Abstract The alteration in free amino acid patterns in various responsive tissues and in plasma has been investigated 4 h after the administration of a relatively low dose of cortisone acetate. In liver, in which cortisone evokes elevated tyrosine transaminase ( L -tyrosine: 2-oxoglutarate aminotransferase, EC 2.6.1.5) levels, a marked depression in the level of tyrosine and an elevation in concentrations of glutamate, aspartate, and alanine are evident. In the liver, in which protein synthesis is augmented under corticoid influences, the concentrations of numerous other amino acids were depressed following cortisone administration. In tissues in which cortisone produces a decrease in protein content, namely, thymus and muscle, the steroid elicited smaller elevations in the concentrations of many amino acids in addition to marked increments in the amounts of glutamate, aspartate, and alanine. Thus the alterations in amino acid patterns in tissues responsive to glucocorticoid seem to be consequent to the hormonally induced elevation of tyrosine α-ketoglutarate transaminase in the liver and to protein loss from lymphoid tissues and muscle.

Tissue-specific control of α2u globulin gene expression: Constitutive synthesis in the submaxillary gland

Synthesis of alpha 2u globulin, previously thought to occur only in the male rat liver, has now been demonstrated in the submaxillary salivary gland. Unlike liver, submaxillary synthesis of alpha 2u globulin mRNA is constitutive--that is, independent of the endocrine state, age and sex. Liver and submaxillary alpha 2u globulin mRNAs are of similar size, and their 5 ends map to the same region of the gene. Isoelectric focusing of in vitro translation products revealed that submaxillary mRNA encodes a more acidic subset of alpha 2u globulins than does liver. Salivary alpha 2u globulin mRNA manifests 5% nucleotide divergence, encoding 20 amino acid substitutions, which specifies a more acidic polypeptide than its hepatic counterpart. Thus the liver and submaxillary gland synthesize alpha 2u globulin from different sets of genes that are subject to very different developmental and hormonal control.

The half-lifetime of induced tryptophan peroxidase in vivo☆

Abstract Rat liver tryptophan peroxidase induction permits investigation of both protein synthetic and catabolic rates in vivo. Substrate or hormonally induced enzyme decreases in concentration according to first-order kinetics with a t 1 2 of 2.3 hr. This enzyme protein turnover is much more rapid than the reported mean turnover of total liver proteins. The kinetics of enzyme synthesis and catabolism were not influenced by a large extrahepatic tumor nor by 8-azaguanine or 6-mercaptopurine.

Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody

A procedure is presented for the purification of specific mRNAs, which exploits the ability of antibodies prepared against a native protein to bind to the nascent polypeptide on the polysome. Rather than precipitating these soluble antibody-polysome complexes with anti-antibody, which can lead to nonspecific trapping of polysomes, we have linked the anti-antibody to an insoluble matrix. Thus, the antibody-polysome complex binds to the anti-antibody support and nonspecific polysomes can easily be removed by several washes. We have found para-aminobenzyl cellulose (PAB cellulose), to be a suitable matrix for this purpose. This support can bind large quantities of anti-antibody and it displayed no detectable nonspecific affinity for polysomes or RNA. Using this procedure, we have obtained an apparently homogeneous preparation of ovalbumin mRNA.

The sequential stimulation of uracil-rich and guanine-rich RNA species during cortisone induction of hepatic enzymes

Abstract A mixture of 5- 3 H-uridine and 8- 14 C-guanine was employed to evaluate hepatic RNA synthesis at various times following a single administration of cortisone. During the first hour of hormone action, which precedes hepatic enzyme induction, uridine-rich RNA was selectively synthesized; subsequently a hormonal stimulation of guanine-rich RNA synthesis occurred. These findings are compatible with early hormonal stimulation of the synthesis of DNA-like RNA (high A+U) followed by synthesis of ribosomal RNA (high G+C). These double-labeled tracer experiments provide some insight into the biochemical processes underlying hormonal enhancement of hepatic enzyme synthesis and the subsequent augumentation of hepatic ribosomal content.

Metabolic effects of glucocorticoids as related to enzyme induction

Abstract The concept that glucocorticoid induction of the hepatic amino acid metabolizing enzymes, tyrosine-α-ketoglutarate transaminase and tryptophan pyrrolase, assumes an important role in the processes by which this hormone manifests many of its regulatory functions has been investigated from several points of view. In a search for the mechanisms by which glucocorticosteroids effect induction of these enzymes, inquiry into the effects of cortisone on the biosynthetic pathway leading to RNA was undertaken. It was found that incorporation in vivo of isotopically labeled precursors into liver RNA was rapidly and markedly enhanced after administration of a single low dose of cortisone, with a time course parallel to that of glucocorticoid induction of increased enzyme protein and activity. However, the following evidence has been accumulated which suggests that little, if any, of the observed stimulation in isotopic incorporation into RNA after cortisone administration is attributable to enhanced elaboration of RNA molecules from their nucleotide precursors: (1) the magnitude of cortisone induced stimulation of incorporation of various labeled precursors into liver RNA varies markedly, glycine- 14 C > inorganic 32 P > rotate- 3 H; (2) cortisone administration rapidly elevates incorporation in vivo of glycine- 14 C into liver acid soluble adenine nucleotides to a greater extent and with similar kinetics as incorporation of this precursor into RNA; (3) the stimulation of isotope incorporation into RNA of all liver subcellular compartments are of similar orders of magnitude and time course; (4) accumulation of increased quantities of hepatic RNA is observed only many hours after large doses of cortisone, at a time when enzyme induction has already subsided; (5) in vitro studies provide no evidence of glucocorticosteroid intervention in the synthesis of RNA on the DNA template, since neither binding of glucocorticoids with purified rat liver DNA nor alteration of the secondary structure of native DNA by these steroids could be detected. The only isotopic evidence suggesting possible enhancement of RNA elaboration following glucocorticoid administration is the observation of a slight stimulation in incorporation of orotate- 3 H into hepatic RNA which exceeds a yet smaller stimulation in incorporation of this isotope into a pyrimidine nucleotide precursor pool. That stimulation of hepatic purine biosynthesis does occur as a marked and early response to glucocorticoids has been established. Investigation of the mechanism of hormonal regulation of purine metabolism has revealed that intraperitoneal administration to adrenalectomized rats of l -glutamate, l -alanine, l -leucine, their d -counterparts, inorganic ammonium salts and l -glutamine, all mimic cortisone in the stimulation of glycine- 14 C incorporation into acid-soluble adenine. It has been accordingly proposed that glucocorticoid induction of transaminase activity may result in sequential expansion in the routes of amino nitrogen transport, leading to the observed enhancement in purine nucleotide biosynthesis—“purinoneogenesis”—as well as gluconeogenesis and increased urea output, all of which are characteristic of the cortisonized animal. Changes in liver amino acid patterns following cortisone administration are consistent with this working hypothesis: liver tyrosine levels are depressed; concentrations of glutamate and aspartate, amino acids pivotal in transamination, are elevated. Extension of these studies to extra hepatic target tissues, has revealed that simultaneous with stimulated glycine-2- 14 C incorporation into protein and acid-soluble purine in the liver, cortisone evokes a marked depression in these metabolic pathways in the lymphoid tissues, thymus and spleen.