Interaction of RNA with transformed glucocorticoid receptor. I. Isolation and purification of the RNA

The glucocorticoid receptor (GR) from mouse AtT-20 pituitary tumor cells, when transformed using a variety of in vitro protocols, yields a DNA-binding RNA-containing 6 S form. In order to better understand the physiological role of RNA interaction with the transformed GR, we have isolated and purified the putative RNA from AtT-20 cells. [3H]Triamcinolone acetonide-labeled cytosolic GR was transformed, using Sephadex G-25 filtration, to yield the RNA-containing 6 S GR. The transformed 6 S GR was separated on DEAE-cellulose into the 4 S GR (eluting at about 100 mM KCl) while its associated RNA eluted at 0.30-0.45 M KCl. The addition of only these RNA fractions to the 4 S GR can reconstitute 6 S GR as shown on 5-20% sucrose gradients. RNA (0.3-0.45 M KCl fractions) was further purified by hydroxylapatite chromatography, and the bound RNA (eluted at approximately 70 mM PO4(-2)) was then loaded onto preparative 5-20% sucrose gradients to separate RNA on the basis of size (sedimentation rate). A uniform class of RNA sedimenting at 4 S was obtained and then adsorbed to oligo(dT)-cellulose columns. The unbound fraction (poly(A-)) was capable of shifting 4 S GR to 6 S. Using these chromatographic procedures about 90% of the cellular RNA, incapable of reconstituting the 6 S GR from the 4 S form, was eliminated. The 4 S GR was covalently cross-linked with the purified RNA (termed PIVB RNA) using formaldehyde. The resulting cross-linked GR X RNA complexes were shown to sediment at the density of ribonucleoprotein (1.38 g/cm3) in CsCl gradients and at the 6 S position in high salt sucrose gradients. The hydrolysis of PIVB RNA with ribonuclease A prevented the formation of high salt-resistant ribonucleoprotein complexes, indicating that the GR may be in close contact with PIVB RNA. Electrophoresis of the PIVB RNA on 5% agarose-formaldehyde-denaturing gels yielded one major band with a molecular size of approximately 75 bases. It thus appears that an endogenous 4 S RNA (PIVB RNA) of about 25 kDa specifically interacts with the monomeric 4 S GR to yield the 6 S GR.     

Effects of cholinergic drugs on longitudinal muscle contractions of Fasciola hepatica

Acetylcholine, cholinergic agonists and acetylcholinesterase inhibitors significantly decrease the amplitude and frequency of spontaneous longitudinal muscle contractions in Fasciola hepatica. In order of their effects on the inhibition of muscle contractions, the cholinergic agonists can be ranked as nicotine greater than carbachol greater than acetylcholine. High calcium ion concentration also causes a significant inhibition of contractions. Atropine, a cholinergic antagonist that acts on muscarinic receptors, significantly increases the amplitude and frequency of spontaneous contractions and completely reverses the effects of cholinomimetic drugs, including nicotine. In adult F. hepatica, the levels of acetylcholine and its precursor choline are 3.14 +/- 0.55 and 13.75 +/- 3.72 pmol/mg wet weight, respectively. The activities of choline acetyltransferase, specific acetylcholinesterase and the nonspecific cholinesterase are 1.25 +/- 0.19, 238.0 +/- 13.0, and 83.0 +/- 33.0 nmol/hr/mg protein, respectively.     

Hormonal regulation of skeletal muscle glycogen synthase through covalent phosphorylation

Studies have been initiated to determine the hormonal regulation of glycogen synthase in rabbit skeletal muscle. It was found that glycogen synthase purified from control animals was quite highly phosphorylated (2.35 mol phosphate/mol synthase subunit) with 40% of the phosphate in the trypsin-sensitive or COOH-terminal domain, and 60% in the trypsin-insensitive or NH2-terminal domain. The phosphorylation state of synthase was elevated (3.9 mol/mol) by epinephrine injection and in the diabetic condition. With epinephrine, about 76% of the additional phosphate was incorporated in the trypsin-sensitive domain, which strongly supports the contention that this hormone acts through the cyclic AMP (cAMP)-dependent protein kinase. In the synthase purified from diabetic rabbits, 90% of the additional phosphate was in the trypsin-insensitive domain. Insulin treatment of the diabetics resulted in specific dephosphorylation of the trypsin-insensitive domain. These results indicate that in this system insulin is not acting by inhibition of the cAMP-dependent protein kinase.     

Localization of99mTc in bone by means of autoradiography

The uptake of two different preparations of^99mTechnetium-methylene diphosphonate in fetal rat calvaria is compared. The localization of^99mTc after administration of^99mTc(Sn)-MDP and^99mTc-MDP showed equal distribution in autoradiography.     

Neuromuscular, anaerobic, and aerobic performance characteristics of elite power athletes

Various aspects of neuromuscular, anaerobic, and aerobic performance capacity were investigated in four powerlifters, seven bodybuilders, and three wrestlers with a history of specific training for several years. The data (means +/- SD) showed that the three subject groups possessed similar values for maximal isometric force per unit bodyweight (50.7 +/- 9.6, 49.3 +/- 4.1, and 49.3 +/- 10.9 N/kg, respectively). However, significant (P less than 0.05) differences were observed in the times for isometric force production, so that e.g., times to produce a 30% force level were shorter for the wrestlers and bodybuilders (28.3 +/- 3.1 and 26.4 +/- 6.6 ms) than that (53.3 +/- 23.7 ms) for the powerlifters. Utilization of elastic energy by the wrestlers was significantly (P less than 0.05) better than that of the other two subject groups, as judged from differences between the counter-movement and squat jumps at 0, 40, and 100 kg's loads. No differences were observed between the groups in anaerobic power in a 1-min maximal test, but the values for VO2 max were higher (P less than 0.05) among the wrestlers and bodybuilders (57.8 +/- 6.6 and 50.8 +/- 6.8 ml X kg-1 X min-1) as compared to the powerlifters (41.9 +/- 7.2 ml X kg-1 X min-1). Within the limitations of the subject sample, no differences of a statistical significancy were observed between the groups in fibre distribution, fibre areas, or the area ratio of fast (FT) and slow (ST) twitch fibres in vastus lateralis.(ABSTRACT TRUNCATED AT 250 WORDS)