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We have recently cloned an amino acid transporter from the human placental choriocarcinoma cell line JAR which, when functionally expressed in HeLa cells, induces an amino acid transport activity with characteristics known to be associated with the amino acid transport system B(0) (R. Kekuda, P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, S. Sinha, T.L. Yang-Feng, F.H. Leibach, and V. Ganapathy, J. Biol. Chem. 271, 18657-18661, 1996). The presence of the amino acid transport system B(0) (ATB(0)) has however not been previously described in these cells by functional studies. In the present investigation, we have obtained evidence for the existence of ATB(0) in JAR cells and delineated the functional characteristics of the transporter. The identifying characteristics include Na(+)-dependence and preference for neutral amino acids. In addition, we have used the JAR cells as a model system to investigate the regulatory aspects of ATB(0). Treatment of the cells with the neuroprotective agent aurintricarboxylic acid (ATA) for 16 h leads to a significant increase in ATB(0) activity. This increase is associated with enhanced maximal velocity of the transporter and with increased steady state levels of the transporter mRNA. The effect of ATA is blocked by the tyrosine kinase inhibitor genistein. ATA treatment results in increased tyrosine phosphorylation of two major proteins, 180 kDa and 140 kDa in size. The 180 kDa protein is likely to be the epidermal growth factor (EGF) receptor because exposure of the cells to EGF also leads to enhanced tyrosine phosphorylation of a protein of similar molecular size. Furthermore, the effects of ATA on ATB(0) activity and on ATB(0) mRNA levels can be reproduced by EGF. Treatment of the cells with EGF for 24 h results in a significant increase in ATB(0) activity and this effect is associated with an increase in the maximal velocity of the transporter and with an increase in the steady state levels of the transporter mRNA. These data suggest that ATA influences ATB(0) activity in JAR cells most likely by activating the EGF receptor through tyrosine phosphorylation. It is concluded that the human placental choriocarcinoma cells functionally express the amino acid transport system B(0) and that the expression of the system in these cells is stimulated by EGF.

Messenger RNA was extracted from polysomes of rat ascites hepatoma, AH 7974, which contains both beta- and gamma-actins, and was translated in nuclease-treated reticulocyte lysate. The isoactins, beta and gamma, synthesized in vitro were characterized by (1) a high affinity to DNAase I-agarose; (2) polymerization with actin purified from bovine brain; (3) coelectrophoresis with bovine brain actin on two-dimensional gel, and (4) peptide mapping of each isoactin by partial digestion with papain (EC 3.4.22.2) followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum conditions with respect to the concentration of RNA, Mg2+, and K+ for the synthesis of beta- and gamma-isoactins were identical: 300 micrograms/ml, 1.5 mM, and 100 mM, respectively. Aurintricarboxylic acid and 7-methyl-GMP (7MeGMP) inhibited the synthesis of beta- and gamma-actins to the same extent. These results strongly suggest that there is very little possibility of differential translational control of each isoactin gene. When polysomal RNA was separated by sucrose gradient centrifugation, both beta- and gamma-actin mRNAs appeared as a sharp peak at the region slightly heavier than 18 S RNA.

When hen oviduct cytosol samples containing progesterone receptor complexed to [3H]progesterone were incubated with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose of ATP-Sepharose was abolished under similar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that aurintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest that use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.

The ribosomal fraction from HeLa cells treated at elevated temperature was centrifuged in Cs2SO4. 1. The bulk of the RNA labeled in the presence of a low dose of actinomycin D banded at p = 1.36 g/cm3. 2. This fraction contained heterogeneous RNA and proteins with molecular weights close to those of proteins found in polysomal messenger ribonucleoproteins. 3. This ribonucleoprotein bound ribosomes in conditions were deproteinized RNA did not. 4. The binding was inhibited by aurintricarboxylic acid, ethylene diamine tetraacetic acid and sodium deoxycholate, but not by Triton X-100.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.

The extent to which the polyribosome content in splenic extracts is affected by the level of endogenous ribonuclease activity was investigated. Ribonuclease activity, free and inhibited, was determined under ionic conditions optimal for preservation of polyribosomes. The results show that ribonuclease activity in control spleens varies mainly with the amount of ribonuclease inhibitor, indicating that free ribonuclease activity is stable. In rapidly proliferating splenic tissue the enzymatic activity is regulated by changes in both the amount of inhibitor and enzyme available. The ribonuclease versus ribonuclease-inhibitor balance was found to parallel the proportion of polyribosomes in a polysome profile.

Actinomycin D inhibits hemoglobin synthesis in rabbit reticulocyte lysates by affecting initiation of globin polypeptide chains. To obtain information on the distribution of actinomycin-sensitive structures among the components of the translation machinery, a reticulocyte lysate is incubated with [3H]actinomycin. The tritium label is found associated with ribosomes actively engaged in protein synthesis and with a 15 S cytoplasmic particle. During cell-free incubation or when initiation is inhibited by aurintricarboxylic acid, the labeling of the 15 S particles increases, suggesting that they are relased from ribosomes into the cytoplasm and play a role in the initiation process. The 15 S cytoplasmic particles contain a DNAase-sensitive component, probably complexed with ribonucleic acids and proteins. A possible role of these DNA-containing particles in the initiation step of translation is discussed.

Avian Myeloblastosis Viral (AMV) core component was isolated and shown to synthesize AMV proteins in vitro. This reaction was linearly dependent on viral core concentration, proceeded linearly with time, and was inhibited by puromycin and aurintricarboxylic acid. The proteins synthesized in vitro co-electrophoresed and co-chromatographed with known proteins, and were immunoprecipitated by total and monospecific antibodies to known AMV proteins.

Commercially available, as well as synthetically prepared, samples of aurintricarboxylic acid (a widely employed potent inhibitor of protein nucleic acid interactions) consist mostly of a heterogeneous collection of polymers, as shown by fractionation schemes utilizing both dialysis and ultrafiltration, and by molecular weight measurements. 13C-NMR studies suggest that the polymeric material is of the phenol-formaldehyde type; inhibitory assays that depend on the formation of a protein-nucleic acid complex revealed that potency varied directly with the molecular weight of the polymer. Fractions of molecular weight 400 were essentially inactive.

Crude aurintricarboxylic acid synthesized by the conventional method was fractionated into 8 components (A1-A8) by silica gel thin layer chromatography. Some of the components were isolated and their effects were tested on nuclear swelling and on in vitro RNA synthesis and DNA synthesis using isolated polymerases. Only two components with low RF (A7 and A8) induced strong nuclear swelling. The other components were almost inactive. Formaurin-dicarboxylic acid, a contaminating polyanion of crude aurintricarboxylic acid, was synthesized separately. It was as effective as the two active components in nuclear swelling and inhibited RNA and DNA polymerases on naked DNA template. However, it stimulated RNA synthesis on chromatin template probably by dissociating histones from DNA. Unfractionated aurintricarboxylic acid showed the same effects at higher concentrations. A preliminary analysis indicated one of the inactive components (A4) is genuine aurintricarboxylic acid. The results suggest that the observed activity of crude aurintricarboxylic acid is due to some contaminating substances, one of which is formaurindicarboxylic acid.

A specific ribosome-associated protein kinase (protein kinase II) and phosphoprotein(s) from the ribosomal KCl wash fraction termed "PPx" have been isolated from plasmacytoma, and tested for their ability to bind to poly(A) and to different plasmacytoma polynucleotides. The nitrocellulose filter binding technique was used to measure RNA-protein interaction. Protein kinase II and PPx preferentially bound mRNA compared to poly(A). They did not bind ribosomal RNA, soluble RNA or DNA. The optimal conditions (temperature, time, protein concentration, ionic strength) for mRNA-protein interaction were determined. Ribosomal protein kinase (protein kinase II) phosphorylated PPx proteins which bound to mRNA represented at least two bands as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (Mr = 90 000 and 80 000). The high affinity of protein kinase II and PPx for mRNA suggests that they may function in regulating protein synthesis.