An integrative genomics screen uncovers ncRNA T-UCR functions in neuroblastoma tumours.

Different classes of non-coding RNAs, including microRNAs, have recently been implicated in the process of tumourigenesis. In this study, we examined the expression and putative functions of a novel class of non-coding RNAs known as transcribed ultraconserved regions (T-UCRs) in neuroblastoma. Genome-wide expression profiling revealed correlations between specific T-UCR expression levels and important clinicogenetic parameters such as MYCN amplification status. A functional genomics approach based on the integration of multi-level transcriptome data was adapted to gain insights into T-UCR functions. Assignments of T-UCRs to cellular processes such as TP53 response, differentiation and proliferation were verified using various cellular model systems. For the first time, our results define a T-UCR expression landscape in neuroblastoma and suggest widespread T-UCR involvement in diverse cellular processes that are deregulated in the process of tumourigenesis.

Neomycin is an agonist at a polyamine site on the N-methyl-D-aspartate receptor.

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.

Comparison of binding at strychnine-sensitive (inhibitory glycine receptor) and strychnine-insensitive (N-methyl-D-aspartate receptor) glycine binding sites.

We compared, for a number of ligands to the two receptors, the displacement of [3H]strychnine binding to the glycine-gated chloride channel of spinal cord and brainstem synaptic membranes to the displacement of [3H]glycine binding to the NMDA receptor complex of hippocampal and cortex synaptic membranes. Glycine and beta-alanine are recognized by both receptors. In the NMDA receptor glycine antagonists, the kynurenic acids, most of the quinoxalinediones, and the (R)-enantiomer of HA-966 had little affinity at the strychnine-sensitive site. Surprisingly, the quinoxalinedione widely used as an AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor antagonist, NBQX (2,3-dihydro-6-nitro-sulfamoylbenzo[f]quinoxaline-2,3-dione) displaced [3H]strychnine binding (IC50 = 11 microM) and to a lesser extent [3H]glycine binding (IC50 = 119 microM). Of the compounds tested, only strychnine, brucine, taurine and (S)-HA-966 were more potent displacers of [3H]strychnine than of glycine binding. Generally, the two glycine recognition sites appear to have remarkably different structural requirements.

An antagonist/partial agonist at the polyamine recognition site of the N-methyl-D-aspartate receptor that alters the properties of the glutamate recognition site.

The effects of N-(3-aminopropyl)-1,10-diaminodecane (APDA10) on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex were investigated. In the presence of 100 microM glutamate and 100 microM glycine, APDA10 had biphasic effects on the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten5,10-imin e (MK-801) to NMDA receptors on well washed synaptic plasma membranes. The maximal stimulation of binding by APDA10 was less than that seen with spermine. In the presence of glutamate and glycine, APDA10 attenuated the stimulatory effect of spermine and the inhibitory effect of 1,10-diaminodecane. In the nominal absence of glutamate and glycine, APDA10 had no effect on the binding of [3H]MK-801, but antagonized the stimulatory effect of spermine on the binding of [3H] MK-801. These data suggest that APDA10 acts as a mixed antagonist/partial agonist at the polyamine recognition site, and that the partial agonist properties of APDA10 are dependent on the activation state of the receptor complex. An increase in the potency of the glutamate site antagonists D-2-amino-5-phosphonovaleric acid and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid for inhibiting the binding of [3H]MK-801 was seen in the presence of APDA10. APDA10 also increased the affinity of binding of [3H]3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid to the NMDA receptor complex but had no effect on the binding of [3H]glycine. These data suggest that the polyamine APDA10 may alter the properties of the glutamate recognition site on the NMDA receptor complex.

Effects of multiple doses of moricizine hydrochloride on its pharmacokinetics and hepatic microsomal enzymes in rats.

We studied the influence of chronic moricizine hydrochloride (MRZ) treatment on the drug's pharmacokinetics and on drug metabolizing enzyme activities in rats. Separate groups of 8 rats (4 males and 4 females) were treated with 40 and 100 mg/kg oral MRZ once daily for 7 days and saline control for 7 days prior to the preparation of hepatic microsomal enzyme suspensions. Depending on the substrate, treatments with multiple oral MRZ increased or decreased hepatic microsomal enzyme activities. For the pharmacokinetic study, rats (4 males and 4 females) were treated with 40 mg/kg oral MRZ once daily for 7 days. A comparison of MRZ pharmacokinetics obtained on day 1 relative to day 7 revealed that both AUC0-t and AUC0-infinity increased about 7-fold in males and 2-fold in females. Cmax also increased about 5-fold from day 1 to day 7 in males. These increases in blood concentrations and AUC's are likely due to enzyme inhibition. Results obtained from female rats on days 1, 4 and 7 suggest that metabolic changes probably occur after the 4th day of dosing. Therefore, chronic MRZ treatment affected its pharmacokinetics and hepatic metabolizing enzyme activities in rats.

Stereoselectivity for the (R)-enantiomer of HA-966 (1-hydroxy-3-aminopyrrolidone-2) at the glycine site of the N-methyl-D-aspartate receptor complex.

HA-966 (1-hydroxy-3-aminopyrrolidone-2) is an antagonist at the glycine allosteric site of the N-methyl-D-aspartate receptor ionophore complex. Unlike presently known glycine antagonists, HA-966 is chiral. We report stereoselectivity for the (R)-enantiomer at the glycine antagonist site. In [3H]glycine binding, the (R)-enantiomer has an IC50 of 4.1 +/- 0.6 microM. The racemic mixture has an IC50 of 11.2 +/- 0.5 microM, whereas (S)-HA-966 has an IC50 greater than 900 microM. In glycine-stimulated [3H]1-[1-(2- thienyl)cyclohexyl]piperidine binding, the (R)-enantiomer inhibits with an IC50 of 121 +/- 61 microM, whereas the racemic mixture has an IC50 of 216 +/- 113 microM and (S)-HA-966 is inactive. The inhibition by (R)-HA-966 can be prevented by the addition of glycine. (R)-HA-966 and racemic HA-966, but not (S)-HA-966, also prevent N-methyl-D-aspartate cytotoxicity in cortical cultures. The (R)-enantiomer and, less potently, the (S)-enantiomer inhibit N-methyl-D-aspartate-evoked [3H]norepinephrine release from rat hippocampal slices (IC50 values of about 0.3 mM and 1.6 mM, respectively), but only the inhibition by (R)-HA-966 is reversed by added glycine. In glutamate-evoked contractions of the guinea pig ileum, (R)-HA-966 causes a glycine-reversible inhibition (IC50 of about 150 microM), whereas (S)-HA-966 is much less potent (IC50 of greater than 1 mM). These results demonstrate stereoselectivity of the glycine antagonist site of the N-methyl-D-aspartate receptor complex in a variety of tissues and assays. The stereoselectivity also confirms the specificity of N-methyl-D-aspartate receptors in glutamate-evoked contractions of the guinea pig ileum, and supports their similarity to central N-methyl-D-aspartate receptors.

Stereoselective enhancement by (R)-HA-966 of the binding of [3H]CPP to the NMDA receptor complex.

The enantiomers of the strychnine-insensitive glycine antagonist, HA-966 (1-hydroxy-3-amino-pyrrolidone-2), stereoselectively enhance binding of the N-methyl-D-aspartate (NMDA) competitive antagonist, [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) to rat brain synaptosomal membranes. The enhancement by the more potent (R)-HA-966 is competitively inhibited by the glycine antagonist 7-chlorokynurenic acid and noncompetitively by the polyamine spermine. Thus, (R)-HA-966, apparently at the glycine site, enhances the binding of antagonist to the NMDA receptor, possibly through a mechanism partially in common with that of spermine.

Induction of deer antlers by transplanted periosteum. I. Graft size and shape.

When discs of frontal periosteum from presumptive antler sites of 6-8 month old male fawns of the fallow deer are grafted beneath the foreleg skin, they will differentiate into pedicle bones and induce small antlers in the overlying integument. These antlers shed their velvet in the fall, and in succeeding years are replaced by larger outgrowths not exceeding 7 cm in length. Periosteal transplants 1.5 cm in diameter gave rise to ectopic antlers in 100% of the grafts, while discs measuring 1.05 cm, 0.75 cm and 0.4 cm did so in only 20% of the cases. Conversely, the donor sites produced antlers in 20-23% of the cases following removal of 1.05 cm or 1.5 cm of periosteum, while 80% and 100% grew antlers after deletions of 0.75 cm and 0.4 cm discs of periosteum, respectively. Semicircular grafts of periosteum induced antler development in most cases, especially when derived from the lateral halves of the antlerogenic region on the frontal bone. These findings confirm that the histogenesis of a deer's first pedicle and antler resides in the frontal periosteum over an area about 1.5 cm wide. They also show that leg skin is capable of antlerogenic development under the inductive influence of frontal periosteum, and that integumental wounding may enhance inductive interactions.