Identification of an essential proximal sequence element in the promoter of the telomerase RNA gene of Tetrahymena thermophila.

Telomerase is a ribonucleoprotein reverse transcriptase that synthesizes and maintains telomeric DNA. Studies of telomeres and telomerase are facilitated by the large number of linear DNA molecules found in ciliated protozoa, such as Tetrahymena thermophila. To examine the expression of telomerase, we investigated the transcription of the RNA polymerase III-directed gene encoding the RNA subunit (TER1) of this enzyme. A chimeric gene containing the Glaucoma chattoni TER1 transcribed region flanked by 5' and 3' Tetrahymena regions was used to identify promoter elements following transformation of Tetrahymena cells. Disruption of a conserved proximal sequence element (PSE) located at -55 in the Tetrahymena TER1 5' flanking region eliminated expression of the chimeric gene. In addition, mutation of an A/T-rich element at -25 decreased expression markedly. A gel mobility shift assay and protein-DNA cross-linking identified a PSE-binding polypeptide of 50-60 kDa in Tetrahymena extracts. Gel filtration analysis revealed a native molecular mass of approximately 160 kDa for this binding activity. Our results point to a similar architecture between ciliate telomerase RNA and metazoan U6 small nuclear RNA promoters.

Molecular cloning of a cDNA encoding human SPH-binding factor, a conserved protein that binds to the enhancer-like region of the U6 small nuclear RNA gene promoter.

Many vertebrate small nuclear RNA gene promoters contain an SPH motif in their distal control regions that can confer transcriptional stimulation by RNA polymerase II or RNA polymerase III. Using the human U6 gene SPH motif as a probe, we isolated a cDNA encoding human SPH-binding factor (hSBF) from a HeLa cell expression library. The coding region of hSBF is almost identical to ZNF143, a 626 amino acid, seven zinc finger protein of previously unknown function. Furthermore, the predicted amino acid sequence of hSBF is highly homologous to Xenopus laevis and mouse Staf proteins, that bind to SPH motifs and stimulate transcription of selenocysteine tRNA gene promoters. Recombinant hSBF expressed in vitro or from Escherichia coli bound specifically to the human U6 gene SPH motif as shown by DNase I footprinting and electrophoretic mobility shift assays using various mutant SPH sites as competitors. Antibodies prepared against recombinant hSBF inhibited assembly of native SBF-DNA complexes. Immunodepleted HeLa S100 transcription extract no longer supported elevated levels of transcription by RNA polymerase III from a U6 promoter containing an SPH motif, whereas addition of recombinant hSBF protein to the immunodepleted extract reconstituted stimulated transcription.

Intermediate efficacy mu opioids: examination of their morphine-like stimulus effects and response rate-decreasing effects in morphine-tolerant rats.

The present study examined the effects of morphine, the intermediate efficacy mu opioids (-)-pentazocine, (-)-metazocine, proxorphan, levallorphan, (-)-NANMY (N-allylnormetazocine) and (-)-cyclazocine, and the mu antagonist naloxone 1) in rats responding under a FR (fixed-ratio) 30 schedule before, during and after a chronic morphine regimen, and 2) in rats trained to discriminate 10.0 [10-MS (morphine sulfate)] or 3.0 mg/kg (3-MS) of morphine from saline. Under the FR30 schedule, chronic administration of morphine produced tolerance to morphine's rate-decreasing effects and conferred cross-tolerance to (-)-metazocine, proxorphan and (-)-pentazocine. However, the effects of these intermediate efficacy mu opioids could be differentiated from those of morphine on the basis of their 1) shallow dose-effect curves, and 2) large differences in the degree to which tolerance developed in individual rats. In the drug discrimination procedure, (-)-metazocine, proxorphan and (-)-pentazocine produced high levels of substitution for the 3-MS stimulus and intermediate levels for the 10-MS stimulus. In contrast to the pattern of substitution observed with morphine in the 10-MS group, the effects of these drugs were characterized by 1) shallow dose-effect curves, 2) large individual differences in the lowest dose of each drug that substituted completely for the 10-MS stimulus and 3) the failure to obtain complete substitution for the 10-MS stimulus in all of the rats tested. The behavioral profile obtained with the intermediate efficacy mu opioids (-)-NANM, levallorphan and (-)-cyclazocine was indicative of opioids with intrinsic efficacy lower than that of (-)-pentazocine, (-)-metazocine and proxorphan. Under the FR30 schedule, chronic administration of morphine produced an enhanced sensitivity to the rate-decreasing effects of (-)-NANM and levallorphan but not (-)-cyclazocine. In the drug discrimination procedure, (-)-NANM, levallorphan and (-)-cyclazocine produced high levels of substitution for the 3-MS stimulus and low levels for the 10-MS stimulus. Like naloxone, these drugs produced a dose-related attenuation of the 10-MS stimulus. The results of the present study suggest that the relative order of intrinsic efficacy among the opioids tested is: morphine > (-)-metazocine = (-)-pentazocine = proxorphan > (-)-cyclazocine = levallorphan = (-)-NANM > naloxone.

Gastric emptying of enteric-coated tablets.

To evaluate the gastric emptying time of pharmaceutical dosage forms in a clinical setting, a relatively simple dual-radionuclide technique was developed. Placebo tablets of six different combinations of shape and size were labeled with indium-111 DTPA and enteric coated. Six volunteers participated in a single-blind and crossover study. Tablets were given in the morning of a fasting stomach with 6 oz of water containing 99mTc pertechnetate and continuously observed with a gamma camera. A scintigraph was obtained each minute. The results suggested that the size, shape, or volume of the tablet used in this study had no significant effect in the rate of gastric emptying. The tablets emptied erratically and unpredictably, depending upon their time of arrival in the stomach in relation to the occurrence of interdigestive myoelectric contractions. The method described is a relatively simple and accurate technique to allow one to follow the gastric emptying of tablets.