Effects of opioids and sedatives on survival in an Australian inpatient palliative care population.

AIMS: To assess whether opioid and sedative medication use affects survival (from hospice admission to death) of patients in an Australian inpatient palliative care unit. BACKGROUND: Retrospective audit. Newcastle Mercy Hospice--a tertiary referral palliative care unit. All patients who died in the hospice between 1 February and 31 December 2000. METHODS: Length of survival from hospice admission to death, and the median and mean doses of opioids and sedatives used in the last 24 h of life. Comparison of these with published studies outside of Australia. RESULTS: In this study, the use of opioids, benzodiazepines and haloperidol did not have an association with shortened survival and the only statistical significant finding was an increased survival in patients who were on 300 mg/day or more of oral morphine equivalent (OME). The proportion of patients requiring greater than or equal to 300 mg OME/day (at 28%) was higher than published studies, but the mean dose of 371 mg OME/day was within the range of other studies. The proportion of patients receiving sedatives (94%) was higher than other studies, but the median dose of parenteral midazolam equivalent of 12.5 mg per 24 h was lower than other studies from outside Australia. CONCLUSIONS: There was no association between the doses of opioids and sedatives on the last day of life and survival (from hospice admission to death) in this population of palliative care patients.

A CBF5 mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing.

In the budding yeast, Saccharomyces cerevisiae, actively transcribed tRNA genes can negatively regulate adjacent RNA polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated silencing is independent of the orientation of the tRNA gene and does not require direct, steric interference with the binding of either upstream pol II factors or the pol II holoenzyme. A mutant was isolated in which this form of silencing is suppressed. The responsible point mutation affects expression of the Cbf5 protein, a small nucleolar ribonucleoprotein protein required for correct processing of rRNA. Because some early steps in the S. cerevisiae pre-tRNA biosynthetic pathway are nucleolar, we examined whether the CBF5 mutation might affect this localization. Nucleoli were slightly fragmented, and the pre-tRNAs went from their normal, mostly nucleolar location to being dispersed in the nucleoplasm. A possible mechanism for tRNA gene-mediated silencing is suggested in which subnuclear localization of tRNA genes antagonizes transcription of nearby genes by pol II.

Potent inhibition of human immunodeficiency virus type 1 (HIV-1) gene expression and virus production by an HIV-2 tat activation-response RNA decoy.

Tat activation-response region (TAR) decoys have been developed for use in gene therapy for people infected with human immunodeficiency virus type 1 (HIV-1). When a TAR RNA decoy is overexpressed, it will bind Tat, thus leaving less of this crucial protein to bind to and activate the natural transcriptional promoter of HIV-1. Previous TAR decoy constructs have used HIV-1 TAR. However, recent epidemiological and biological data began to suggest that the TAR region from the human immunodeficiency virus type 2 (HIV-2) may suppress HIV-1 transcription and hence replication. We created a vector which overexpresses TAR-2 under the control of the human U6 small nuclear RNA gene promoter and here show that the U6-TAR-2 decoy construct potently inhibits both HIV-2 and HIV-1 gene expression. Further, this decoy construct is able to markedly suppress HIV-1 replication. Thus, we have directly proven that TAR-2 can suppress HIV-1 replication and suggest that the HIV-2 TAR decoy may prove useful for combating HIV-1 infection.

Expression of small, therapeutic RNAs in human cell nuclei.

Effective intracellular expression of small RNA therapeutics depends on a number of factors. The RNA, whether antisense, ribozyme, or RNA aptamer, must be efficiently transcribed, stabilized against rapid degradation, folded correctly, and directed to the part of the cell where it can be most effective. To overcome a number of these problems we have been testing expression cassettes based on the human tRNA(met) and U6 snRNA promoters, in which transcripts encoding small RNA inserts are protected against attack from the 3' and Transient expression in cultured cells results in 10(9)-2 x 10(7) full-length transcripts per cell, depending partially on the promoter construct used but also on the nature of the insert RNA 5' gamma-Phosphate methylation (capping) depended, as expected, on the inclusion of specific U6 snRNA sequences from positions +19 to +27. In situ localization of the transcripts shows that both tRNA and U6 promoter transcripts give primarily punctate nuclear patterns, and that capping of transcripts is not required for nuclear retention. Several different insert RNAs directed against HIV-1 were tested by cotransfection with HIV-1 provirus and assay for subsequent viral reverse transcriptase production. These include antisense RNA, hairpin and hammerhead ribozymes, and RNA ligands (aptamers) for Tat and Rev RNA binding proteins. Results show that Rev-binding RNAs efficiently block HIV-1 gene expression, whereas other RNAs have little or no effected when expressed in these cassettes.

Yeast expression vectors using RNA polymerase III promoters.

A series of Saccharomyces cerevisiae--Escherichia coli shuttle vectors is described in which small RNAs can be stably expressed in yeast from two different promoters for RNA polymerase III transcription. The vectors are available in either high- or low-copy-number forms with either URA3, HIS3, or TRP1 selection markers, and are based on a previously described set of plasmid vectors [Sikorski and Hieter, Genetics 122 (1989) 19-27]. Transcripts have structured pre-tRNA or RPR1 leaders fused to RNA corresponding to inserted sequences. Levels of RNA accumulation are dependent on plasmid copy number and the type of transcript.

Gal4 protein binding is required but not sufficient for derepression and induction of GAL2 expression.

The Saccharomyces cerevisiae GAL2 gene upstream activator sequence (UAS) region was examined for protein bound in vivo by chromatin footprinting at high resolution. Gal4 transcriptional activator protein binds to the two consensus UAS sites whether GAL2 expression is induced, uninduced, or repressed by growth with different carbon sources. Although wild type strains show loss of the Gal4 protein-specific footprint in repressing media containing glucose, constitutive high level expression of Gal4 protein restores the GAL2 UAS footprints without fully derepressing GAL2 transcription. Thus binding of the Gal4 activator to target sites in the DNA is required but not sufficient for GAL2 derepression and induction. Gal4-independent protein-DNA complexes were also detected in the region, including one over the previously noted centromere-binding protein (CP1) site upstream of the Gal4 complexes.