Characterization of DNA-directed RNA polymerases in isolated macronuclei of the ciliated protozoan Tetrahymena thermophila. Effects of purified ornithine decarboxylase and amine compounds.

The possible regulatory interactions of purified ornithine decarboxylase with DNA-directed RNA polymerases in isolated macronuclei from the ciliated protozoan Tetrahymena thermophila were studied. It has been found that highly purified ODC (specific activity 10.2 mumols CO2 x h-1 x mg-1), even at activities of 37,500 nmol CO2 x h-1 per ml failed to alter RNA polymerase activity in the in vitro transcription assay in the presence or absence of the substrate L-ornithine at 20mM. The naturally occurring di- and polyamines putrescine, spermidine, and spermine stimulated in-vitro-transcription in isolated macronuclei more at optimal Mg2+/Mn(2+)-concentrations than at suboptimal concentrations, suggesting that polyamines act via a mechanism which is distinct from that of the inorganic cations. Of the monovalent amine compounds tested, (NH4)+ at high concentrations between 40 and 50mM slightly stimulated activity whereas the onset of stimulation by the organic amine compounds, piperidine and cyclohexylamine, was inversely related to the hydrophobicity of each particular compound. In the series of divalent amines, the correct distance between the N-atoms appeared to be very important since ethylenediamine and piperazine did not stimulate significantly but did inhibit at concentrations above 5 mM. 1,3-Diaminopropane stimulated slightly but inhibited above 10 mM, whereas the 1,4-diamino compounds putrescine and 1,4-diaminocyclohexane (DAC) were equally potent stimulators with the more hydrophobic one, DAC, reaching the maximum at lower concentrations than putrescine. For the trivalent amines, the influence of correct spacing seems not to be as important: N-(2-aminoethyl)piperazine stimulated very similar to spermidine.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of cytosolic sexual steroid receptors in autochthonous methylnitrosourea-induced rat mammary carcinoma following application of 2-chloroethylnitrosocarbamoyl-L-alanine linked to oestradiol or dihydrotestosterone.

This study concentrated on the influence of 2-chloroethylnitrosocarbamoyl-L-alanine (CNC-L-ala) linked to oestradiol (CNA-L-ala-E2) or dihydrotestosterone (CNC-L-ala-DHT) in position 17 of the respective steroid hormone on tumour growth and receptor kinetics of methylnitrosourea-induced rat mammary carcinoma. Both compounds almost completely arrested logarithmically growing mammary carcinoma of Sprague-Dawley rats: in the first week CNC-L-ala-E2 blocked the growth of these tumours by 92% compared to untreated control animals while, in animals treated with the physically equimolar mixture of CNC-L-ala and oestradiol (positive control), tumour growth was inhibited by 51% only. CNC-L-ala-DHT arrested the tumour growth in the first week by 95%, while the respective positive control (CNC-L-ala plus dihydrotestosterone) effected a growth inhibition of 71% compared to the untreated control. These results correlate well with the influence of both drugs on the cytosolic receptor content of sexual steroid hormones in the tumours. CNC-L-ala-E2 depleted the content of oestradiol receptors and kept it down for a week, while concomitantly the content of progesterone receptors increased considerably and that of androgen receptors showed a short-lived decrease. CNC-L-ala-DHT depleted androgen receptors as well as progesterone receptors. The content of androgen receptors remained low for a week, while that of progesterone receptors recovered within 8 days. The content of oestrogen receptors showed a moderate decrease.

Polyamine effects on DNA-directed RNA polymerases in the ciliate Tetrahymena thermophila. In vivo- and in vitro-experiments suggesting highly specific regulative interactions.

Stimulation of growth in resting cultures of the ciliated protozoan Tetrahymena thermophila stimulated putrescine formation in a manner coordinated to transcription and partly to DNA-polymerisation. The stimulation of polyamine biosynthesis involved increased formation of the precursor L-ornithine from L-arginine as well as stimulation of the regulative key enzyme ornithine decarboxylase. In order to characterize the interrelationships between polyamines and transcription, which were suggested by these in vivo-assays, in vitro-assays were performed employing isolated pure macronuclei from Tetrahymena thermophila. The results obtained were: i) The diamine putrescine and the polyamines spermidine and spermine stimulated the incorporation of [4-14C]UTP into RNA time- and concentration dependently. This stimulation was not due to changes in the ionic strength nor to substitution for divalent cations (e.g. Mg2+ or Mn2+). The di- and polyamines did not alter the Km of RNA polymerases for the substrates, e.g. UTP. ii) Purified yeast-RNA, when added to the in vitro transcription system at concentrations capable of stimulating purified ornithine decarboxylase from Tetrahymena inhibited the RNA polymerases. The inhibitory effect of RNA on the polymerases was partly antagonized by spermidine and spermine but not by putrescine whereas the residual polymerase activity was stimulated by all three bases. iii) The stimulating effects of the di- and polyamines were synergistic but not absolutely additive, suggesting different targets for their actions. iv) Stimulation of RNA polymerases by putrescine, spermidine, or spermine after inhibition of particular enzymes by alpha-amanitin allowed to distinguish the effects on the three polymerases (I, II, and III): putrescine was not specific for any of the polymerases; spermidine was most active stimulating polymerase I and spermine was most active stimulating polymerase II, less active stimulating polymerase I but strongly inhibitory to polymerase III. v) The results obtained in the previous experiments were confirmed by electrophoretic analysis of the products formed in the in vitro transcription assays which furthermore showed that the differences between the experiments with or without polyamines were most pronounced if partly denatured calf thymus DNA was present in the assay mixture. This finding and the inhibition by RNA suggest that the factor influenced most by the polyamines is the binding of the RNA polymerases to the DNA target.

Manual and computerized cumulative reporting systems for the clinical microbiology laboratory.

A manual and a computerized system that produce cumulative updated reports from the clinical microbiology laboratory are described. Each system gives the physician a report that is clearly formatted, cumulative, readily updated, and written in conversational terms with minimal abbreviations. The report formats and updating sequences are nearly identical, so that one system can easily replace or back up the other. The cost and complexity of the hardware and software for the computerized system are modest, so that these are suitable for the moderate-sized hospital laboratory processing fewer than 10,000 specimens per year. Also, the laboratory personnel in our community-based nonteaching hospital were able to develop, set up, and support these systems without external consultation or purchased services. Therefore, the improved quality of reporting based on these types of systems can now be available to all laboratories without regard to size or workload.