Inhibition of colony formation in cultured hepatocellular carcinoma cells of the rat was used to test the efficacy of inhibitors of de novo pyrimidine biosynthesis as potential anticancer drugs. N-(phosphonacetyl)-L-aspartic acid (PALA) (10 and 100 micrograms/ml) and 5-aza-5,6-dihydroorotic acid (DHOX) (100 micrograms/ml) inhibited the formation of colonies and these inhibitions were completely reversed by inclusion of 0.1 mM uridine, the end product of de novo pyrimidine biosynthesis, in the culture medium. With some lots of fetal bovine serum where PALA and DHOX had little effect on inhibiting colony formation, addition of 0.1 mM cytidine restored the inhibitory characteristics of PALA and, to some extent, DHOX. The results demonstrate that cytidine levels modulate the inhibitions of hepatoma colony formation by both PALA and DHOX and that co-administration of these drugs together with cytidine provides a simple expedient to increase drug efficacy.

A method, utilizing brine shrimp (Artemia salina Leach), is proposed as a simple bioassay for natural product research. The procedure determines LC (50) values in microg/ml of active compounds and extracts in the brine medium. Activities of a broad range of known active compounds are manifested as toxicity to the shrimp. Screening results with seed extracts of 41 species of Euphorbiaceae were compared with 9KB and 9PS cytotoxicities. The method is rapid, reliable, inexpensive, and convenient as an in-house general bioassay tool.

Galsky et al. (10, 11) have reported that the inhibition and growth of crown gall tumors, initiated on potato discs by Agrobacterium tumefaciens, apparently has good agreement with 3PS in vivo antileukemic activity in mice. We have now modified and evaluated this assay for its potential use as a prescreen and fractionation monitor of plant extracts for 3PS activity. The modified assay was performed on a series of natural compounds and plant extracts (known to have 3PS activity) and on extracts of seeds of 41 Euphorbiaceae species. The results suggest that the potato disc assay is a safe, simple, rapid, in-house, low cost, prescreen for 3PS antitumor activity; it detects some false positives, but few false negatives; it is statistically much more predictive (p = 0.002) of 3PS activity than either the 9KB (p = 0.140) or the 9PS (p = 0.114) cytotoxicity assays. Its extended use could easily obviate the expense and extensive need for animals in the initial stages of antitumor screening and plant fractionation.

Several cutaneous fiber types project directly to the gracile nucleus of the cat. Under barbiturate anesthesia, terminal excitability changes on single fibers of identified type were produced by electrical and natural stimulation of the ipsilateral hind limb but not by stimulation of other limbs. The time course of primary afferent depolarization evoked by electrical stimulation of the sciatic nerve was similar for all fiber types. Light brushing of hairs on the hind limb was effective in producing primary afferent depolarization on all fiber types. Rubbing the skin did not produce significantly greater excitability changes. Comparison of periphery-to-nucleus conduction time and onset time of primary afferent depolarization for individual fibers showed no clear relationship between these two parameters, although some fibers with long conduction times also had a late onset of primary afferent depolarization. The data suggest that under barbiturate anesthesia, ipsilateral peripheral inputs activate a single interneuron pool which directs primary afferent depolarization onto fibers of all types reaching the gracile nucleus directly.

The time required for impulse conduction from the skin to the gracile nucleus has been determined for primary afferent fibers in the sural and accessory sural nerves of the cat. Fibers were classified into types on the basis of their receptor properties. Fibers of a particular type were found to have a characteristic range of conduction times. Fibers of the same type in different nerves were found to have similar skin-to-nucleus conduction times despite differences in conduction distance. These data enable one to predict the temporal characteristics of the input to the nucleus evoked by an abrupt stimulus at the skin. Conduction times from the sciatic nerve to the gracile nucleus were also determined. Comparison was made between the temporal dispersion of the input evoked by electrical stimulation of the sciatic nerve and the first spike latencies of post-synaptic cells in the nucleus excited by the same stimulus. The range of arrival times of the input closely matched the distribution of postsynaptic first spike latencies.