DNA topoisomerase I was purified to near homogeneity from a clonal line of human lymphoblastic leukemia cells, RPMI 8402, that is resistant to camptothecin, a cytotoxic alkaloid from Camptotheca acuminata, and compared with that of the parent wild-type cells. As assayed by relaxation of the supercoiled plasmid DNA and by formation of enzymelinked DNA breaks, the purified enzyme from the resistant cells was shown to be >125-fold as resistant to camptothecin as the wild-type enzyme, comparable to a cellular resistance index of about 300. Therefore, the cellular resistance appears to be due to the resistance of the enzyme. The amount of the immunoreactive enzyme protein in whole extract appeared to be reduced to less than half that of the wild-type enzyme. These results establish that DNA topoisomerase I is the cellular target of camptothecin and that DNA topoisomerase I is essential for the survival of mammalian cells.The DNA topoisomerases are enzymes that catalyze the concerted breakage and rejoining of the DNA backbone and thereby are presumed to participate in various genetic processes (1-5). The type I topoisomerases transiently cut and reseal one DNA strand so that the linking number changes by steps of one. Genetic and functional studies of the enzymes have been largely limited to prokaryotes and the lower eukaryote yeast. Viable mutants ofEscherichia coli defective in the topA gene encoding topoisomerase I were isolated (6, 7), but these proved to have mutations in DNA gyrase genes that compensated for the mutation in topA (8-10). This finding suggested an essential role for the enzyme in regulating the degree of supercoiling of DNA by counteracting the activity of the type II enzyme. In contrast, however, topoisomerase I-deficient mutants of yeast were isolated and shown to be viable, although they possessed the wild-type allele of topoisomerase II (11,12). The effect of the topoisomerase I mutation, however, was manifested by an additional mutation in topoisomerase II, implicating the complementary role of the latter (12).Topoisomerase I was previously found associated with transcriptionally active chromatin in mammalian cells (13,14). It also appears to be catalytically active on transcriptionally active genes in Drosophila polytene chromosomes (15) as well as on nucleolus-associated ribosomal genes (16-19). These experiments suggest a functional role for the enzyme in transcriptional events involving either RNA polymerase I or II.The availability of mutants and specific inhibitors of this enzyme as was the case in prokaryotes might help dissect and establish the role of this enzyme in DNA metabolism. We previously reported that heparin is a potent inhibitor of a mammalian DNA topoisomerase I (20, 21), but its broad specificity limits its usefulness for this purpose. Camptothecin (CPT) is a cytotoxic alkaloid isolated from Camptotheca acuminata (22, 23), which has a strong antitumor activity against a wide range of experimental tumors. CPT inhibits RNA and DNA synthesis and causes rapid and rev...

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Using a conventional thermal chemical vapor deposition (CVD) system, ethanol vapor was enclosed in a reactor, i.e., no flow, with Co/Mo dip-coated quartz substrates to synthesize submillimeter long vertically aligned single-walled carbon nanotube (SWNT) films successfully. The no-flow CVD method yielded an increase in film thickness of up to 0.11 mm compared with the normal flowing gas method for 30 min of synthesis. The thermal decomposition of ethanol was investigated using Fourier transform infrared spectroscopy and quadrupole mass spectrometry. At CVD temperatures, ethanol decomposed mainly into ethylene, acetylene, acetaldehyde, and water. Some of these molecules could enhance SWNT growth.

A radioimmunoassay method for endothelin was developed. Antisera raised against endothelin 1 showed significant crossreaction with endothelin 2 and 3 (45 and 13%, respectively). Considerable endothelin immunoreactivity was shown to be present in the cerebrospinal fluid of patients with a subarachnoid hemorrhage, ranging from 0.3 pmol/l cerebrospinal fluid to 4.5 pmol/l cerebrospinal fluid, though no endothelin immunoreactivity was observed in the cerebrospinal fluid of controls and patients with cerebral infarction, subdural haematoma or brain tumours. Endothelin immunoreactivity was also observed in two out of five cerebrospinal fluid samples from patients with cerebral bleeding. Reverse phase high performance liquid chromatography showed that the main immunoreactive component in cerebrospinal fluid appeared to elute at the same position. There was, however, an immunoreactive component which eluted at the same position as endothelin 3. These results may support the idea that endothelin immunoreactivity in the cerebrospinal fluid originate mainly from endothelial and neural tissues and that endothelin may contribute to the generation of the vasospasm often observed in subarachnoid hemorrhage, a conclusion based on the exceptionally high endothelin immunoreactivity in cerebrospinal fluid observed in patients with subarachnoid haemorrhage.