Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin.

DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.     

APOLAR EMBRYOS OF FUCUS RESULTING FROM OSMOTIC AND CHEMICAL TREATMENT

Embryos of the brown alga Fucus vesiculosas L. were grown as populations in glass petri dishes in seawater at 15 C in continuous low-intensity unilateral fluorescent illumination for periods up to 2 weeks. A quantitative estimate of increase in nuclear number was made from acetocarmine squash preparations of samples taken at 12-or-24 hr intervals. Over the period of 2-6 days embryos showed a doubling time of about 12-18 hr. Under normal seawater culture conditions each embryo formed a single rhizoid. When grown in seawater supplemented with sugar concentrations above 0.4 m , Fucus embryos developed as multicellular spherical embryos lacking rhizoids. In 0.6 m sucrose-seawater, 97% of the embryos were apolar at 2 days; only 37% were apolar at 4 days, many having recovered from the sucrose inhibition. Some embryos remained apolar after growth in 0.6 m sucrose for 2 weeks. Nuclear counts showed that sucrose-seawater markedly inhibited the rate of cell division. Other sugars including D-glucose, D-fructose, D-galactose and the sugar alcohol D-mannitol were also effective. When apolar embryos grown in sucrose-seawater were returned to seawater, embryo growth resumed at the normal seawater rate, judged from nuclear counts. Such embryos formed multiple rhizoids, varying from two to eight rhizoids per embryo, which developed on the embryo quadrant or half away from the unilateral light. Each of the multiple rhizoids originated from a single small cell in the periphery of the multicellular spherica embryo. Thus the rhizoid-forming stimulus apparently had been subdivided among a number of the cells of the apolar embryos. The implications of this finding are discussed. Attempts to produce multiple rhizoids by treatment of embryos with indoleacetic acid or 2,4-dichlorophen-oxyacetic acid failed. However, embryos treated with 10⁻⁴ M or 5 × 10⁻⁵ m 2,3,5-triiodobenzoic acid formed 40 and 30% multiple rhizoids, respectively, suggesting that some chemical, perhaps hormonal, mechanism is involved in polarization and rhizoid initiation in Fucus embryogenesis.     

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Environmental Biology of Fishes -     

Macromolecular synthesis in organ cultures of neonatal rat lung

Organ cultures of newborn rat lungs synthesize and accumulate DNA, RNA, collagen and noncollagenous proteins almost at a linear rate for at least 5 days. During this period the synthesis of collagen consistently exceeds the synthesis of noncollagenous proteins in a pattern similar to neonatal lung growth in vivo. Although some morphological characteristics of lung architecture are distorted after culture, fundamental structural similarities to lungs growing in intact animals are retained. When these cultures are maintained in atmospheres rich in oxygen, increased collagen synthesis is observed, a response similar to that of lungs in intact animals exposed to high oxygen concentrations in vivo. Our studies suggest that lung organ cultures may be a suitable system for investigating the biochemical aspects of lung tissue-environmental interaction. These studies were supported in parts by NIH Grant HL-19668, a contract (68-03-2005) from the U.S. Environmental Protection Agency, and grants from the California Lung Association.     

Lineage analysis of the olfactory epithelium using a replication-incompetent retrovirus

We have analysed the lineage of olfactory receptor neurons usinga replication-incompetent retrovirus injected beneath the olfactoryepithelium of young rats. There are two major types of clustersof infected cells seen at 5–40 days after infection: (i)horizontal basal cells (HBCs); (ii) variable numbers of globosebasal cells (GBCs), and immature and mature sensory neurons.Olfactory nerve lesion increased the frequency of the globose/sensoryneuron clusters, as well as the number of cells/cluster, butdid not change the number of HBC clusters or cells/cluster.No clusters contained sustentacular cells. These data indicatethat, at least in young rats: (i) HBCs are not precursors ofolfactory neurons; (ii) there is a lineage path from GBCs tomature neurons; and (iii) sustentacular cells arise from a separatelineage.     

Intermediate sucrose octa-acetate sensitivity suggests a third allele at mouse bitter taste locus Soa and Soa-Rua identity

Mice have been characterized as either tasters or non-tastersof the bitter compound sucrose octa-acetate(SOA). However, 11of 17 supposedly non-taster inbred strains were found to avoid1 mM SOA. All 17 strains were indifferent to 0.1 mM SOA. Tasterstrains avoided both concentrations. The intermediate phenotypewas dubbed demitaster. A consistent phenotypic dominance orderwas found in crosses among both inbred and outbred strains (taster> non-taster > demitaster). Demitasters were found (withtasters) in an outbred strain showing monogenic segregationfor SOA avoidance. This, plus monogenic segregation in a back-crossof taster to demitaster inbred strains, suggested a third alleleat the Soa locus (Soa^c). Demitaster allelism was supported bythe strong associations found in 15 strains between the threeSOA phenotypes and HindIII restriction fragment patterns forthe closely linked Prp (proline rich protein) loci. SOA demitasterstrains were also intermediate in raffinose undeca-acetate (RUA)avoidance. Furthermore, B6.SW-Soa² congenic mice avoided notonly SOA, but RUA and eight other acetylated sugars. A previouslyproposed separate RUA-sensitivity gene (Rua) thus appeared tobe redundant.