DNA-mediated transfer of cAMP resistance in CHO cells

Chinese hamster ovary (CHO) strain 10215 carries a dominant mutation which confers resistant to cAMP by virtue of an altered catalytic subunit of the cAMP-dependent protein kinase (Evain et al., 1979). This mutation was transferred to wild-type CHO cells by DNA-mediated gene transfer. Based on the absence of cAMP growth inhibition, seven transformant colonies were isolated. One of these, 11586, was studied in detail. This transformant showed the same phenotype as the mutant, including resistance to the morphological changes and growth inhibitory effects of 1 mM 8-Br-cAMP, reduced total cAMP dependent protein kinase activity and lowered sensitivity of the kinase to cAMP activation. When the cAMP-dependent protein kinase was fractionated on a DEAE-cellulose column, the transformant was lacking in type II cAMP dependent protein activity, to the same degree as the mutant. The transformant and mutant, but not wild-type cells, also failed to phosphorylate a 52,000-dalton protein in a cAMP-dependent manner. These characteristics support the conclusion that the gene for the mutant cAMP-dependent protein kinase has been transferred. The ability to transfer this gene by DNA-mediated transfer suggests that this methodology may be useful for the molecular isolation of the gene encoding the catalytic subunit of cAMP-dependent protein kinase.     

Amino acid structure and "difficult sequences" in solid phase peptide synthesis.

Deprotection peak profiles have been determined as a measure of internal aggregation during Fmoc-polyamide continuous flow solid phase synthesis. The results have been correlated with amino-acid structure and discussed in terms of minimising aggregation during synthesis.     

Incremental sampling methodology for petroleum hydrocarbon contaminated soils: volume estimates and remediation strategies

Current environmental assessments for petroleum hydrocarbon (PHC) contaminated sites are dependent on discrete soil sampling to estimate the degree and extent of contamination, leading to unreliable and non-reproducible results. Incremental sampling methodology (ISM) involves collecting and combining samples within a targeted area and holds promise for being a cost-effective, representative, and reproducible sampling strategy for contaminated site characterization. We hypothesized that traditional Phase II Environmental Site Assessments (ESA) discrete and ISM sampling protocols were not mutually exclusive, and the two approaches can be used to formulate a responsible land management strategy. Results gathered through ISM were compared to those from Phase II ESA for two PHC contaminated sites in Canada. Both methods indicated the sites were impacted with PHC beyond Saskatchewan Tier I guidance, however, the delineation of the PHC plume differed by as much as 75% for the heavier hydrocarbons. The Phase II ESA methods had higher incidences of false positive results and an overestimation of contamination at depth. A laboratory experiment confirmed that ISM does not “dilute” the samples as to cause underestimation, whereby the hydrocarbon concentrations for a single combined sample was equivalent to the mean of 30 discrete samples. Based on our results, sites should undergo risk assessment based on the estimates of the Phase II ESA results using vapor phase logs to estimate contaminant extent. If exposure pathways cannot be eliminated through the risk assessment process, remediation planning based on the ISM results is justified given the demonstrated cost-effectiveness, representativeness, and reproducibility.     

Isolation of α-tubulin genes from the human malaria parasite, Plasmodium falciparum: sequence analysis of α-tubulin

As a step towards identifying exploitable differences between host and parasite at the molecular level, we have isolated and sequenced genomic clones encompassing an entire alpha-tubulin gene (designated alpha-tubulin I) from the human malaria parasite, Plasmodium falciparum. The gene, which contains two introns, encodes a product with a predicted length of 453 amino acid residues (50.3 kD). The protein sequence shows a high degree of homology to other alpha-tubulins, particularly that of the coccidian parasite, Toxoplasma gondii (94%), whose gene carries introns in identical positions. Only one copy of the alpha-tubulin I gene itself was found, although a second gene designated alpha-II was also identified which is closely related but which differs at both the nucleotide and amino acid sequence levels. The alpha-I and beta-tubulin genes were found to reside on different chromosomes.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.