British American Tobacco (Canada)

Abstract Protein transfer in tobacco smoke has been studied using the protease, Savinase™, as a model protein. Mainstream and sidestream smoke were collected from cigarettes to which Savinase had been added at various concentrations. Savinase was extracted from the smoke condensate with an organic solvent system before being precipitated and further identified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblotting. The detection limit of the method, based on addition of Savinase to the smoke condensate, was 25 µg in mainstream and 100 µg in sidestream smoke. At a Savinase concentration of 6000 µg per gram of tobacco, the methodology allows the detection of protein transfer as low as 0.009% and 0.054% in mainstream and sidestream smoke, respectively. Using this approach, it was shown that there is no detectable Savinase in the mainstream and sidestream smoke of filtered and unfiltered cigarettes containing up to 6000 µg of Savinase per gram of tobacco. These facts strongly suggest that there is no significant transfer of protein from tobacco into cigarette smoke.

To determine whether aqueous humor flare, measured by use of laser flaremetry, was proportional to aqueous humor protein concentration and to use laser flaremetry to evaluate disruption of the blood-aqueous barrier (BAB) in cats. 30 healthy adult cats. Laser flaremetry values for all eyes were compared with aqueous humor protein concentrations determined by use of a Coomassie blue microprotein assay. Laser flaremetry was then performed on both eyes before (0 hours) and 4, 8, and 26 hours after initiation of topical application of 2% pilocarpine (q 8 h) to 1 eye of 9 cats or paracentesis of the anterior chamber of 1 eye of 8 cats. Intraocular pressure and pupil size were also determined. Aqueous humor protein concentration was extrapolated from flare values by use of linear regression. There was a linear relationship between flare values and aqueous humor protein concentrations. Topical application of 2% pilocarpine and paracentesis of the anterior chamber caused a breakdown of the BAB that was detected by use of laser flaremetry. The highest mean flare readings after application of pilocarpine or paracentesis were 24.4 and 132.8 pc/ms, respectively, which corresponded to aqueous humor protein concentrations of 85.5 and 434.9 mg/dl, respectively. Paracentesis of the anterior chamber resulted in a more severe breakdown of the BAB in cats than topical application of 2% pilocarpine. Laser flaremetry may be a useful clinical method to detect increases in aqueous flare and, hence, disruptions of the BAB in cats.

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of 'Delgold' tobacco, a BC5F8 near isogenic line (NIL) carrying the resistance gene in a 'Delgold' background, and 'PB19', the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between 'Delgold' and 'PB19', indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between 'Delgold' and the NIL. All of these markers proved to be unlinked with the resistance gene in F2 linkage tests. Of the remaining three RAPD markers polymorphic between 'Delgold' and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.

Leaf disks of Nicotiana tabacum var. Delcrest were supplied with α-C14-labelled cinnamic, p-coumaric, and caffeic acids, p-coumaroyl-quinic acid, and the 1-glucose esters of cinnamic and p-coumaric acids. Considerable incorporation of radioactivity into chlorogenic acid occurred in 40 hours from all substrates except caffeic acid. In particular, p-coumaric acid was found to be a good precursor of chlorogenic acid in contrast to the findings of other workers with potato tuber tissue. More p-coumaric acid than cinnamic acid is converted to chlorogenic acid, although the reverse holds for conversion to p-coumaroyl-quinic acid. Chlorogenic acid synthesis from caffeic acid involves breakdown and resynthesis of the acid supplied, leading to the incorporation of radioactivity into other positions in the caffeic acid molecule and into the quinic acid moiety. Cinnamic and p-coumaric acids, on the other hand, are incorporated essentially intact into both p-coumaroyl-quinic and chlorogenic acids. These findings are discussed in relation to the pathway of chlorogenic acid biosynthesis postulated for potato tuber tissue.