Development and commercial use of afla-Guard(®), an aflatoxin biocontrol agent.

A biopesticide, afla-guard(®), has been developed for controlling aflatoxin contamination in peanuts. This product provides the means of introducing a competitive, non-aflatoxigenic strain ofAspergillus flavus into soils where peanuts are being grown. The introduced strain competitively excludes toxigenic strains naturally present from invading developing peanuts. The biocontrol technology was made commercially available in 2004 by Circle One Global, Inc., upon receiving U.S. Environmental Protection Agency section 3 registration of afla-guard(®) as a biopesticide. The product was applied to approximately 2000 ha of peanuts in Georgia and Alabama during the 2004 crop year. Application of afla-guard(®) changed the composition ofA. flavus soil populations from an average 71.1% toxigenic strains in untreated fields to only 4.0% in treated soils. Analyses of farmer's stock peanuts being delivered at seven different locations showed a consistent reduction in aflatoxin contamination in peanuts from fields treated with afla-guard(®). Over all locations, aflatoxin averaged 78.9 ng/g in untreated peanuts compared with 11.7 ng/g in treated peanuts, an 85.2% reduction. Peanuts from treated and untreated fields were stored together in separate warehouse bins at two different locations. Aflatoxin analyses at the Unadilla, GA location showed that 48.4% of shelled edible lots from untreated fields contained unacceptable levels of aflatoxin (>15 ng/g). At the Dawson, GA location, 15.8% of shelled lots from untreated fields contained >15 ng/g. At both locations, no shelled edible lots from treated fields contained >15 ng/g. Mean aflatoxin concentrations in edible peanuts from untreated and treated fields at Unadilla were 36.2 and 0.9 ng/g, respectively. At Dawson the respective means were 7.2 and 2.2 ng/g.

Implementing change in the National Health Service.

This paper will outline the current changes being imposed on the National Health Service. The literature on change management will be employed to propose some guidelines for health service managers. The National Health Service (NHS) spent much of the 1980s and 1990s learning about the transition from administration to management and must now make the transition from management to leadership. The emphasis is now focused less on doing and more on being.

The effect of the one-chain to two-chain conversion in tissue plasminogen activator: characterization of mutations at position 275.

Tissue plasminogen activator (t-PA) is homologous to other serine proteases and contains an apparent activation cleavage site at arginine 275. It has been demonstrated that this arginine-275 can be replaced with either glutamic acid (Tate, K. M., Higgins, D. L., Holmes, W. E., Winkler, M. E., Heyneker, H. L., and Vehar, G. A. Biochemistry 26, 338-343, 1987) or glycine (Peterson, L. C., Johannessen, M., Foster, D., Kumar, A., and Mulvihill, E. Biochim. Biophys. Acta 952, 245-254, 1988; Boose, J. A., Kuismanen, E., Gerard, R., Sambrook, J. and Gething, M.-J. Biochemistry 28, 635-643, 1989) so that the product of the plasminogen activation reaction, plasmin, can no longer hydrolyze the one-chain form of t-PA to the two-chain form. These "one-chain" t-PA variants had diminished activity, compared to wild-type t-PA, in the absence of a cofactor, but in the presence of the fibrin(ogen) cofactor the two variants had activity similar to wild-type t-PA. In order to compare the effects of all possible substitutions, t-PA variants with each of the other nineteen amino acids besides arginine at position 275 were produced by site-directed mutagenesis. All were recovered from cell culture supernatants completely in the one-chain form, except for R275 (wild-type) and R275K, which were partially converted to the two-chain form. These latter two species could be completely converted to the two-chain form by plasmin. In addition, these two forms showed significantly more plasminogen activating activity in the absence of a fibrin(ogen) cofactor, compared to the other 18 variants. In the presence of a cofactor, all of the t-PA mutants had plasminogen activating activity equivalent to wild-type t-PA, except for R275C. The R275C t-PA had comparatively less clot lysis and fibrin binding activity as well. Presumably the new cysteine in this variant was involved in a mixed disulfide or caused misfolding of the molecule resulting in decreased activity. The difference in the plasminogen activating activity of one- and two-chain forms of t-PA was investigated by determining the apparent Michaelis constants and the apparent turnover numbers for R275E t-PA, which remains in the one-chain form throughout the assay, and two-chain R275 t-PA. The kinetic constants were measured in both the presence and the absence of plasmin-digested fibrinogen.(ABSTRACT TRUNCATED AT 400 WORDS)

The incorporation of a fluorescent probe into the active sites of one- and two-chain tissue-type plasminogen activator.

Dansyl-glutamyl-glycyl-arginyl chloromethyl ketone (DEGR-CK) was shown to inactivate both one- and two-chain human, recombinant tissue-type plasminogen activator (t-PA). The interaction of DEGR-CK with both forms of t-PA was accompanied by an identical increase in the fluorescence intensity and a blue shift in the wavelength of maximum emission, which suggests that the environment of the incorporated DEGR is similar in both one- and two-chain t-PA. The kinetics of the interaction of t-PA with DEGR-CK could be followed by both loss of activity and increase in fluorescence. The second order rate constants (k2/Ki) obtained with these two methods agreed quite well. With two-chain t-PA the values were 42 X 10(4) M-1 min-1 and 46 X 10(4) M-1 min-1 by the activity loss and fluorescence methods, respectively. With one-chain t-PA the results were 2.5 X 10(4) M-1 min-1 and 3.1 X 10(4) M-1 min-1. The rate at which one-chain t-PA is inactivated by DEGR-CK is 15 times lower than the rate with two-chain t-PA. The results demonstrated, however, that the cleavage of the one-chain protein to the two-chain form is not required for reactivity with DEGR-CK. This fluorescently labeled t-PA should be useful in probing the interactions of one- and two-chain t-PA with other proteins.