Pest protection conferred by a Beta vulgaris serine proteinase inhibitor gene.

Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 30 kDA recombinant BvSTI protein on Western blots. In gel trypsin inhibitor activity assays revealed a major clear zone that corresponded to the BvSTI proteinase inhibitor that was not detected in the untransformed control plants. BvSTI-transgenic plants were bioassayed for resistance to five lepidopteran insect pests. Spodoptera frugiperda, S. exigua and Manduca sexta larvae fed BvSTI leaves had significant reductions in larval weights as compared to larvae fed on untransformed leaves. In contrast, larval weights increased relative to the controls when Heliothis virescens and Agrotis ipsilon larvae were fed on BvSTI leaves. As the larvae entered the pupal stage, pupal sizes reflected the overall larval weights. Some developmental abnormalities of the pupae and emerging moths were noted. These findings suggest that the sugar beet BvSTI gene may prove useful for effective control of several different lepidopteran insect pests in genetically modified tobacco and other plants. The sugar beet serine proteinase inhibitor may be more effective for insect control because sugar beet is cropped in restricted geographical areas thus limiting the exposure of the insects to sugar beet proteinase inhibitors and build up of non-sensitive midgut proteases.

Effects of thrombolytic agents on tympanostomy tubes occluded by blood clots.

OBJECTIVE: To investigate the efficacy of various topical applications in opening a clotted tympanostomy tube (TT) using an in vitro model. DESIGN: In vitro clinical trial. INTERVENTIONS: Fresh human blood was allowed to clot in the lumen of TTs. Seven agents were tested: 0.9% saline (control), 1-mg/mL alteplase, 100-U/mL unfractionated heparin, 3% hydrogen peroxide (H(2)O(2)), 3% acetic acid, 5% acetic acid, and a mixture of 3% H(2)O(2) and 3% acetic acid. Each agent was added twice a day for 14 days to TTs that were incubated and humidified to simulate ear canal conditions. The tubes were analyzed with binocular microscopy to determine the status of the obstruction. RESULTS: A total of 16 trials per agent, including a saline control, were performed. The saline control, alteplase, and heparin failed to open any TTs in any of the trials. Compared with the control, H(2)O(2) also was not effective (P = .23). Acetic acid was increasingly effective, with a 3% concentration completely clearing 5 of 16 tubes and a 5% concentration completely clearing 11 of 16 tubes (P = .006). The addition of 3% H(2)O(2) to 3% acetic acid did not significantly increase clearance (P = .21). CONCLUSIONS: Thrombolytic agents and H(2)O(2) were not effective in resolving TTs that were clotted with blood in an in vitro environment simulating the ear canal. Increasing concentrations of acetic acid are increasingly effective in this capacity.

Biolistic transformation of highly regenerative sugar beet (Beta vulgaris L.) leaves.

Leaves of greenhouse-grown sugar beet (Beta vulgaris L.) plants that were first screened for high regeneration potential were transformed via particle bombardment with the uidA gene fused to the osmotin or proteinase inhibitor II gene promoter. Stably transformed calli were recovered as early as 7 weeks after bombardment and GUS-positive shoots regenerated 3 months after bombardment. The efficiency of transformation ranged from 0.9% to 3.7%, and stable integration of the uidA gene into the genome was confirmed by Southern blot analysis. The main advantages of direct bombardment of leaves to regenerate transformed sugar beet include (1) a readily available source of highly regenerative target tissue, (2) minimal tissue culture manipulation before and after bombardment, and (3) the overall rapid regeneration of transgenic shoots.