What was observed in the sugar beet background except for the 30 kDa protein. The smaller proteins may represent modified or partially degraded forms of the 30 kDa BvSTI protein subjected to proteolytic enzymes of the host plant. A possibility that these proteins represent newly induced proteinase inhibitors of N. benthamiana cannot be excluded. A number of the independently drived BvSTI-transgenic LEE011 plants were bioassayed for resistance to several lepidopteran insects that are pests of tobacco. Fall armyworm, beet armyworm, tobacco hornworm, tobacco budworm and black Vorinostat 149647-78-9 cutworm cause significant yield losses in hundreds of economically important crops and all, with the exception of tobacco hornworm and budworm, infest sugar beet. The sugar beet root maggot was not included in this study since its host range is limited and does not include Nicotiana spp. The major digestive proteases utilized by the lepidopteran insects in this study have been reported to be predominantly in the serine class of proteases. Therefore, presence of the recombinant BvSTI proteinase inhibitor has the potential to deter insect feeding or inhibit digestion of ingested food thus reducing the overall larval weights as compared to larvae feeding on untransformed control plants. When the BvSTI-transgenic plants were fed to fall armyworm, beet armyworm and tobacco hornworm larvae, significant reductions in larval weights were observed, with some pupae and emerging moths displaying developmental abnormalities. Fall armyworm larvae weighed 19�C51%, 34�C66% and 59�C71% less at 3, 6 and 8 days of feeding, respectively, as compared to control larvae. Except for the smaller pupae sizes that corresponded to the reduced larval weights and a lighter brown color, no significant differences in development or mortality rates were noted. The beet armyworm pupae and emerging moth sizes similarly reflected the reduced weights of the larvae fed the BvSTI transgenic leaves. In addition, many of the pupae did not emerge as moths and of the ones that did, developmental abnormalities were often noted. Tobacco hornworm larvae were also significantly smaller than the control larvae and the resulting pupae and moth sizes correlated with the reduced larval weights. Larval weights after 6 days of feeding on the BvSTI transgenic plants were about 50�C60% lower than those fed on control untransformed plants. In contrast, black cutworm and tobacco budworm larvae fed on BvSTI transformed plants accumulated biomass faster than those fed on the control foliage. Black cutworm larval weighs were more than double those of the control larvae at 3 and 5 days of feeding. After 7 days, the larvae weighed almost 50% more than the control larvae. No differences in larval mortality were noted and pupae and moth sizes reflected larval weights. Similar responses were observed with tobacco budworm larvae fed on BvSTI leaves. On the average, the larvae were 10 to 50% heavier than the control larvae. Larval mortality rates were up to 5 times those of the control larvae and emerging moths displayed varying degrees of abnormal wing development after feeding on the BvSTI transformants. Increases in larval weights feeding on proteinase inhibitor transformed plant materials have been reported by others. Faster biomass accumulation of Colorado potato beetle feeding on potato transformed with a rice cysteine proteinase inhibitor gene was reported.

Aerobic glycolysis is common particularly in aggressive tumors, though the causative relationship remains unknown. The specificity of SDH loss in PGL has led to the hypothesis that it is succinate accumulation, not just TCA cycle dysfunction, that is pathogenic. As described above, the detection of approved LOPAC drugs disulfiram and dequalinium among compounds differentially toxic to sdh2D mutant yeast cells is interesting and provocative. These agents have been proposed as anti-malarials, with disulfiram as a potential inhibitor of AZD6244 606143-52-6 alcohol dehydrogenase and dequalinium potentially increasing reactive oxygen species, a stress already known to be higher in sdh2D mutant yeast. Interestingly, six of the ten compounds identified by the main HTS are potentially chemically reactive as Michael acceptors. Related compounds are sometimes categorized as pan assay interference “PAINS” compounds that can be problematic in HTS by interference with fluorescent detection or other technical problems. However, it must be noted that these compounds were identified here in a simple differential growth screening protocol that avoids nonspecific effects commonly associated with PAINS compounds. In fact, the compounds shown in Fig. 6 reflect potentially reactive agents as have been enjoying a resurgence in medicinal chemistry through covalent mechanisms including thiol-reactivity. With respect to possible mechanisms of differential toxicity to sdh2D mutant yeast cells, the most obvious a priori targets include the unique reliance of sdh2D mutant cells on glycolysis and the potentially stressful increases in intracellular ROS and Su. If each of these characteristics holds for SDH mutant human PGL tumor cells, rational approaches might be imagined. In the simplest case, PGL cells could be starved by providing only nonfermentable carbon sources if metabolism in other tissues could be supported by ketogenic agents. Our HTS screening results add insights into possible clinical tactics against SDH mutant cells such s SDH-mutant PGL. Our observation that alcohol dehydrogenase inhibitors are differentially toxic to sdh2D mutant yeast is interesting and provocative. Disulfiram is an approved anti-alcoholism medication shown to inhibit both FTY720 aldehyde dehydrogenase and alcohol dehydrogenase. Disulfiram is also an anti-malarial, noteworthy because, like PGL cells, P. falciparum cells lack a TCA cycle and rely on glycolysis. Moreover, we have now shown that four of the ten leading differential inhibitors of sdh2D mutant yeast growth are inhibitors of yeast alcohol dehydrogenase. This result points to glycolysis inhibition as an interesting approach in human PGL. In the absence of a TCA cycle, NAD + recycling to NADH might become limiting for PGL tumor cells. Yeast uses pyruvate decarboxylase to convert pyruvate to acetaldehyde and then alcohol dehydrogenase to regenerate NAD + by reduction of acetaldehyde to ethanol at the expense of NADH. Mammals accomplish the same NAD + regeneration by reducing pyruvate to lactate using lactate dehydrogenase. Thus, by analogy with the results of this high-throughput screen, an interesting target for therapeutic inhibition of SDH mutant PGL would be lactate dehydrogenase. We confirm this concept here by showing that human cells deficient in SDH activity are differentially sensitive to the lactate dehydrogenase inhibitor oxamate. This provocative result points to the potential value of lactate dehydrogenase inhibitors in paraganglioma therapy.

Over-expression of heterologous PI genes in crop plants has resulted in enhanced resistance to a wide spectrum of insect pests. However, the ongoing challenge of the PI based insect control strategy is the need to discover and characterize new and novel PIs to address the inherent and induced XAV939 complexity of the insect gut proteases. PIs such as those derived from non-host plants to which the targeted insect has had minimal or no prior exposure can generally be most useful for enhancing insect resistance in engineered plants. In a study of sugar beet root defense responses, a single serine PI gene was identified among the more than 150 sugar beet genes whose expression was found to be modulated by a dipteran pest of sugar beet, the root maggot. Expression of the BvSTI gene was determined to be up-regulated by mechanical- and insect-wounding in sugar beet lines used in breeding for root maggot resistance. The observed lack or reduced accumulation and activity of BvSTI PI in tissues of susceptible and less resistant lines emphasized the potentially important role of the BvSTI PI in insect pest defense mechanisms. In this study, the prospect of over-expressing the sugar beet BvSTI gene for control of lepidopteran insect pests in genetically modified N. benthamiana was investigated. Serine proteases that include trypsin-, chymotrypsin- and elastase-like have been well-documented as comprising the major midgut proteolytic activities in lepidopteran insects. Homozygous T2 populations of transgenic N. benthamiana plants carrying a single copy of the BvSTI transgene construct exhibited phenotypes that were similar to the normal untransformed plants. Elevated levels of BvSTI gene transcripts driven by the constitutive CaMV35S promoter were detected in all analyzed T2 homozygous plants. Presence of the recombinant BvSTI proteins in the T2 transformants was confirmed on SCH727965 Western blots with BvSTIspecific antibody that cross-reacted with low quantities of peptides in the range of 22�C25 kDa and 30 kDa that was previously observed in sugar beet. These finding suggests that processing and modification of the recombinant BvSTI protein may be different in the tobacco background as compared to its regulation in sugar beet. Detection of low levels of recombinant PI protein has been reported by others. Independently derived apple transformants with increased resistance to the light-brown apple moth had low levels of the recombinant PI protein. It has also been shown that feeding inhibition did not necessarily increase beyond that observed with low protein concentrations in studies where recombinant PI proteins were fed to larvae. Because the detected signal on Western blots was weak this suggests a possible high turnover and/or modification of the BvSTI protein in N. benthamiana irrespective of the high transcript and trypsin protein activity levels in the BvSTI transformants. A unique and distinct clear zone of at about 30 kDa was detected in all five homozygous BvSTI transformants by an in gel trypsin activity assay. Two additional activity zones corresponding to proteins of approximately 28 and 26 kDa were also visible in the transformed lines but not in the untransformed control plants. No apparent cross reactivity of the BvSTI specific antibody with the 28 and 26 kDa trypsin inhibitory proteins was observed on Western blots although one can argue that the 26 kDa protein is similar to that in the range of 21�C25 kDa.