Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is a plant alkaloid that inhibits alpha- and beta-glucosidase in fibroblast extracts [Saul, R., Chambers, J. P., Molyneux, R. J., & Elbein, A. D. (1983) Arch. Biochem. Biophys. 221, 593-597]. In the present study, castanospermine also proved to be a potent inhibitor of glycoprotein processing by virtue of the fact that it inhibits glucosidase I. Thus, when influenza virus was raised in the presence of castanospermine, at 10 micrograms/mL or higher, 80-90% of the viral glycopeptides were susceptible to the action of endoglucosaminidase H, whereas in the normal virus 70% of the glycopeptides are resistant to this enzyme. The major oligosaccharide released by endoglucosaminidase H from castanospermine-grown virus migrated like a hexose10GlcNac on a calibrated Bio-Gel P-4 column. This oligosaccharide was characterized as a Glc 3 Man 7 GlcNAc on the basis of various enzymatic treatments, as well as by methylation analysis of the [2-3H]-mannose-labeled or [6-3H]galactose-labeled oligosaccharide. The presence of three glucose residues in the oligosaccharide was also confirmed by periodate oxidation studies of the [6-3H]galactose-labeled hexose10GlcNAc. Castanospermine did not inhibit the incorporation of [3H]leucine or [14C]alanine into protein in MDCK cells at levels as high as 50 micrograms/mL. In addition, influenza virus produced in the presence of this alkaloid were fully infective and apparently produced in similar amounts to that of control cells, as determined by plaque counts. Castanospermine did, however, cause considerable changes in cell surface properties, since MDCK cells grown in 10 micrograms/mL castanospermine were able to bind twice as much [3H]concanavalin A as were control cells.

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A lack of analytically robust and multiplexed assays has hampered studies of the large, branched phosphosignaling network responsive to DNA damage. To address this need, we developed and fully analytically characterized a 62-plex assay quantifying protein expression and post-translational modification (phosphorylation and ubiquitination) after induction of DNA damage. The linear range was over 3 orders of magnitude, the median inter-assay variability was 10% CV and the vast majority (∼85%) of assays were stable after extended storage. The multiplexed assay was applied in proof-of-principle studies to quantify signaling after exposure to genotoxic stress (ionizing radiation and 4-nitroquinoline 1-oxide) in immortalized cell lines and primary human cells. The effects of genomic variants and pharmacologic kinase inhibition (ATM/ATR) were profiled using the assay. This study demonstrates the utility of a quantitative multiplexed assay for studying cellular signaling dynamics, and the potential application to studies on inter-individual variation in the radiation response.

Castanospermine, an inhibitor of a-glucosidase activity, was injected into rats to determine its effects in vivo. Daily injections of alkaloid, at levels of 0.5 mg/g of body weight, or higher, for 3 days decreased hepatic a-glucosidase to 40% of control values, whereas a-glucosidase in brain was reduced to 25% of control values and that in spleen and kidney was reduced to about 40%. In liver, both the neutral (pH 6.5) and the acidic (pH 4.5) a-glucosidase activities were inhibited, but the former was more susceptible. On the other hand, 13-Nacetylhexosaminidase activity was elevated in the livers of treated animals, whereas f8-galactosidase activity was unchanged and a-mannosidase activity was somewhat inhibited. Livers of treated animals were examined by light and electron microscopy and compared to control animals to determine whether changes in morphology had occurred. In treated animals fed normal rat chow, the hepatocytes were smaller in size and simplified in structure, whereas the high-glucose diet lessened these alterations. Furthermore, in those animals receiving castanospermine at 1.0 mg or higher per g of body weight for 3 days, there was a marked decrease in the amount of glycogen in the cytoplasm, while a large number of lysosomes were observed that were full of dense, granular material. That this dense material was indeed glycogen was shown by the fact that it disappeared when blocks of fixed tissue were pretreated with a-amylase. Glycogen levels in liver, as measured either colorimetrically or enzymatically, were somewhat depressed at the higher levels of castanospermine.Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) is an indolizine alkaloid that is found in the seeds of the Australian tree Castanospermum australe (1). These seeds have been reported to be toxic to animals and to cause various symptoms, including gastrointestinal upset. We recently found that this alkaloid was a potent inhibitor of lysosomal a-and ,3-glucosidase but had no effect on a number of other glycosidases (2). In addition, castanospermine also inhibits the neutral glucosidase(s) that participates in the processing of the oligosaccharide chains of the N-linked glycoproteins (3). Thus, in cultured mammalian cells, castanospermine prevents the formation of complex chains and gives rise to glycoproteins having mostly Glc3Man7_9GlcNAc2 structures.Since glucosidases are very important enzymes in a number of metabolic sequences, including glycogen degradation, we were interested in determining whether this alkaloid would affect glycogen metabolism if injected into animals. Based on preliminary experiments, we postulated that castanospermine would inhibit the lysosomal a-glucosidase and produce a lysosomal block leading to the abnormal storage of glycogen. The data presented in this paper show that this alkaloid markedly depressed the activity levels of a-glucosidase in liver and other tissues. Coupled with this decreased activity was the finding that the livers of rats given this alkaloid for several days had...