A toxicological, metabonomic and transcriptional analysis to investigate the property of mulberry 1-deoxynojirimycin against the growth of Samia cynthia ricini.

1-Deoxynojirimycin (DNJ) is a natural d-glucose analogue from mulberry with promising physiological activity in vivo. Up to the present, the antidiabetic effects of DNJ on lowering blood sugar and accelerating lipid metabolism in mammals were broadly reported, but the specific character of DNJ against insects was vastly ignored. In this study, a toxicological test of DNJ againgst eri-silkworm, Samia cynthia ricini was carried out to investigate the potential of DNJ in insect management. Further, a method of nuclear magnetic resonance (NMR) metabonomics and real-time qPCR (RT-qPCR) were performed to analyze the alteration in midgut of eri-silkworm caused by DNJ. The result of toxicology showed that 5% and 10% DNJ could significantly inhibit the development of third-instar larvae on day 1-5, and mass deaths happened in DNJ groups on day 3-5. The quantitative analysis of 1H NMR in fifth-instar larvae showed that trehalose level increased in midgut of 0, 6 and 12 h DNJ groups, while the concentrations of glucose, lactate, alanine, pyruvate, α-ketoglutarate and fumarate were reduced in varying degrees. Meanwhile, principal component analysis (PCA) indicated that there were significant differences in the metabolic profiles among 12 h DNJ groups and the control group. In addition, RT-qPCR results displayed that four genes coding α-glucosidase, trehalase (THL) and lactate dehydrogenase (LDH) were lowered in expression of 12 h DNJ groups. Simultaneously, THL activity was significantly lowerd in 12 h DNJ groups. These mutually corroborated results indicated that the backbone pathways of energy metabolism, including hydrolysis of trehalose and glycogens, glycolysis and tricarboxylic acid (TCA) cycle were significantly inhibited by DNJ. Thus, the specific mechanism of DNJ efficiently suppressing the growth and energy metabolism of eri-silkworm was explored in this study, providing the potential of DNJ as to the production of botanical insecticide.

Amplification of the inhibitory activity of miglitol by monofluorination.

Selective monofluorination of the alpha-glycosidase inhibitor and antidiabetic agent Miglitol at position C2 creates an competitive inhibitor of five times higher activity than the parent compound. Its screening against a panel of human cell lines showed a low cytotoxicity therefore making this compound an interesting candidate for further clinical investigations.

Beta-fructofuranosidase genes of the silkworm, Bombyx mori: insights into enzymatic adaptation of B. mori to toxic alkaloids in mulberry latex.

Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. D-AB1 and DNJ are strong inhibitors of alpha-glucosidases (EC 3.2.1.20); however, they do not affect the activity of beta-fructofuranosidases (EC 3.2.1.26). Although alpha-glucosidase genes are found in a wide range of organisms, beta-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of beta-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mori. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional beta-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or D-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the beta-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry's defense system.

Membrane disruption and cytotoxicity of hydrophobic N-alkylated imino sugars is independent of the inhibition of protein and lipid glycosylation.

The N-alkyl moiety of N-alkylated imino sugars is crucial for therapeutic activities of these compounds as inhibitors of glycosphingolipid (GSL) biosynthesis and as antivirals. The improved potency afforded by a long N-alkyl moiety is coincident with increased compound-induced cytotoxicity. Therefore, in the present study, we examined the mechanism of this cytotoxicity in detail. Despite N-butyl-deoxynojirimycin and N-butyl-deoxygalactonojirimycin inhibiting the glycosylation of ceramide to glucosylceramide, ceramide levels did not increase in HL60 cells treated with these compounds. Long-chain N-alkylated imino sugars were toxic to cells at concentrations considerably lower than the critical micellar concentrations for these compounds and consequently did not solubilize radioactively labelled cellular proteins and lipids. However, membrane disruption and cell fragmentation did increase in a concentration- and chain-length-dependent manner. These results are consistent with previously proposed interactions between surface-active amphiphiles and protein-containing lipid membranes when drug concentrations are below the critical micellar concentration. Taken together, these results demonstrate that the cellular toxicity of hydrophobic N-alkylated imino sugars is due to cell lysis and cell fragmentation and, most importantly, is not related to the beneficial therapeutic effects of these compounds on protein and in lipid glycosylation. This information will aid in the future development of more selective imino sugar therapeutics for the treatment of human disease.

Low-dose N-butyldeoxynojirimycin (OGT 918) for type I Gaucher disease.

The objective of this study was to evaluate the efficacy and safety of low-dose substrate balance therapy with OGT 918 for the treatment of adults with Gaucher disease. Eighteen patients with Gaucher disease from two centers were enrolled in an open-label 6-month study of OGT 918, 50 mg taken three times daily (TID), followed by an optional extended-use phase. Changes in liver and spleen volume at 6 and 12 months, as well as routine hematological and biochemical parameters on a monthly basis, were evaluated. During the extension, dosage was increased to 100 mg TID in patients in one center to improve the response. Seventeen patients completed 6 months; of 16 patients in the extension phase, 13 were evaluable at 12 months. Percentage changes in liver (-5.9%, P = 0.007) and spleen (-4.5%, P = 0.025) volumes and in chitotriosidase levels (-4.6%, P = 0.039) at 6 months were commensurately lower than those reported previously in an open-label trial using 100 mg TID; hemoglobin and platelet counts were not boosted. At 12 months there were further mean decreases from baseline in liver volume (-6.2%, P = 0.037), spleen volume (-10.1%, P < 0.05), and chitotriosidase levels (-15.3%, P < 0.05) as well as mean changes of +1.2 and +14.7% in hemoglobin and platelet concentrations, respectively [correction]. There were no serious adverse effects throughout the 6-month study period; common side effects were diarrhea (94%) and weight loss (67%), comparable to the incidence in the original trial. We conclude that OGT 918 was safe and effective at 50 mg TID, but shows dose dependency in ameliorating parameters of Gaucher disease relative to the results noted in the seminal trial; there was no improvement in the rate of hematological response and no reduction in side effects. Results from the extension wherein some patients were dose increased suggest that 100 mg TID should be the preferred starting regimen for patients with symptomatic type I Gaucher disease.

Imino sugars that are less toxic but more potent as antivirals, in vitro, compared with N-n-nonyl DNJ.

Imino sugar glucosidase inhibitors have selective antiviral activity against certain enveloped, mammalian viruses. Deoxynojirimycins (DNJs) modified by N-alkylation to contain a nine carbon atom side chain (N-n-nonyl-deoxynojirimycin; N-nonyl-DNJ, NN-DNJ) were shown to be, for example, at least 20 times more potent in inhibiting hepatitis B virus (HBV) and bovine viral diarrhoea virus (BVDV) in cell based assays than the non-alkylated DNJ. These data suggested that modification of the alkyl side chain could influence antiviral activity. Previous work has focused on varying side chain length. In this report, the influence of side chain branching and cyclization upon toxicity and antiviral activity was explored. Briefly, using a virus secretion assay for HBV and a single step growth (yield reduction) assay for BVDV, 14 different DNJ-based sugars, possessing various N-alkyl substitutions, were tested for antiviral activity. Of the series, N-methoxy-nonyl-DNJ and N-butyl-cyclohexyl DNJ were determined to have the best selectivity index against BVDV and HBV, with the N-methoxy analogue being the most potent with micromolar antiviral activity. The results of this antiviral survey and the implications for the mechanism of action and ultimate therapeutic potential of the DNJ-based imino sugars is provided and discussed.