1-deoxynojirimycin Research Articles

Hypoglycemic effect of deoxynojirimycin–polysaccharide on high fat diet and streptozotocin-induced diabetic mice via regulation of hepatic glucose metabolism

Type 2 diabetes mellitus (T2DM) is currently considered a worldwide epidemic and finding effective therapeutic strategies against this disease is highly important. A deoxynojirimycin–polysaccharide mixture (DPM) has previously been shown to exert hypoglycemic effects on alloxan- or streptozotocin (STZ)-induced diabetic mice. The purpose of the present study was to evaluate the therapeutic effects and underlying mechanism(s) of DPM on T2DM induced by high fat diet following low-dose STZ treatment in mice. After daily oral treatment of diabetic mice with DPM (150mg/kgb.w.) for 90d, significant decline in blood glucose, pyruvate, triglyceride (TG), aspartate transaminase (AST), alanine transaminase (ALT), creatinine (Cr), lipid peroxide (LPO) and malondialdehyde (MDA) levels as well as evident increases in high density lipoprotein (HDL-c) and hepatic glycogen concentrations were observed. In the first stage, in which DPM was administered for 60d, blood insulin levels did not undergo significant change but a significant decrease in the HOMA-IR index was detected. By contrast, the HOMA-IR index increased significantly in T2MD controls. In the second stage, in which DPM treatment was continued for another 30d, insulin levels significantly increased in DPM-treated mice in comparison with T2DM controls. These results indicate that insulin resistance in the pre-diabetic period and the dysfunction of pancreatic β-cells are ameliorated by DPM treatment. DPM also down-regulated protein levels of insulin receptor (IR) and gluconeogenic enzymes (pyruvate carboxylase, fructose-1, 6-bisphosphatase, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase) in peripheral tissues (liver and/or muscle), but enhanced the expressions of insulin in pancreas, lipoprotein lipase (LPL) and glycolysis enzymes (glucokinase, phosphofructokinase, private kinase and pyruvate decarboxylase E1) in the liver. Furthermore, deoxynojirimycin (DNJ) and polysaccharide (P) were found to increase proliferation of hepatic LO-2 cells and scavenging of radicals in vitro. These results support the results of our biochemical analyses and underscore possible mechanisms underlying the protective effects of DPM on STZ-induced damage to the pancreas and the liver. Taken together, our findings suggest that DPM may be developed as an antihyperglycemic agent for the treatment of diabetes mellitus.

Inhibition of major virulence pathways of Streptococcus mutans by quercitrin and deoxynojirimycin: a synergistic approach of infection control.

ObjectivesTo evaluate the synergistic effect of Quercitrin and Deoxynojirimycin (DNJ) together with their individual inhibitory effect against virulence pathways of Streptococcus mutans.MethodologyMICs of both the compounds were determined by the microdilution method, followed by their in vitrosynergy using checkerboard and time kill assay. The nature of interaction was classified as synergistic on the basis of fractional inhibitory concentration index (FICI) value of ≤0.5. Furthermore, the activity of Quercitrin and DNJ was evaluated individually and in combination against various cariogenic properties of S. mutans UA159 such as acidogenesis, aciduracity, glucan production, hydrophobicity, biofilm and adherence. Moreover, expression of virulent genes in S. mutans was analysed by quantitative RT- PCR (qRT-PCR) and inhibition of F1F0-ATPase, lactate dehydrogenase and enolase was also evaluated. Finally, scanning electron microscopy (SEM) was used to investigate structural obliteration of biofilm.ResultsThe in vitro synergism between Quercitrin and DNJ was observed, with a FICI of 0.313. Their MIC values were found to be 64 μg/ml and 16 μg/ml respectively. The synergistic combination consistently showed best activity against all the virulence factors as compared to Quercitrin and DNJ individually. A reduction in glucan synthesis and biofilm formation was observed at different phases of growth. The qRT-PCR revealed significant downregulation of various virulent genes. Electron micrographs depicted the obliteration of biofilm as compared to control and the activity of cariogenic enzymes was also inhibited.ConclusionsThe whole study reflects a prospective role of Quercitrin and DNJ in combination as a potent anticariogenic agent against S. mutans.

Novel imino sugar α-glucosidase inhibitors as antiviral compounds

Deoxynojirimycin (DNJ) based imino sugars display antiviral activity in the tissue culture surrogate model of Hepatitis C (HCV), bovine viral diarrhoea virus (BVDV), mediated by inhibition of ER α-glucosidases. Here, the antiviral activities of neoglycoconjugates derived from deoxynojirimycin, and a novel compound derived from deoxygalactonojirimycin, by click chemistry with functionalised adamantanes are presented. Their antiviral potency, in terms of both viral infectivity and virion secretion, with respect to their effect on α-glucosidase inhibition, are reported. The distinct correlation between the ability of long alkyl chain derivatives to inhibit ER α-glucosidases and their anti-viral effect is demonstrated. Increasing alkyl linker length between DNJ and triazole groups increases α-glucosidase inhibition and reduces the production of viral progeny RNA and the maturation of the envelope polypeptide. Disruption to viral glycoprotein processing, with increased glucosylation on BVDV E2 species, is representative of α-glucosidase inhibition, whilst derivatives with longer alkyl linkers also show a further decrease in infectivity of secreted virions, an effect proposed to be distinct from α-glucosidase inhibition.

Enhanced production of α-glucosidase inhibitor by a newly isolated strain of Bacillus subtilis B2 using response surface methodology

The strain Bacillus subtilis B2 isolated from a Chinese traditionally fermented food was capable of producing α-glucosidase inhibitor (AGI). On the basis of single-factor experiments, the optimum culture conditions using okara as the substrate were achieved by response surface methodology (RSM). The experimental results lead to the conclusion that the highest yield of AGI was reached at the fermentation time 65.6h, fermentation temperature 39.0°C, Okara concentration 4.5% and pH 7.11. A second-order model developed for the production of AGI prediction exhibits a F-value of 15.8 and a high value for the coefficient of determination (R2=0.933). Under these conditions, the AGI activity of the broth reached 29.4. In comparison with the highest AGI activity (24.3) obtained at the one-factor-at-a-time fermentation condition, the AGI activity increased by 21%. The AGI in the fermentation broth of B. subtilis B2 was also partially purified and determined by HPLC-ELSD. One of effective AGI, deoxynojirimycin (DNJ) has been proved in the fermentation broth, which indicated that the fermented okara might be considered as one of the promising materials for producing the food-derived DNJ products.

Liposome-Mediated Delivery of Iminosugars Enhances Efficacy against Dengue Virus In Vivo

A key challenge faced by promising antiviral drugs, such as iminosugars, is in vivo delivery to achieve effective levels of drug without toxicity. Four iminosugars, all deoxynojirimycin (DNJ) derivatives-N-butyl DNJ (NB-DNJ), N-nonyl DNJ, N-(9-methoxynonyl) DNJ, and N-(6'-[4″-azido-2″-nitrophenylamino]hexyl)-1-DNJ (NAP-DNJ)-potently inhibited both the percentage of cells infected with dengue virus and release of infectious virus from primary human monocyte-derived macrophages, demonstrating their efficacy in primary cells. In a lethal antibody-dependent enhancement mouse model of dengue pathogenesis, free NB-DNJ significantly enhanced survival and lowered viral load in organs and serum. Liposome-mediated delivery of NB-DNJ, in comparison with free NB-DNJ, resulted in a 3-log(10) reduction in the dose of drug sufficient to enhance animal survival. The optimizing of the effective dose in this way could liberate the therapeutic potential of many cytotoxic antivirals against both dengue virus and a wide array of other viruses.

A novel SGLT is expressed in the human kidney

Selective inhibitors of sodium-glucose cotransporter 2 (SGLT2)-mediated reabsorption of glucose in the proximal tubule of the kidney are being developed for the treatment of diabetes. SGLT2 shares high degree of homology with SGLT3; however, very little is known about the expression and functional role of SGLT3 in the human kidney. Indeed, the SGLT2 inhibitors that are currently in clinical trials might affect the expression and/or the activity of SGLT3. Therefore, the present study examined the expression of SGLT3 mRNA and protein in human kidney and in a human proximal tubule HK-2 cell line. The results indicated that human SGLT3 (hSGLT3) message and protein are expressed both in vivo and in vitro. We also studied the activity of hSGLT3 protein following its over-expression in mammalian kidney-derived COS-7 cells and in HK-2 cells treated with the imino sugar deoxynojirimycin (DNJ), a potent agonist of hSGLT3. Over-expression of hSGLT3 in COS-7 cells increased intracellular sodium concentration by 3-fold without affecting glucose transport. Activation of hSGLT3 with DNJ (50μM) increased sodium uptake in HK-2 cells by 5.5 fold and this effect could be completely blocked with SGLT inhibitor phlorizin (50μM). These results suggest that SGLT3 is expressed in human proximal tubular cells where it serves as a novel sodium transporter. Up-regulation of the expression of SGLT3 in the proximal tubule in diabetic patients may contribute to the elevated sodium transport in this segment of the nephron that has been postulated to promote hyperfiltration and renal injury.

Effect of deoxynojirimycin derivatives on morphogenesis of hepatitis C virus

Viral hepatitis C is a dangerous, widespread human disease. The choice of drugs for treatment of chronic hepatitis C virus (HCV) infection is limited, and prophylactic vaccines do not exist. Thus, the development of new antiviral strategies and substances is an issue of great importance. The targeting of viral morphogenesis might be used as an alternative approach to existing strategies of HCV blocking. The glycosylation of viral envelope proteins is an important step of viral particle morphogenesis, which determines the correct assembly of HCV virions. Derivatives of a glucose analog deoxynojirimycin (DNJ) act as an α-glucosidase inhibitor and can impair the assembly of structural proteins and HCV particle formation. In the present work, the effects of alkylated DNJ derivatives, N-pentyl-DNJ and N-benzyl-DNJ, on HCV morphogenesis were studied in a model system of insect cells that produce three viral structural proteins with the formation of virus-like particles. It was shown that DNJ derivatives impair the intracellular N-glycosylation of HCV envelope glycoproteins. At the concentration of 1 mM, these substances cause an increase in the levels of gpE1 and gpE2 glycoproteins and a decrease in their electrophoretic mobility, apparently due to the inhibition of α-glucosidase in the endoplasmic reticulum and the accumulation of hyperglycosylated N-glycans in HCV glycoproteins. The interaction of the latter with calnexin results in the formation of unproductive dimers and blocks the productive assembly of virus-like particles.

Design, Synthesis, and Biological Evaluation of N-Alkylated Deoxynojirimycin (DNJ) Derivatives for the Treatment of Dengue Virus Infection

We recently described the discovery of oxygenated N-alkyl deoxynojirimycin (DNJ) derivative 7 (CM-10-18) with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising but had an EC(50) against DENV in BHK cells of 6.5 μM, which limited its use in in vivo. Compound 7 presented structural opportunities for activity relationship analysis, which we exploited and report here. These structure-activity relationship studies led to analogues 2h, 2l, 3j, 3l, 3v, and 4b-4c with nanomolar antiviral activity (EC(50) = 0.3-0.5 μM) against DENV infection, while maintaining low cytotoxicity (CC(50) > 500 μM, SI > 1000). In male Sprague-Dawley rats, compound 3l was well tolerated at a dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (F = 92 ± 4%).

Inhibition of glycoprotein synthesis in the endoplasmic reticulum as a novel anticancer mechanism of (−)-epigallocatechin-3-gallate

(-)-Epigallocatechin-3-gallate (EGCG) has been found to trigger the unfolded protein response (UPR) likely due to the inhibition of glucosidase II, a key enzyme of glycoprotein processing and quality control in the endoplasmic reticulum (ER). These findings strongly suggest that EGCG interferes with glycoprotein maturation and sorting in the ER. This hypothesis was tested in SK-Mel28 human melanoma cells by assessing the effect of EGCG and deoxynojirimycin (DNJ) on the synthesis of two endogenous glycoproteins. Both tyrosinase and vascular endothelial growth factor (VEGF) protein levels were remarkably reduced despite unaltered mRNA expression in EGCG- or DNJ-treated cells compared to control. The hindrance of tyrosinase and VEGF protein synthesis could be prevented by proteasome inhibitor, lactacystine. Collectively, our results support that glucosidase II inhibitor EGCG interferes with protein processing and quality control in the ER, which diverts tyrosinase, VEGF, and likely other glycoproteins towards proteasomal degradation. This mechanism provides a novel therapeutic approach in dermatology and might play an important role in the antitumor effect or hepatotoxicity of EGCG.

Apposite Insulin-like Growth Factor (IGF) Receptor Glycosylation Is Critical to the Maintenance of Vascular Smooth Muscle Phenotype in the Presence of Factors Promoting Osteogenic Differentiation and Mineralization

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

Improving Anti-?-Glucosidase Activity of Douchi Koji Using a Newly Isolated Strain of Bacillus subtilis B2

In attempt to develop an effective strategy to obtain douchi with anti-?-glucosidase functionality, Bacillus subtilis B2 was used as the initial microorganism to produce bacterial-type douchi koji capable of inhibiting ?-glucosidase activity in the current study. Various fermentation conditions, such as soybean type, fermentation time and humidity, resulted in different anti-?-glucosidase activity of the douchi koji. Regardless of soybean or black bean, the douchi koji exhibited a drastic increase in anti-?-glucosidase activity during the first 48 h of fermentation and kept a slight increase from 48 h to 96 h. The anti-?-glucosidase activities of both soybean and black bean kojis displaying the lowest at the relative humidity of 70 percent, rose up to the highest at the humidity of 90 percent and then showed a slight decrease. Under the same fermentation conditions, it is found that the soybean douchi koji exhibited higher ?-glucosidase inhibitory activity than that of black bean douchi koji. Maltose, sucrose and glucose were reduced drastically in soybean and black soybean koji after 96 h of fermentation. One of the most effective ?-glucosidase inhibitors, deoxynojirimycin (DNJ), with promising biological activity was observed both in the soybean koji and black soybean koji, which provide an inspiring strategy to produce DNJ-derived foods with low content of saccharides for the diabetic patients.

A derivatization procedure for the simultaneous analysis of iminosugars and other low molecular weight carbohydrates by GC–MS in mulberry (Morus sp.)

Different derivatization procedures were assayed to simultaneously analyse iminosugars such as deoxynojirimycin (DNJ) or fagomine and other carbohydrates of low molecular weight by gas chromatography coupled to mass spectrometry (GC–MS) in Morus sp. Both oximation+trimethylsilylation and oximation+acetylation allowed the separation of target compounds, whereas trimethylsilyl (TMS) and acetylated derivatives showed several coelutions. Nevertheless, oximation+acetylation were discarded for giving inaccurate results for ketoses due to their incomplete derivatization. Different conditions for the conversion into trimethylsilyl oximes (TMSO) were assayed, the best results being achieved using hexamethyldisilazane with trifluoroacetic acid as silylation agent. Contents of iminosugars (DNJ, fagomine and pipecolic acid derivatives) and other carbohydrates such as mono and disaccharides, myo-inositol and galactinol isomers in mulberry extracts (fruits, leaves and branches) were determined by GC–MS using the TMSO procedure.

671 NO EVIDENCE FOR DRUG RESISTANCE BY IMINOSUGAR-BASED HCV ASSEMBLY INHIBITORS IN TISSUE CULTURE

At present, neither a selective antiviral therapy nor a vaccine is available for HCV, and the current treatment is only effective in a fraction of patients. Given the recent demonstration that antiviral compounds targeting the NS5B polymerase and the NS3/4A protease lead to rapid selection for resistance, iminosugar derivatives as HCV assembly inhibitors were characterized for drug resistance. It has been previously shown that deoxynojirimycin (DNJ)-containing iminosugars are potent HCV antivirals inhibiting ER alpha-glucosidases I and II. Long alkyl chain derivatives such asNN-DGJ inhibit the HCV p7 ion channel activityin vitro. The aim of this study was to characterize the drug resistance profile of an inhibitor targeting cellular enzymes (NB-DNJ) and of the p7 inhibitor NN-DGJ in comparison to the NS5B polymerase inhibitor 2’C-methyladenosine (2CMA).

Daya Hambat Hidrolisis Karbohidrat Oleh Ekstrak Daun Murbei

Inhibition hydrolysis of carbohydrate by mulberry leaves extractABSTRACT. Mulberry leaves has a great potential as animal feeds because of its high nutrient content, but has deoxynojirimycin (DNJ) active matter. It is potential to inhibit carbohydrate hydrolysis process, come to monosaccharides. The objective of this experiment is to study the inhibiting ability of mulberry leave extract in carbohydrate hydrolysis process. The kinds of carbohydrates were using glucose, maltose, sucrose and starch. This experiment used twenty four of 60 days old male mice (Mus musculus). Diet and water were given ad libitum. Treatment were allocated ina factorial completely randomized design with three replications and two factors containing of completely mulberry leaves extract and variance of carbohydrates. Variable observed were feed consumtion, feed digestibility, body weight gain and blood glucose. The data were analyzed with univariate analysis of variance. The result showed that inclusion of mulberry leaves extract had decrease body weight (P 0,05) and reduce blood glucose (P 0,05).

Improved cellular inhibitors for glycoprotein processing α-glucosidases: biological characterisation of alkyl- and arylalkyl-N-substituted deoxynojirimycins

A series of N-alkyl- and N-arylalkyl-DNJ compounds have been evaluated for their efficacy for inhibition of endoplasmic reticulum resident α-glucosidases in cells. A recently developed free oligosaccharide (FOS) assay allowed the products of glucosidase inhibition to be quantified and compounds compared for relative inhibitory activity. A N-alkyl chain of one to six carbon atoms provided a flexible linker between deoxynojirimycin (DNJ) and a phenyl, cyclohexyl or cyclopentyl group to explore the requirements for glucosidase inhibition. The most effective compounds were those in which the linker contained four to six carbon atoms and a phenyl group. These compounds all significantly inhibited α-glucosidase I at concentrations of 100 μM following addition to cells for 24 h whereas DNJ was without effect. Inhibition of α-glucosidase II was evident by all inhibitors, consistent with a previously identified mechanism of action of imino sugar inhibitors in cells.

943 NOVEL GLUCOSIDASE INHIBITORS WITH ANTI-HBV AND ANTI-HCV ACTIVITY

Glucosidase inhibitors (Gluc-I) have antiviral properties against hepatitis B and C viruses. We have previously shown that glucosidase inhibitors derived from glucose (i.e., deoxynojirimycin (DNJ) derivatives) could inhibit HBV and HCV morphogenesis in cellulo via the inhibition of ER-glucosidases resulting in the perturbation of the folding and assembly of viral envelope glycoprotein. A pro-drug of castanospermine, another potent Gluc-I, has proven beneficial activity in a phase-II clinical trial on HCV-infected patients in combination with PegIFN/ribavirin. One of the main side effects of Gluc-I results from the inhibition of gastrointestinal (GI) osidases. Therefore the development of Gluc-I with a better selectivity toward ER-glucosidases would be of interest. Here we report the anti-HBV and anti-HCV activity of two alpha-1-C-alkyl-1-deoxynojirimycin derivatives, i.e. butyl and nonyl forms, (CBDNJ and CN-DNJ), which differ from N-butyl-DNJ (NB-DNJ) and N-nonyl-DNJ (NN-DNJ) by the position of the alkyl chain on the ring. These molecules have different selectivity patterns against GI-related osidases compared to previously studied DNJ derivatives. We have used BVDV, HCVpp, and HCVcc models, as well as HepG2.2.15 cells or HBV-transfection systems to determine anti-HCV and anti-HBV activities. We showed that CB-DNJ and CN-DNJ have similar EC50 and CC50 than NB-DNJ and NN-DNJ against BVDV, HBV and HCV, while displaying slightly reduced EC90. We confirmed that the antiviral effect was not due to the inhibition of genome synthesis, but was rather due to the inhibition of morphogenesis, secretion and infectivity of viruses. The reduced EC90 of CB-DNJ and CN-DNJ derivatives over parental molecules was due to a reduced inhibition of ER-glucosidases in cellulo. The research of novel glucosidase inhibitors with improved selectivity towards ER glucosidases should be pursued.

Deoxynojirimycin enhanced the transglycosylation activity of a glycosidase from the China white jade snail

A β- d-glycosidase (G I) from the China white jade snail showed non-Michaelis–Menten mode in catalyzing the reaction using pNPGlu and pNPFuc as the substrate and monitoring the released pNP. We determined quantitatively both the transglycosidic and hydrolytic products of pNPGlu and pNPFuc solvolysis for the detailed kinetic analysis on G I-catalyzed hydrolysis and transglycosylation reaction. The inhibition kinetic studies using deoxynojirimycin (DNJ) and butanol as inhibitors were preceded. DNJ only inhibited competitively the hydrolysis of cellobiose and pNPGlu while “activated” the transglycosylation of pNPGlu and pNPFuc. This was evident from the increased V max tr value with no change of the apparent K m tr. In contrast, butanol exhibited a competitive inhibition to the transglycosylation reaction and non-competitive inhibition to the hydrolysis. The results indicated that the non-Michaelis–Menten kinetic behavior was caused by the co-occurrence of substrate transglycosylation reaction. This study provided a simple method to increase the transglycosylation yield by using DNJ to inhibit hydrolysis.

1-Deoxynojirimycin을 생산하는 Bacillus subtilis S10 배양액의 혈당강하 효과

본 연구는 α-glucosidase의 강력한 효소저해제인 1-deoxynojirimycin(DNJ)을 고효율로 생산하는 균주의 선발 및 동정, 배양최적화 및 균 배양액의 혈당강하효과 등에 관하여 조사하였다. 먼저 토양으로부터 α-glucosidase에 대해 강력한 저해능을 나타내는 9균주를 대상으로 이 중에서 DNJ 생산성이 가장 우수한 한 개 균주를 최종 선발하였고, 이 균주를 16S rDNA 염기서열분석으로 균주동정을 하여 Bacillus subtilis S10이라 명명하였다. 본 균주의 배양액을 이온교환수지법(Amberlyst 15 H? form, Dowex 1×2-100 OH? form, Amberlite CG-50 NH₃? form)에 의해 DNJ를 정제하고, 정제된 DNJ를 LC-MS로 분석한 결과 표준 DNJ와 동일한 물질임을 확인하였다. DNJ 대량생산을 위한 S10균주의 배양최적탄소원 및 최적질소원과 이들의 최적농도를 조사한 결과 각각 1% galactose, 1.6% polypeptone임이 밝혀졌으며, 확립된 최적화 조건에서의 DNJ 생산량은 0.75 g/L이었다. 고혈당 유도 마우스를 대상으로 각종 혈당강하제의 효과를 알아보기 위해 혈당치를 비교한 결과 무처리구의 혈당치 510±10.9 ㎎/dL에 비해 acarbose 처리구는 139.4±33.1 ㎎/dL, 누에분말 처리구는 209.1±19.6 ㎎/dL, 그리고 S10 균주 배양액 처리구는 208.6±39.8 ㎎/dL로써 무처리구에 비해 각각 72.7%, 59.0%, 그리고 59.1%의 혈당 억제율을 나타냈다. 이상의 연구결과 S10균주 배양액은 누에분말과 같은 수준의 고혈당 억제 및 완화효과가 있음이 확인되었으며 향후 S10균주의 대량생산을 통해 식후혈당조절을 위한 건강기능식품 개발이 기대된다 하겠다.

Reduction of the infectivity of hepatitis C virus pseudoparticles by incorporation of misfolded glycoproteins induced by glucosidase inhibitors

Folding and assembly into complexes of some viral glycoproteins are exquisitely sensitive to endoplasmic reticulum (ER) alpha-glucosidase inhibition, which prevents the trimming of glucose from N-linked glycans. Derivatives of deoxynojirimycin (DNJ) iminosugars, which are potent alpha-glucosidase inhibitors, were shown to have antiviral activity against bovine viral diarrhea virus, a pestivirus related to hepatitis C virus (HCV). The aim of this study was to determine whether these inhibitors would affect HCV infectivity and to provide novel insights on their mechanism of action. The overall antiviral activity of glucosidase inhibitors was shown by using the two most relevant models currently available: the cell-culture model enabling complete replication of the HCV JFH1 strain in Huh7.5 cells, and infectious HCV pseudotyped particles (HCVpp) produced in HEK-293T cells that display functional E1-E2 glycoprotein complexes. By using the latter model, it is shown that the inhibition of alpha-glucosidases by iminosugars results in the misfolding and misassembly of HCV glycoprotein pre-budding complexes. This inhibition of the assembly of E1-E2 in the ER of transfected HEK-293T cells leads to a reduction in the incorporation of E1-E2 complexes into HCVpp. More importantly, it is demonstrated that the infectivity of HCVpp that are released under treatment is reduced and that this reduction in infectivity is due to the incorporation of misfolded envelope glycoproteins in secreted particles. These properties suggest the potential usefulness of DNJ derivatives in combating HCV infection.

Pharmacological Enhancement of Mutated α-Glucosidase Activity in Fibroblasts from Patients with Pompe Disease

We investigated the use of pharmacological chaperones for the therapy of Pompe disease, a metabolic myopathy due to mutations of the gene encoding the lysosomal hydrolase α-glucosidase (GAA) and characterized by generalized glycogen storage in cardiac and skeletal muscle. We studied the effects of two imino sugars, deoxynojirimycin (DNJ) and N-butyldeoxynojirimycin (NB-DNJ), on residual GAA activity in fibroblasts from eight patients with different forms of Pompe disease (two classic infantile, two non-classic infantile onset, four late-onset forms), and with different mutations of the GAA gene. We demonstrated a significant increase of GAA activity (1.3-7.5-fold) after imino sugar treatment in fibroblasts from patients carrying the mutations L552P (three patients) and G549R (one patient). GAA enhancement was confirmed in HEK293T cells where the same mutations were overexpressed. No increase of GAA activity was observed for the other mutations. Western blot analysis showed that imino sugars increase the amount of mature GAA molecular forms. Immunofluorescence studies in HEK293T cells overexpressing the L552P mutation showed an improved trafficking of the mutant enzyme to lysosomes after imino sugar treatment. These results provide a rationale for an alternative treatment, other than enzyme replacement, to Pompe disease.