Nantenine as an acetylcholinesterase inhibitor: SAR, enzyme kinetics and molecular modeling investigations.

Nantenine, as well as a number of flexible analogs, were evaluated for acetylcholinesterase (AChE) inhibitory activity in microplate spectrophotometric assays based on Ellman's method. It was found that the rigid aporphine core of nantenine is an important structural requirement for its anticholinesterase activity. Nantenine showed mixed inhibition kinetics in enzyme assays. Molecular docking experiments suggest that nantenine binds preferentially to the catalytic site of AChE but is also capable of interacting with the peripheral anionic site (PAS) of the enzyme, thus accounting for its mixed inhibition profile. The aporphine core of nantenine may thus be a useful template for the design of novel PAS or dual-site AChE inhibitors. Inhibiting the PAS is desirable for prevention of aggregation of the amyloid peptide Aß, a major causative factor in the progression of Alzheimer's disease (AD).

Potency of siRNA versus shRNA mediated knockdown in vivo.

The intracellular delivery of small interfering RNA (siRNA) is a therapeutic strategy to transiently block gene expression. Two silencing RNA strategies utilize either synthetic double stranded RNA or plasmid DNA encoding a short hairpin RNA (shRNA). In the present study, we have quantitatively compared the potency of siRNA (siLuc1) and shRNA (pShagLuc) mediated knockdown of luciferase expression in vivo using hydrodynamic dosing and bioluminescence imaging (BLI). Following hydrodynamic coadministration of siLuc1 or pShagLuc with a plasmid encoding luciferase (pGL3), mice were analyzed for transgene expression by BLI. The knockdown of luciferase expression by siLuc1 or pShagLuc was observed at 3 h and persisted for 3 days. The potency of siLuc1 and pShagLuc was equivalent with maximal effect at 10 microg coadministered with 1 microg of pGL3 resulting in >80% knockdown. Combined dosing of siLuc1 and pShagluc (5 microg each) with 1 microg of pGL3 resulted in >99% knockdown. Analysis of the data established that shRNA was significantly more potent than siRNA at mediating knockdown when compared on a mole basis. The combination of hydrodynamic dosing and BLI to measure siRNA or shRNA mediated knockdown of luciferase provide an attractive in vivo quantitative method to test formulations that target the liver.

Synthetic PEGylated glycoproteins and their utility in gene delivery.

PEGylated glycoproteins (PGPs) were synthesized by copolymerizing a Cys-terminated PEG-peptide, glycopeptide, and melittin peptide. Compositionally unique PGPs were prepared by varying the ratio of PEG-peptide (20-90%) and melittin (0-70%) with a constant amount of glycopeptide (10%). The PGPs were purified by RP-HPLC, and characterized for molecular weight and polydispersity by GPC-HPLC and SDS-PAGE and for composition by RP-HPLC following reduction to form monomeric peptides. PGPs formed DNA condensates of 200-300 nm in diameter that were administered to mice via the tail vein. Biodistribution studies confirmed their primary targeting to liver hepatocytes with a DNA metabolic half-life of 1 h. Upon stimulation by hydrodynamic dosing with saline, PGP DNA (5 microg) mediated luciferase expression in the liver detected by bioluminescence imaging (BLI) after 24 h. The level of gene expression mediated by PGP DNA was 5000-fold less than direct hydrodynamic dosing of an equivalent amount of DNA and was independent of the mol percent of melittin incorporated into the polymer, but dependent on the presence of galactose on PGP. The results establish the ability to prepare three-component gene delivery polymers that function in vivo. Further design improvements in fusogenic peptides for gene delivery and for the simultaneous use of a nuclear targeting strategy will be necessary to approach levels of expression mediated by the direct hydrodynamic dosing of DNA.

Quantitative bioluminescence imaging of transgene expression in vivo.

Bioluminescent imaging (BLI) is a widely used in vivo method to determine the location and relative intensity of luciferase expression in mice. Luciferase expression is observed following an i.p. dose of d-luciferin, resulting in bioluminescence that is detected in anesthetized mice by a charge-coupled device camera. To establish whether BLI could be used as a quantitative measurement of non-viral-mediated luciferase expression, precise quantities of plasmid DNA encoding the luciferase gene were hydrodynamically dosed in mice. The results established a linear correlation between the DNA dose and the BLI response measured in liver which spanned five orders of magnitude. The level of luciferase expression was found to be a direct function of d-luciferin dose. The time course of luciferase expression and the influence of multidosing of substrate were measured by BLI. The recovery of luciferase from the liver of hydrodynamically dosed mice allowed calibration of the BLI measurements. The results establish BLI's limit-of-detection at 20 pg of luciferase per liver following a hydrodynamic dose of 100 pg of plasmid DNA. These results demonstrate that BLI is both sensitive and linear and should allow for the direct comparison of the efficiency of gene transfer vectors that target the liver.

Proximate analysis and some antinutritional factor constituents in selected varieties of Jamaican yams (Dioscorea and Rajana spp.).

Two wild (Dioscorea polygonoides and Rajana cordata) and seven cultivated varieties of Jamaican yams (Dioscorea spp.) were analyzed for their proximate composition and the levels of antinutritional factors. The protein level range was 47.8 +/- 2.6 to 88.0 +/- 2.5 g/kg dry weight. The lowest level was seen in D. cayenensis. The range for the dietary fiber content in the tubers was 16.3 +/- 0.7 to 63.5 +/- 0.4 g/kg dry weight. The wild yam varieties recorded higher levels. Saponins level was <600 mg/kg dry weight in all the tubers analyzed except for bitter yam (2962.5 +/- 60.5 mg/kg dry weight). Total phenol content ranged from 1.3 +/- 0.1 to 79.3 +/- 6.1 g/kg while total condensed tannin content ranged from 0.1 +/- 0.0 to 26.7 +/- 3.8 g/kg dry weight. Samples that showed high levels of phenols also had high levels of condensed tannins. All the samples analyzed contained low levels of lectins and no alkaloids were detected. The levels of antinutritional factors did not clearly delineate the wild varieties from the edible varieties.

Hypoglycemic effects of steroidal sapogenins isolated from Jamaican bitter yam, Dioscorea polygonoides.

In this study, three steroidal sapogenins (Delta3 diosgenin, diosgenin, and pennogenin) and the phytosterols, stigmasterol and beta-sitosterol were isolated from Jamaican bitter yam, Dioscorea polygonoides. Their effects on fasting blood glucose and intestinal amylase and ATPases in streptozotocin-induced diabetic rats were studied. The diabetic rats (fed supplemented and unsupplemented diets) lost weight significantly compared to the normal group. There was a significant increase in the activity of alpha-amylase in the proximal region of the small intestinal mucosa of diabetic rats fed sapogenin extract or commercial diosgenin. However, this did not result in increased fasting blood glucose. Instead, supplementation of the diet with bitter yam sapogenin extract significantly decreased fasting blood glucose compared to the diabetic group. Supplementation of the diet with bitter yam sapogenin extract or commercial diosgenin significantly reduced Na+-K+-ATPase activity in all three regions compared to the diabetic control group. Commercial diosgenin supplementation resulted in a significant increase in Ca2+ ATPase activity in proximal region compared to the diabetic control and bitter yam sapogenin extract groups. The effect of bitter yam sapogenin extract or commercial diosgenin on intestinal Na+-K+-ATPase activity could account for their hypoglycemic properties. However, there was adverse effect on the body weight.