Monitoring the correction of glycogen storage disease type 1a in a mouse model using [(18)F]FDG and a dedicated animal scanner

Monitoring gene therapy of glycogen storage disease type 1a in a mouse model was achieved using [(18)F]FDG and a dedicated animal scanner. The G6Pase knockout (KO) mice were compared to the same mice after infusion with a recombinant adenovirus containing the murine G6Pase gene (Ad-mG6Pase). Serial images of the same mouse before and after therapy were obtained and compared with wild-type (WT) mice of the same strain to determine the uptake and retention of [(18)F]FDG in the liver. Image data were acquired from heart, blood pool and liver for twenty minutes after injection of [(18)F]FDG. The retention of [(18)F]FDG was lower for the WT mice compared to the KO mice. The mice treated with adenovirus-mediated gene therapy had retention similar to that found in age-matched WT mice. These studies show that FDG can be used to monitor the G6Pase concentration in liver of WT mice as compared to G6Pase KO mice. In these mice, gene therapy returned the liver function to that found in age matched WT controls as measured by the FDG kinetics in the liver compared to that found in age matched wild type controls.     

Cyotomedical therapy for insulinopenic diabetes using microencapsulated pancreatic beta cell lines

Current therapy for type 1 diabetes mellitus involves a daily regimen of multiple subcutaneous or intramuscular injections of recombinant human insulin. To achieve long-term insulin delivery in vivo, we investigated the applicability of cytomedical therapy using beta TC6 cells or MIN6 cells, both of which are murine pancreatic beta cell lines that secrete insulin in a subphysiologically or physiologically regulated manner, respectively. We examined this therapy in the insulinopenic diabetic mice intraperitoneally injected with beta TC6 cells or MIN6 cells microencapsulated within alginate-poly(L)lysine-alginate membranes (APA-beta TC6 cells or APA-MIN6 cells). The diabetic mice treated with APA-beta TC6 cells fell into hypoglycemia, whereas those injected with APA-MIN6 cells maintained normal blood glucose concentrations for over 2 months without developing hypoglycemia. In addition, we also conducted an oral glucose tolerance test using these mice. The blood glucose concentrations of normal and of diabetic mice injected with APA-MIN6 cells similarly changed over time, although the blood insulin concentration increased later in the injected diabetic mice than in the former. These results suggest that cytomedicine utilizing microencapsulated pancreatic beta cell lines with a physiological glucose sensor may be a beneficial and safe therapy with which to treat diabetes mellitus.     

Aphidicolin induces 6-thioguanine resistant mutants in human diploid fibroblasts

Cytotoxic and mutagenic effects of aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta, were studied in human diploid VH-10 fibroblasts. The cells were treated (2 or 4h) with APC at concentration ranges of 10-40 microM. The effect of APC on cell survival after 4 h treatment was significantly higher than after 2 h treatment. The mutagenicity of APC was investigated at the HPRT locus, and the frequency of HPRT mutants was estimated by selection in medium containing 6-thioguanine (6-TG). Treatment of fibroblast cells with 20 microM of APC for 2 or 4 h resulted approximately in 5 or 10 times increase of 6-TG resistant mutant frequencies, respectively, compared to untreated control cells.The cell cycle analyses performed during the expression time (9-12 days) have shown that after 2 and 4h treatment with APC the cells were blocked in G2 phase during the majority of the expression period, compared to control cells. Four days after the treatment, the amount of cells in G2 phase increased about two-fold (28.6-31.8% compared to 13.5% in the untreated cells). The mode of cell death during the expression time was via necrosis, rather than apoptosis, which was demonstrated by fluorescein-diacetate (FDA)-staining and terminal dUTP nick end labeling (TUNEL)-method.     

Acylated flavonol diglucosides from Lotus polyphyllos

Three acylated flavonol diglucosides, kaempferol 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; quercetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside; isorhamnetin 3-O-β-(6″-O-E-p-coumaroylglucoside)-7-O-β-glucoside were isolated from the whole plant aqueous alcohol extract of Lotus polyphyllos. The known 3,7-di-O-glucosides of the aglycones kaempferol, quercetin and isorhamnetin were also characterized. All structures were established on the basis of chemical and spectral evidence.     

Isoflavone glycosides from Derris scandens

Five isoflavone glycosides, named derriscandenosides A–E (1–5), were isolated from the stems of Derris scandens, together with ten known compounds comprising one isoflavone, two benzoic acid derivatives, three glucosyl isoflavones and four rhamnosyl-(1→6)-glucosyl isoflavones. The structures of the glycosides were assigned on the basis of spectroscopic data, especially of the acetate derivatives. Three known rhamnosyl-(1→6)-glucosyl isoflavones isolated from a crude fraction were retested for hypotensive activity with varying results.     

Procathepsin D interacts with prosaposin in cancer cells but its internalization is not mediated by LDL receptor-related protein

The cell surface binding, endocytosis, and lysosomal routing of procathepsin D (procath-D) in cancer cells are mostly independent of the mannose-6-phosphate (M6P) receptors. In an attempt to define the receptor involved, we intracellularly cross-linked procath-D with a 68-kDa protein that we identified with specific antibodies as prosaposin in human breast and ovarian cancer cell lines. In cancer cells, this protein-protein interaction was resistant to ammonium chloride or M6P treatment, indicating that it was independent of the M6P receptors. A similar interaction also occurred in the breast cancer cell culture medium between the secreted prosaposin and procath-D. Since these two precursors can be endocytosed, we then determined whether they were interacting with the same cell surface receptor. In fibroblasts, we confirmed that the endocytosis of these two proteins was different since it was generally mediated by the M6P receptors for procath-D and mostly by LRP (LDL receptor-related protein) for prosaposin. In breast cancer cells, prosaposin endocytosis was not detected, in contrast to procath-D endocytosis, suggesting that the majority of procath-D is not internalized as a complex with prosaposin. Moreover, RAP (receptor-associated protein), a ligand inhibiting LRP-mediated endocytosis, prevented internalization of prosaposin in 49-F rat fibroblasts, but did not affect procath-D M6P-independent internalization in MDA-MB231 cells. We conclude that in breast cancer cells, even though procath-D interacts intracellularly and extracellarly with prosaposin, it is endocytosed independent of prosaposin by a receptor different from the M6P receptors and the LRP.     

Constituents of Lepidium meyenii 'maca'.

The tubers of Lepidium meyenii contain the benzylated derivative of 1,2-dihydro-N-hydroxypyridine, named macaridine, together with the benzylated alkamides (macamides), N-benzyl-5-oxo-6E,8E-octadecadienamide and N-benzylhexadecanamide, as well as the acyclic keto acid, 5-oxo-6E,8E-octadecadienoic acid. The structure elucidation of the isolated compounds was based primarily on 1D and 2D NMR spectroscopic analyses, including 1H-1H COSY, 1H-13C HMQC, 1H-13C HMBC and 1H-1H NOESY experiments, as well as from 1H-15N NMR HMBC correlations for macaridine and N-benzylhexadecanamide.     

Sesquiterpenoids of Torilis japonica fruit

From the methanolic extract of Torilis japonica D. C. fruit (Umbelliferae), two eudesmane-type sesquiterpenoids were isolated together with five previously described sesquiterpenoids. From the results of spectral analyses, they were characterized as 4(15)-eudesmene-1beta,5alpha-diol and 4alpha,15-epoxyeudesmane-1beta,6alpha-diol, respectively. The absolute stereostructures of these sesquiterpenoids were elucidated by the modified Mosher's method.     

Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae,Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4omega6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3omega6, 20:3omega6 and AA, decreased, indicating an inhibition of the Delta12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 degrees C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 degrees C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.