Human lysosomal alpha-mannosidases exhibit different inhibition and metal binding properties.

Two structurally-related members of the lysosomal mannosidase family, the broad substrate specificity enzyme human lysosomal alpha-mannosidase (hLM, MAN2B1) and the human core alpha-1, 6-specific mannosidase (hEpman, MAN2B2) act in a complementary fashion on different glycosidic linkages, to effect glycan degradation in the lysosome. We have successfully expressed these enzymes in Drosophila S2 cells and functionally characterized them. hLM and hEpman were significantly inhibited by the class II alpha-mannosidase inhibitors, swainsonine and mannostatin A. We show that three pyrrolidine-based compounds designed for selective inhibition of Golgi alpha-mannosidase II (GMII) exhibited varying degrees of inhibition for hLM and hEpman. While these compounds inhibited hLM and GMII similarly, they inhibited hEpman to a lesser extent. Further, the two lysosomal alpha-mannosidases also show differential metal dependency properties. This has led us to propose a secondary metal binding site in hEpman. These results set the stage for the development of selective inhibitors to members of the GH38 family, and, henceforth, the further investigation of their physiological roles.

Clinical and analytical studies of sheep dosed with various preparations of Astragalus lusitanicus.

Dosing different preparations and extracts of Astragalus lusitanicus to lambs showed the fresh plant or its dry powder were highly toxic while the ethyl acetate or methanol extract did not cause toxicosis, suggesting the toxic principle is an extremely water soluble compound. The animals alternated excitement and depression, with cardiac and respiratory disorders terminally. Alpha-mannosidase inhibition was not detected in blood of dosed lambs, but an inhibitory activity was in tissues from lambs given the fresh plant or its powder. There was increased aspartate aminotransferase and creatine kinase activity, suggesting skeletal muscle and neurological effects. Thin-layer chromatography and the alpha-mannosidase inhibition assay did not detect swainsonine in ethyl acetate, methanol or water: methanol plant extracts.

Analysis of swainsonine and its early metabolic precursors in cultures of Metarhizium anisopliae.

The alpha-mannosidase inhibitor swainsonine is produced by the filamentous fungus Metarhizium anisopliae. The primary metabolite pathway from which it is derived is known to be that leading to lysine. In order to effect improvements in the yield of swainsonine it is of interest to study the changes in the intracellular levels of lysine and its biosynthetic intermediates, as well as swainsonine itself, which accompany changes in culture conditions or in the genetics of the microbe. Czapek-Dox defined medium has been used for these studies. A reversed-phase, high performance liquid chromatography procedure was developed for the analysis of lysine, saccharopine, alpha-aminoadipic acid and pipecolic acid in mycelial extracts. The method is based upon precolumn derivatization with 9-fluorenylmethyl chloroformate (FMOC), a reagent known to be useful for the derivatization of amino-containing compounds. Elution with an acetate buffer/acetonitrile gradient effected separation of the four metabolites which were quantified by UV absorption at concentrations from 1 to 20 microg ml(-1). Swainsonine concentrations were determined using a previously described enzyme-based method, but applied now to intracellular as well as extracellular samples. Analysis of mycelial extracts from the end of swainsonine accumulation in medium supplemented with L-lysine revealed the accumulation of pipecolic acid and to a lesser extent lysine compared to control mycelium. Controlling the culture medium pH to 9.0 resulted in a drop in swainsonine yield accompanied by an increase in intracellular pipecolic acid levels. Spontaneous mutants tolerant to the presence of the toxic lysine analogue 2-aminoethylcysteine (AEC) were isolated in an attempt to generate lysine over-producers, which might be expected to produce more swainsonine. Surprisingly, four independently isolated mutants produced lower yields of swainsonine, but accumulated higher levels of saccharopine. The tolerance to AEC therefore appears to be due to a reduction in the diversion of saccharopine into swainsonine biosynthesis, allowing the biosynthesis of sufficient lysine to overcome AEC competition.

Effects of graded levels of bentonite on serum clinical profiles, metabolic hormones, and serum swainsonine concentrations in lambs fed locoweed (Oxytropis sericea).

To determine which clay minerals have the potential to bind swainsonine, an in vitro screening procedure was conducted. Twenty compounds were screened in one replicated in vitro trial. A commercially available bentonite bound approximately 10% swainsonine and was chosen for use in a subsequent lamb feeding trial. Twenty fine-wool lambs (30.5 +/- .7 kg) were assigned to one of five treatments (four lambs/treatment). Treatments included 1) positive control, 100% sorghum sudangrass hay, 2) 85% sorghum sudangrass:15% locoweed (Oxytropis sericea, 430 ppm [DM basis] of swainsonine) +0 g of bentonite, 3) Treatment 2 + 14 g of bentonite, 4) Treatment 2 + 28 g of bentonite, and 5) Treatment 2 + 42 g of bentonite. Lambs were fed the experimental diets for 35 d and were then fed the positive control diet for an additional 21 d. Lambs were weighted and blood was collected via jugular venipuncture weekly from d 0 through 56. On d 35, additional blood samples were collected 1, 2, 4, and 8 h after feeding. Weekly blood samples were analyzed for serum clinical chemistry profiles, and additional blood samples collected on d 35 were analyzed for serum metabolic hormones and serum swainsonine concentrations. Within 1 wk, serum alkaline phosphatase and glutamic oxaloacetic transaminase activities increased markedly (P < .05) in lambs fed locoweed. Serum insulin, growth hormone, and prolactin concentrations were not affected by feeding locoweed, but serum triiodothyronine and thyroxine concentrations were decreased by approximately 50% (P < .05) in lambs fed locoweed.(ABSTRACT TRUNCATED AT 250 WORDS)