Protein Degradation and Quality Control in Cells from Laforin and Malin Knockout Mice

Lafora disease is a progressive myoclonus epilepsy caused by mutations in the EPM2A or EPM2B genes that encode a glycogen phosphatase, laforin, and an E3 ubiquitin ligase, malin, respectively. Lafora disease is characterized by accumulation of insoluble, poorly branched, hyperphosphorylated glycogen in brain, muscle, heart, and liver. The laforin-malin complex has been proposed to play a role in the regulation of glycogen metabolism and protein quality control. We evaluated three arms of the protein degradation/quality control process (the autophago-lysosomal pathway, the ubiquitin-proteasomal pathway, and the endoplasmic reticulum (ER) stress response) in mouse embryonic fibroblasts from Epm2a^−/−, Epm2b^−/−, and Epm2a^−/− Epm2b^−/− mice. The levels of LC3-II, a marker of autophagy, were decreased in all knock-out cells as compared with wild type even though they still showed a slight response to starvation and rapamycin. Furthermore, ribosomal protein S6 kinase and S6 phosphorylation were increased. Under basal conditions there was no effect on the levels of ubiquitinated proteins in the knock-out cells, but ubiquitinated protein degradation was decreased during starvation or stress. Lack of malin (Epm2b^−/− and Epm2a^−/− Epm2b^−/− cells) but not laforin (Epm2a^−/− cells) decreased LAMP1, a lysosomal marker. CHOP expression was similar in wild type and knock-out cells under basal conditions or with ER stress-inducing agents. In conclusion, both laforin and malin knock-out cells display mTOR-dependent autophagy defects and reduced proteasomal activity but no defects in the ER stress response. We speculate that these defects may be secondary to glycogen overaccumulation. This study also suggests a malin function independent of laforin, possibly in lysosomal biogenesis and/or lysosomal glycogen disposal.     

Enhancement of taxol production from endophytic fungus Fusarium redolens

The optimization of taxol production by Fusarium redolens by one factor at a time (OFAT) approach led to production of 70 μg/L of taxol. With sucrose and NH₄NO₃ as the carbon and nitrogen sources and medium volume (V _m ) to flask volume (V _f ) ratio of 0.2, a greater taxol production was attained. NH₄NO₃, MgSO₄?7H₂O and NaOAc at 6.25, 0.63, and 1.25 g/L, were the significant factors for attaining the highest taxol production. The optimization of culture variables led to the production of taxol from 66 to 198 μg/L, which is three fold higher than that in the unoptimized medium. Current study results suggested the success of Response Surface Methodology in enhancing the production of fungal taxol.     

Diversity and antimitotic activity of taxol-producing endophytic fungi isolated from Himalayan yew

Endophytic fungi represent an under explored resource of novel lead compounds and have the capacity to produce diverse classes of plant secondary metabolites. Here, we investigated the endophytic fungal diversity of taxol-producing endophytes from Taxus baccata L. ssp. wallichiana (Zucc.) Pilger and also tested the antimitogenic effect of fungal taxol using potato disc tumor assay. A total of 60 fungal endophytes were isolated from the inner bark (phloem-cambium) of T. baccata ssp. wallichiana, collected from different locations of the northern Himalayan region. Two key genes, DBAT (10-deacetylbaccatin III-10-O-acetyl transferase) and BAPT (C-13 phenylpropanoid side chain-CoA acyltransferase), involved in taxol biosynthesis were used as molecular markers for the screening of taxol-producing strains. Five representative species gave positive amplification hits by molecular marker screening with the bapt gene. These fungi were characterized and identified based on morphological and molecular identification. The taxol-producing capability of these endophytic fungi was validated by HPLC-MS. Among the five taxol-producing fungi, the highest yield of taxol was found to be 66.25 μg/l by Fusarium redolens compared with those of the other four strains.