Thermostable amylolytic enzymes from a cellulolytic fungusMyceliophthora thermophila D14 (ATCC 48 104)

Summary The production of amylolytic enzymes by a thermophilic cellulolytic fungus,Myceliophthora thermophila D14 was investigated by batch cultivation in Czapek-Dox medium at 45° C. Among various nitrogenous compounds used, NaNO₃ and KNO₃ were found to be the best for amylase production. Starch, cellobiose and maltose induced the synthesis of amylase while glucose, fructose, galactose, lactose, arabinose, xylose, sorbitol, mesoinositol and sucrose did not. Calcium ions had the most stimulating effect on enzyme formation amongst many ions investigated. The synthesis of amylolytic enzymes was dependent on growth and occurred predominantly in the mid-stationary phase. The enzyme was active in a broad temperature range (50° C–60° C) and displayed activity optima at 60° C and pH 5.6.     

Chromatographic separation of DNA dependent RNA polymerases and molecular properties of RNA polymerase II from a Leishmania Spp

Multiple forms of DNA-dependent RNA polymerases have been isolated and characterized from Leishmania strain UR6 promastigotes. RNA polymerases from this organism fail to resolve into multiple forms by conventional chromatography on DEAE-Sephadex A25, but could be separated by a modification of the method using CM-Sephadex C25. The CM-Sephadex bound enzyme is resistant toamanitin even up to a concentration of 250g/ml. The activity which flows through CM-Sephadex further resolves into two forms upon chromatography on DEAE-Sephadex A25. These forms are sensitive to -amanitin to different extent. Enzyme activity in peak I is 50% inhibited by 3g/ml and in peak II by 50g/ml of the drug respectively. The enzyme in peak I has been further purified by heparin agarose and fast performance liquid chromatography (FPLC) on MonoQ. The enzyme has Stoke's radius of 70å, a sedimentation coefficient of 17.6S and an f/fo of 1.35. Analysis of ammonium sulfate and met n peak I, relative activities with Mn+2 versus Mg+2 and template specificities gave results similar to those reported for other type II RNA polymerases in eukaryotes. The MonoQ purified enzyme resolves into 16 polypeptides on denaturing polyacrylamide gel and densitometric analysis suggests that 9 major bands are present in the stoichiometry expected of RNA polymerase subunits having molecular weights: 154000; 104000; 77000; 64000; 52000; 48000; 46000; 45000 and 39000 respectively.     

Technical communication: A simple and economically viable medium for the growth of Agrobacterium radiobacter for the production of d-amino acid

A simple and cost effective medium for the growth and d-amino acid production by Agrobacterium radiobacter from hydantoin is reported. The effectiveness of this medium is compared with other synthetic media.     

Synthesis and NMR Assignment of the Two Diastereomers of 8,6′-Cyclo-2′,6′-Dideoxyadenosine

We herein present the first synthesis and characterization of the two C5′ diastereomers of 8,6′-cyclo-2′,6′-dideoxyadenosine. Starting from commercially available 2′-deoxyadenosine, the target cyclonucleosides were synthesized in 11 linear steps. Following a zinc-mediated cyclization reaction to form the seven-membered ring, the stereochemistry of the newly formed chiral center was established using two-dimensional NOESY NMR experiments.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids for the free supplemental resource.]     

Multidrug Resistance-associated Protein-1 (MRP-1)-dependent Glutathione Disulfide (GSSG) Efflux as a Critical Survival Factor for Oxidant-enriched Tumorigenic Endothelial Cells

Endothelial cell tumors are the most common soft tissue tumors in infants. Tumor-forming endothelial (EOMA) cells are able to escape cell death fate despite excessive nuclear oxidant burden. Our previous work recognized perinuclear Nox-4 as a key contributor to EOMA growth. The objective of this work was to characterize the mechanisms by which EOMA cells evade oxidant toxicity and thrive. In EOMA cells, compared with in the cytosol, the nuclear GSSG/GSH ratio was 5-fold higher. Compared to the ratio observed in healthy murine aortic endothelial (MAE) cells, GSSG/GSH was over twice as high in EOMA cells. Multidrug resistance-associated protein-1 (MRP-1), an active GSSG efflux mechanism, showed 2-fold increased activity in EOMA compared with MAE cells. Hyperactive YB-1 and Ape/Ref-1 were responsible for high MRP-1 expression in EOMA. Proximity ligand assay demonstrated MRP-1 and YB-1 binding. Such binding enabled the nuclear targeting of MRP-1 in EOMA in a leptomycin-B-sensitive manner. MRP-1 inhibition as well as knockdown trapped nuclear GSSG, causing cell death of EOMA. Disulfide loading of cells by inhibition of GSSG reductase (bischoloronitrosourea) or thioredoxin reductase (auranofin) was effective in causing EOMA death as well. In sum, EOMA cells survive a heavy oxidant burden by rapid efflux of GSSG, which is lethal if trapped within the cell. A hyperactive MRP-1 system for GSSG efflux acts as a critical survival factor for these cells, making it a potential target for EOMA therapeutics.     

Induction and regulation of alpha-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14 (ATCC 48104).

The alpha-amylase enzyme synthesis was higher when M. thermophila D-14 (ATCC 48104) was grown in culture medium incorporated with starch or other carbohydrates containing maltose units. Maximum enzyme production was attained with 1% starch followed by a gradual decrease with increasing concentration. Marked decrease in alpha-amylase synthesis occurred with the addition of glucose to the culture medium and this decreasing activity was proportional to the concentration of glucose. The enzyme synthesis was resumed as soon as the glucose concentration fell below a critical level. The addition of cAMP did not eliminate the repressive activity of glucose. The findings suggest that extracellular alpha-amylase synthesis in M. thermophila D-14 was inducible and subject to catabolite repression.