Douglas J. H. Olson

The Molecular Cloning of Artemisinic Aldehyde Δ11(13) Reductase and Its Role in Glandular Trichome-dependent Biosynthesis of Artemisinin in Artemisia annua

At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the Δ11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde Δ11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small α,β-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.

Structural and functional changes in human insulin induced by methylglyoxal

Elevated methylglyoxal (MG) levels have been reported in insulin‐resistance syndrome. The present study investigated whether MG, a highly reactive metabolite of glucose, induced structural and functional changes of insulin. Incubation of human insulin with MG in vitro yielded MG‐insulin adducts, as evidenced by additional peaks observed on mass spectrometric (MS) analysis of the incubates. Tandem MS analysis of insulin B‐chain adducts confirmed attachment of MG at an arginine residue. [3H]‐2‐deoxyglucose uptake by 3T3‐L1 adipocytes was significantly and concentration‐dependently decreased after the treatment with MG‐insulin adducts, in comparison with the effect of native insulin at the same concentrations. A significant decrease of glucose uptake induced by MG‐insulin adducts was also observed in L8 skeletal muscle cells. MG alone had no effect on glucose uptake or the transcriptional expression of insulin receptor. Unlike native insulin, MG‐insulin adducts did not inhibit insulin release from pancreatic β‐cells. The degradation of MG‐insulin through liver cells was also decreased. In conclusion, MG modifies insulin by attaching to internal arginine residue in β‐chain of insulin. The formation of this MG‐insulin adduct decreases insulinmediated glucose uptake, impairs autocrine control of insulin secretion, and decreases insulin clearance. These structural and functional abnormalities of insulin molecule may contribute to the pathogenesis of insulin resistance.—Jia, X., Olson, D. J. H., Ross, A. R. S., Wu, L. Structural and functional changes in human insulin induced by methylglyoxal. FASEB J. 20, E871–E879 (2006)

Differential expression of proteins in the wild type and 7B-1 male-sterile mutant anthers of tomato (Solanum lycopersicum): A proteomic analysis

In the 7B-1 male-sterile mutant of tomato, pollen development breaks down prior to meiosis in microspore mother cells (MMCs). We have used the proteomic approach to identify differentially expressed proteins in the wild type (WT) and mutant anthers with the objective of analyzing their roles in normal pollen development and in male sterility. By using 2-DE and DIGE technologies, over 1800 spots were detected and of these 215 spots showed 1.5-fold or higher volume ratio in either WT or 7B-1 anthers. Seventy spots, either up-regulated in WT, or in 7B-1, were subjected to mass spectrometry and 59 spots representing 48 distinct proteins were identified. The proteins up-regulated in WT anthers included proteases, e.g., subtilase, proteasome subunits, and 5B-protein with potential roles in tapetum degeneration, FtsZ protein, leucine-rich repeat proteins, translational and transcription factors. In 7B-1 anthers, aspartic protease, superoxide dismutase, ACP reductase, ribonucleoprotein and diphosphate kinase were up-regulated. Also, cystatin inhibitory activity was high in the mutant and correlated with the expression of male sterility. Other proteins including calreticulin, Heat shock protein 70, glucoside hydrolase, and ATPase, were present in both genotypes. The function of identified proteins in tapetum and normal pollen development, and in male sterility is discussed.

Modification of Akt1 by methylglyoxal promotes the proliferation of vascular smooth muscle cells

Methylglyoxal (MG), a reactive dicar‐bonyl molecule, can modify protein to form advanced glycation endproducts. Increased MG level has been implicated in proliferative vascular diseases, but the underlying mechanisms are not clear yet. The serine/ threonine kinase, Akt, regulates multiple signaling pathways that control cell proliferation. Using mass spectrometric analysis, we have detected the modification of Akt1 by MG at Cys77. This structural modification increased Akt1 phosphorylation at Ser473 and Thr308. Akt1 phosphorylation and activity were also increased by MG treatment (<50 µM) in cultured vascular smooth muscle cells (VSMCs). MG treatment of VSMCs led to increased DNA synthesis (EC50=5.8 µM), cell proliferation, phosphorylation of p21 and glycogen synthase kinase‐3α/β (GSK‐3α/β), and increased cyclin‐dependent kinase 2 (CDK2) activity. These effects of MG were significantly inhibited by silencing Akt1 or by an Akt inhibitor. Overexpression of Akt1 Cys77Ser mutant in HEK‐293 cells increased cell proliferation and DNA synthesis, concurrent with an increase in Akt1 activity, which could not be further augmented by MG treatment. It is concluded that MG‐induced VSMC proliferation is mediated by the activation of Akt1 via the modification of Akt1 at Cys77.—Chang, T., Wang, R., Olson, D. J. H., Mousseau, D. D., Ross, A. R. S., Wu, L. Modification of Akt1 by methylglyoxal promotes the proliferation of vascular smooth muscle cells. FASEB J. 25, 1746–1757 (2011). www.fasebj.org

Glycinebetaine in oilseed rape and flax leaves: Detection by liquid chromatography/continuous flow secondary ion-massspectrometry

Abstract Glycinebetaine [betaine; (Me) 3 N + CH 2 COO − ] is likely to contribute to osmoregulation in those plants that accumulate significant amounts of it, and hence the interest in engineering its synthesis in ‘nonaccumulators’. Although only large amounts of betaine, as found in the chenopods Kochia scoparia (298.6 μmol g −1 dry wt) and Salicornia rubra (162 μmol g −1 dry wt), would be physiologically significant, its presence in flax ( Linum usitatissimum , 4 μmol g −1 dry wt) and rapeseed ( Brassica napus ; 4.9 μmol g −1 dry wt) indicates a genetic potential for betaine synthesis in these important plants. The liquid chromatography/continuous flow secondary ion-mass spectrometry method described here offers a 10-fold increase in sensitivity over other methods, and allows for detection of as little as 5 pmol μl −1 of underivatized betaine. The sensitivity of this method was further demonstrated by quantitation in the range of 50–1000 pmol μl −1 .