The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis

ActVA-Orf6 monooxygenase from Streptomyces coelicolor that catalyses the oxidation of an aromatic intermediate of the actinorhodin biosynthetic pathway is a member of a class of small monooxygenases that carry out oxygenation without the assistance of any of the prosthetic groups, metal ions or cofactors normally associated with activation of molecular oxygen. The overall structure is a ferredoxin-like fold with a novel dimeric assembly, indicating that the widely represented ferredoxin fold may sustain yet another functionality. The resolution (1.3 A) of the enzyme structure and its complex with substrate and product analogues allows us to visualize the mechanism of binding and activation of the substrate for attack by molecular oxygen, and utilization of two gates for the reaction components including a proton gate and an O(2)/H(2)O gate with a putative protein channel. This is the first crystal structure of an enzyme involved in the tailoring of a type II aromatic polyketide and illustrates some of the enzyme-substrate recognition features that may apply to a range of other enzymes involved in modifying a polyketide core structure.     

Differences in purity of commercial samples of hemicholinium-3: effects on high affinity choline uptake, neuromuscular transmission, acetylcholinesterase and carbachol and acetylcholine contractions.

Commercial samples of hemicholinium-3 (HC-3) have been found to vary in colour (from white to a sandy-yellow colour) and chemical composition. There were no significant differences between the various HC-3 samples with regards to inhibition of high affinity choline uptake into synaptosomes or inhibition of neuromuscular transmission in the chick biventer cervicis (CBC) preparation. Yellow HC-3 inhibited acetylcholinesterase more than white HC-3 with I₅₀ of 4.8 × 10^?5 M and 3.3 × 10^?4 M, respectively. Carbachol-induced contractions of the CBC preparation were inhibited more by yellow HC-3 than white HC-3; the opposite was true for acetylcholine- induced contractions. The results indicated that there is a minor contaminant in yellow HC-3 other than deanol which was a potent inhibitor of acetylcholinesterase and the carbachol response.     

Stimulation of AMP-activated protein kinase (AMPK) is associated with enhancement of Glut1-mediated glucose transport

In cells expressing only the Glut1 isoform of glucose transporters, we have shown that glucose transport is markedly stimulated in response to hypoxia or inhibition of oxidative phosphorylation, conditions that would be expected to cause a stimulation of AMP-activated protein kinase (AMPK) activity. In the present study we tested the hypothesis that the stimulation of AMPK activity might be accompanied by an enhancement of Glut1-mediated glucose transport. Exposure of Clone 9 cells, 3T3-L1 preadipocytes, and C(2)C(12) myoblasts (cells that express only the Glut1 isoform) to 5-aminoimidazole-4-carboxamideribonucleoside (AICAR), an adenosine analog that stimulates AMPK activity, resulted in a marked increase in the rate of glucose transport (ranging from four- to sixfold) that was accompanied by activation of AMPK. This stimulation of AMPK activity was associated with an increase in the phosphorylation of threonine 172 on the activation loop of its alpha subunit, with the predominant change being in the alpha-2 isoform. Exposure of Clone 9 cells to 5-iodotubercidin, an inhibitor of adenosine kinase, abolished the accumulation of AICAR-5'-monophosphate (ZMP), stimulation of AMPK, and the enhancement of glucose transport in response to AICAR. There was no significant increase in the content of Glut1 in plasma membranes of Clone 9 cells exposed to AICAR. We conclude that stimulation of AMPK activity is associated with enhancement of Glut1-mediated glucose transport, and that the glucose transport response is mediated by activation of Glut1 transporters preexisting in the plasma membrane.     

Diversity of natural enemies in Central Otago,New Zealand apple orchards: a practical measure of sustainability in pest management?

The diversity of the arthropod fauna in apple orchards in Central Otago was recorded from 1994 to 1999 using beating trays, pitfall traps, and sticky traps and the data analysed by the Shannon–Wiener Index. Three different fruit production systems were compared, conventional (CFP), integrated (IFP), and biological (BFP), to determine whether total arthropod diversity and/or the diversity of natural enemies (predators and parasitoids) could be used to measure the sustainability of their pest management practices. The contribution of natural enemies to the diversity indices was also examined to determine if they reflected the key species essential for sustainable pest management. The diversity of natural enemies in beating trays was found to be the measure most sensitive to changes in pest management. The diversity of both the total arthropod fauna and natural enemies was much lower under CFP than under either IFP or BFP, due to the use of broad-spectrum pesticides. This practice made the CFP programme unsustainable and it is no longer used. During the transition from CFP to IFP, the diversity indices rose to become similar to that in the BFP programme by the third season. The introduction of frequent applications of fungicides in BFP, including lime sulphur, appeared to reduce total arthropod and natural enemy diversity. Falls in the diversity indices for natural enemies were shown to reflect reduced numbers, range, and evenness of natural enemies but not necessarily the key species known to be critical for sustainable pest management. It is proposed that a Shannon–Wiener Index for natural enemies in beating trays of 0.2 or less in summer is strongly indicative of unsustainable pest management in Otago apple orchards. On the other hand, indices of 0.3–0.8, as found under IFP and BFP, do not give unequivocal or quantitative indications of the sustainability of pest management. A suite of other measures of sustainability are more useful, notably combining lower pesticide use, reduced pest damage and presence at harvest, the greater roles of key natural enemies, more effective plant resistance to pests and diseases, and higher profitability.     

Study of agreement between LDL size as measured by nuclear magnetic resonance and gradient gel electrophoresis

LDL particle size can be measured by gradient gel electrophoresis (GGE) and NMR. The agreement between the two methods has not been extensively evaluated. Therefore, we measured LDL size by NMR and GGE in 324 individuals (152 with type 1 diabetes and 172 controls). The Spearman correlation between both methods was 0.39 [95% confidence interval (CI) = 0.29, 0.48]. The average difference was 5.38 nm (NMR being smaller), but it increased with increasing LDL size. Less than 50% of people classified as pattern B on GGE were classified as pattern B on NMR (kappa = 0.31; 95% CI = 0.17, 0.45). Agreement was lower for diabetic subjects compared with controls, for women compared with men, and for subjects with triglycerides less than 1.30 mmol/l compared with subjects with triglycerides greater than 1.30 mmol/l. External validation showed that cholesteryl ester transfer rate was related to LDL size on GGE in all subgroups and to LDL size on NMR only in men and nondiabetic subjects. Our findings show that agreement between NMR- and GGE-based LDL size is far from perfect and is not consistent across subgroups of patients. In particular, the two methods should not be assumed to be interchangeable in women and diabetic subjects. Whether NMR or GGE predicts cardiovascular disease risk better has not yet been evaluated.     

Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367. Received 18 March 2001/ Accepted in revised form 09 July 2001     

Agri-environment scheme enhances breeding populations of some priority farmland birds in Northern Ireland

Capsule: The abundance of Tree sparrow Passer montanus, House Sparrow Passer domesticus and Yellowhammer Emberiza citrinella, three priority farmland birds, increased over a 5-year period in response to a targeted agri-environment scheme (AES).

Aims: To assess the effects of a targeted AES on the abundance of a suite of farmland bird species over a 5-year period.

Methods: We compare temporal changes in abundance of 12 farmland bird species of conservation concern on 33 AES and 22 control farms in County Down, Northern Ireland. Five of these species were designated targets for conservation action under the Countryside Management Scheme (CMS).

Results: CMS management was associated with more positive changes in abundance for three of the five target species and more negative changes for one target species (albeit caused mainly by a large reduction at a single farm). CMS management had little influence on the abundance of non-target species or on avian species richness. Farm-scale changes in abundance were generally unrelated to the extent of local CMS provision, the only exception involved House Sparrow and seed-rich winter habitats.

Conclusion: This study demonstrates that generic AES land management can improve the population status of target farmland birds on farms with AES management.