Cytochrome P450105D1 (CYP105D1) from Streptomyces griseus: heterologous expression, activity, and activation effects of multiple xenobiotics.

The open reading frame of CYP105D1, a soluble cytochrome P450 from Streptomyces griseus, was cloned behind the tac promoter of the bacterial expression vector pSPg1910L and expressed in Escherichia coli. The recombinant protein retained normal spectral characteristics having a Soret peak at 448 nm in the reduced carbon monoxide difference spectrum. CYP105D1 was active, obtaining reducing equivalents from endogenous E. coli ferredoxin and ferredoxin reductase redox partners present in E. coli. In vitro activity studies revealed CYP105D1 to catalyse the NADH- and NADPH-dependent oxidation of the xenobiotic substrates benzo[a]pyrene, erythromycin, warfarin, and testosterone. Furthermore, this activity could be stimulated in the presence of either alpha-benzoflavone or beta-benzoflavone in an analogous manner to that reported for mammalian P450 forms including human liver cytochrome P4503A4 (CYP3A4). The system produces an alternative to whole-cell biotransformation of xenobiotic for the production of drug metabolites and an experimental system for probing the structural features of a cytochrome P450 with a broad substrate range.     

Climate change and pollutant impacts on Scottish vegetation

Summary

By the 2050s the UK is projected to be about 1.6°C warmer, when the atmospheric CO₂ concentration will be 525 ppmv. These changes will have profound effects on the Scottish flora and fauna. Vegetation primary productivity will increase, except in dry regions, and the productivity of upland forest plantations may increase by several Yield Classes. The spread of plant species may be less than expected, but a number of slow-growing ‘stress-tolerant’ species, including montane/alpine species, are likely to be lost. Nitrogen deposited as a result of emission of NO_X from vehicles and NH₃ from agriculture is now a major source of acidity, and problems of acidification and eutrophication are linked. Despite reductions in sulphur emissions, critical loads of acid deposition are likely to be exceeded for soils in most of the Scottish uplands until at least 2005. Critical levels affecting tree growth may be exceeded where forests are in cloud for 10% of the time in areas of the Great Glen. Much of the Scottish uplands receives 25–30 kg N ha^-1 yr^-1, which may be causing change in species composition. Background tropospheric ozone concentrations are increasing. Much of the Scottish uplands experiences mean summer ozone concentrations exceeding those in southern England, but with fewer exceedances of critical levels. However, many crops and some sensitive native species are probably being adversely affected.     

Studies on pre-incubation conditions in the seeded agar plate well inhibition assay

Pre-incubation at 4°C increases the zone of bacterial growth inhibition produced by methanolic extracts of freshwater algae in the agar diffusion assay. Preloading of wells with methanol increases the inhibition zone produced by water-insoluble antibiotics.     

A computer program for processing data from amino acid analysis and for the calculation of molecular weights from those data.

A computer program is described for the calculation of the complete amino acid composition of a protein from the analytical data. The program also derives a molecular weight on the basis of the amino acid composition. The use of the program for the determination of the molecular weights of the liver carboxylesterases of chicken, horse, ox, and sheep is described.     

Nitrogen States in Plant Ecosystems: A Viewpoint

Terrestrial ecosystems are considered to be in only two possiblestates: N unsaturated or N saturated. This view lacks rigourand has led to three differing concepts of N saturation: (1)a continuum of changes in N states and processes; (2) a thresholdpoint, when N output increases; and (3) any equilibrium state,when N output equals N input. A simple model of ecosystem Ncontent, input and output is used to point out that, strictly,there are four possible N states of ecosystems: (1) at equilibrium,but N unsaturated and N-limited, so that they will respond toadditional N input with increased N accumulation; (2) not atequilibrium and accumulating N, either in response to additionalN input, or more commonly in nature, when recovering from fire,other disturbance or unfavourable conditions; (3) at equilibriumbut truly N saturated and not N-limited, so that any additionalN input is matched by equal N output; and (4) not at equilibriumand losing N because of disturbance or soil changes inducedby N addition itself or other factors. Most natural ecosystemsare, for most of the time, in one of the two non-equilibriumstates, especially in short-term N-addition experiments. Itis not meaningful to regard them as being either N saturatedor N-limited. Copyright 2000 Annals of Botany Company Nitrogen saturation, deposition, leaching, nitrification, plant ecosystems, forest     

Herd-level risk factors associated with the presence of Phage type 21/28 E. coli O157 on Scottish cattle farms

Background  

E. coli O157 is a bacterial pathogen that is shed by cattle and can cause severe disease in humans. Phage type (PT) 21/28 is a subtype of E. coli O157 that is found across Scotland and is associated with particularly severe human morbidity.     

Three-dimensional distribution of ryanodine receptor clusters in cardiac myocytes

The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 microm in ventricular myocytes and estimated 0.97 microm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 microm in ventricular and 1.69 microm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that approximately 100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed.