A new strategy for the synthesis of glycoproteins

Posttranslational modifications of proteins regulate many biological processes, including metabolism, signal transduction, and gene expression. The synthetic challenges associated with generating homogeneous populations of selectively modified proteins, however, have hindered detailed studies of the effects of these modifications on protein structure and function. Here, we report an approach to the cotranslational synthesis of selectively glycosylated proteins in which the modified amino acid is genetically encoded. We show that myoglobin containing beta-N-acetylglucosamine (GlcNAc)-serine at a defined position can be expressed in Escherichia coli in good yield and with high fidelity. The beta-GlcNAc moiety can be recognized by a saccharide-binding protein, or subsequently modified with a galactosyltransferase to build more complex carbohydrates. This approach should be generally applicable to other posttranslational modifications such as protein phosphorylation, acetylation, and methylation.     

X-ray Absorption Spectroscopic Investigation of Trace Vanadium Sites in Coal

X-ray absorption spectroscopy was used to probe the chemical and structural environment of vanadium in coal. It was found that vanadium exists in at least two environments, in both of which it was coordinated to oxygens. There was no evidence of vanadium in nitrogen (porphyrin) or sulfide environments. It was also found that the vanadium environments in the raw coal did not survive unchanged in a liquefaction process. These findings have implications for coal cleaning processes and for trace element release into the liquefaction process stream.     

Evaluation of an antimicrobial resistance monitoring program for Campylobacter in poultry by simulation

An ideal national resistance monitoring program should deliver a precise estimate of the resistance situation for a given combination of bacteria and antimicrobial at a low cost. To achieve this, decisions need to be made on the number of samples to be collected at each of different possible sampling points. Existing methods of sample size calculation can not be used to solve this problem, because sampling decisions do not only depend on the prevalence of resistance and sensitivity and specificity of resistance testing, but also on the prevalence of the bacteria, and test characteristics of isolation of these bacteria. Our aim was to develop a stochastic simulation model that optimized a national resistance monitoring program, taking multi-stage sampling, imperfect sensitivity and specificity of diagnostic tests, and cost-effectiveness considerations into account. The process of resistance testing of Campylobacter spp. isolated from cloacal swab samples from poultry was modeled using a Markov Chain Monte Carlo model. Different sampling scenarios on the number of flocks to be tested, the number of birds from each flock, and the number of campylobacter colonies submitted to susceptibility testing were evaluated regarding the precision of the resulting prevalence estimate. Precision of the prevalence estimate was defined as the absolute difference between apparent and true prevalence of resistance. A partial budget approach was utilized to find the most cost-effective combination of samples to obtain a defined precision of the prevalence estimate. For a sampling scenario testing 100 flocks, five birds per flock, and one campylobacter colony per sample, the median error of the prevalence estimate was 2.5%, and 95% of the simulations resulted in an error of 7% or less. When the total number of samples was kept constant, maximizing the number of flocks tested, and only testing one bird per flock resulted in the most precise prevalence estimate. Submitting more than one campylobacter colony to resistance testing did not improve the prevalence estimate. Partial budget analysis indicated that the most cost-effective strategy was testing of two birds per flock, and submitting one colony per sample to resistance testing.     

Beneficial effects of earthworms and arbuscular mycorrhizal fungi on establishment of leguminous trees on Pb/Zn mine tailings

Planting trees to stabilize metalliferous mine tailings is a widely used form of land reclamation although substantial soil amendment is invariably required, both to improve the physico-chemical status of the tailings and to ameliorate toxicity prior to planting. Here, we report a glasshouse study of the combined effects of burrowing earthworms (Pheretima guillelmi) and arbuscular mycorrhizal fungi (Glomus spp., AMF) on establishment of a naturally invasive, woody, nitrogen-fixing legume, Leucaena leucocephala, on topsoil-amended Pb/Zn mine tailings. AMF provided the most effective preliminary inoculant, improving N, P and K uptake, but earthworms had more influence improving N nutrition. In most cases, the combined effects of AMF and earthworms were additive and proved to be beneficial to plant growth, plant nutrition and for protection against uptake of toxic metals. AMF influenced metal uptake more than earthworms, but together they reduced mobility of Pb and Zn in soil by as much as 25%. Some minor but significant negative interactions were also evident; for example, earthworms enhanced soil microbial activity but inhibited the beneficial effects of AMF on N₂-fixation. We argue that increased attention to ecological interactions in soil could reduce costs and improve the efficacy of restoring a vegetation cover to land impacted by contaminated spoils.     

Proton motive force involved in protein transport across the outer membrane of Aeromonas salmonicida

Many Gram-negative bacteria export proteins to the exterior. Some of these proteins are first secreted into the periplasm and then cross the outer membrane in a separate step. The source of energy required for the translocation is unknown. Export of the extracellular protein proaerolysin from the periplasm through the outer membrane of Aeromonas salmonicida is inhibited by a proton ionophore and by low extracellular pH. One possible explanation of these results is that a proton gradient across the outer membrane is required for export.     

Selenium-containing allophycocyanin purified from selenium-enriched Spirulina platensis attenuates AAPH-induced oxidative stress in human erythrocytes through inhibition of ROS generation

Both selenium and allophycocyanin (APC) have been reported to show novel antioxidant activities. In this study, a fast protein liquid chromatographic method for purification of selenium-containing allophycocyanin (Se-APC) from selenium-enriched Spirulina platensis and the protective effect of Se-APC on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative stress have been described. After fractionation by ammonium sulfate precipitation, and separation by DEAE-Sepharose ion-exchange and Sephacryl S-300 size exclusion chromatography, Se-APC with purity ratio (A652/A280) of 5.30 and Se concentration of 343.02 μg g(-1) protein was obtained. Se-APC exhibited stronger antioxidant activity than APC by scavenging ABTS (2,2'-azinobis-3-ethylbenzothiazolin-6-sulfonic acid) and AAPH free radicals. The oxidative hemolysis and morphological changes induced by AAPH in human erythrocytes were effectively reversed by coincubation with Se-APC. Lipid oxidation induced by the pro-oxidant agent cupric chloride in human plasma, as evaluated by formation of conjugated diene, was blocked by Se-APC. The accumulation of malondialdehyde, loss of reduced glutathione, and increase in enzyme activities of glutathione peroxidase and reductase induced by AAPH in human erythrocytes were effectively suppressed by Se-APC. Furthermore, Se-APC significantly prevented AAPH-induced intracellular reactive oxygen species (ROS) generation. Taken together, our results suggest that Se-APC demonstrates application potential in treatment of diseases in which excess production of ROS acts as a casual or contributory factor.     

Pre-maturity α-amylase and incipient sprouting in UK winter wheat,with special reference to the variety Rialto

In 1998 and 1999 the UK winter wheat variety Rialto produced unexpected low Hagberg falling numbers that could not be directly linked to sprouting. It was proposed that these reductions in quality could be due to pre-maturity α-amylase activity (PMAA). The problem was not identified during the selection and commercial development stages. Our study tested the hypothesis that the variety Rialto is PMAA-susceptible. Analysis was done on 13 year-location combinations of field grown Rialto. Together, visual and chemical assessments of sprouting and iso-electric focusing of α-amylase isozymes identified several samples with significant α-amylase activity in the apparent absence of sprouting. In addition, tests with α-amylase sensitive Phadebas gel revealed distinctive PMAA discoloration patterns in 10–44% of the grain from the 13 samples, leading to the conclusion that Rialto is PMAA-susceptible. Diurnal temperature range accumulated for an 11 day period during a warm spell in early simulated grain development displayed a significant but negative correlation with the number of grains showing clear PMAA discoloration patterns on Phadebas gel. The number of clear PMAA grains correlated positively with rainfall accumulated over 11 days. These results suggest that PMAA can increase under conditions similar to those conducive to pre-harvest sprouting. It is however also possible that in some instances both PMAA and incipient sprouting could have produced similar patterns of α-amylase activity. In addition to tests with Rialto, Phadebas gel tests were therefore also done with the known high Hagberg varieties Option and Malacca, sprouted in a controlled environment. Results from the additional gel tests suggest that visual and chemical assessments of sprouting in the grain combined with Phadebas gel analysis could identify PMAA more reliably in grain sub-samples than Phadebas gel analysis alone.