The solution structure of the CBM4-2 carbohydrate binding module from a thermostable Rhodothermus marinus xylanase

The solution structure is presented for the second family 4 carbohydrate binding module (CBM4-2) of xylanase 10A from the thermophilic bacterium Rhodothermus marinus. CBM4-2, which binds xylan tightly, has a beta-sandwich structure formed by 11 strands, and contains a prominent cleft. From NMR titrations, it is shown that the cleft is the binding site for xylan, and that the main amino acids interacting with xylan are Asn31, Tyr69, Glu72, Phe110, Arg115, and His146. Key liganding residues are Tyr69 and Phe110, which form stacking interactions with the sugar. It is suggested that the loops on which the rings are displayed can alter their conformation on substrate binding, which may have functional importance. Comparison both with other family 4 cellulose binding modules and with the structurally similar family 22 xylan binding module shows that the key aromatic residues are in similar positions, and that the bottom of the cleft is much more hydrophobic in the cellulose binding modules than the xylan binding proteins. It is concluded that substrate specificity is determined by a combination of ring orientation and the nature of the residues lining the bottom of the binding cleft.     

Repression of both isoforms of disproportionating enzyme leads to higher malto-oligosaccharide content and reduced growth in potato

Two glucanotransferases, disproportionating enzyme 1 (StDPE1) and disproportionating enzyme 2 (StDPE2), were repressed using RNA interference technology in potato, leading to plants repressed in either isoform individually, or both simultaneously. This is the first detailed report of their combined repression. Plants lacking StDPE1 accumulated slightly more starch in their leaves than control plants and high levels of maltotriose, while those lacking StDPE2 contained maltose and large amounts of starch. Plants repressed in both isoforms accumulated similar amounts of starch to those lacking StDPE2. In addition, they contained a range of malto-oligosaccharides from maltose to maltoheptaose. Plants repressed in both isoforms had chlorotic leaves and did not grow as well as either the controls or lines where only one of the isoforms was repressed. Examination of photosynthetic parameters suggested that this was most likely due to a decrease in carbon assimilation. The subcellular localisation of StDPE2 was re-addressed in parallel with DPE2 from Arabidopsis thaliana by transient expression of yellow fluorescent protein fusions in tobacco. No translocation to the chloroplasts was observed for any of the fusion proteins, supporting a cytosolic role of the StDPE2 enzyme in leaf starch metabolism, as has been observed for Arabidopsis DPE2. It is concluded that StDPE1 and StDPE2 have individual essential roles in starch metabolism in potato and consequently repression of these disables regulation of leaf malto-oligosaccharides, starch content and photosynthetic activity and thereby plant growth possibly by a negative feedback mechanism.     

Haplotyping cryptic Adélie penguin taxa using low-cost,high-resolution melt curves

Distinguishing morphologically cryptic taxa, by definition, requires genetic data such as DNA sequences. However, DNA sequences may not be obtained easily for taxa from remote sites. Here we provide the details of a high-resolution melt-curve-based method using taxon-specific primers that can distinguish two taxa of Adélie penguins, and that will be usable in Antarctica when combined with some of the newly developed field-deployable thermal cyclers. We suggest that the wider adoption of field-deployable polymerase-chain-reaction-based techniques will enable faster assignation of haplotype to individuals in situ, and so allow the targeting of observations and sample collection to specimens relevant to the research question. Targeting individuals will also reduce the need to repeatedly handle animals and reduce the time and travel required to complete field work.     

Effects of food abundance and early clutch predation on reproductive timing in a high Arctic shorebird exposed to advancements in arthropod abundance

Climate change may influence the phenology of organisms unequally across trophic levels and thus lead to phenological mismatches between predators and prey. In cases where prey availability peaks before reproducing predators reach maximal prey demand, any negative fitness consequences would selectively favor resynchronization by earlier starts of the reproductive activities of the predators. At a study site in northeast Greenland, over a period of 17 years, the median emergence of the invertebrate prey of Sanderling Calidris alba advanced with 1.27 days per year. Yet, over the same period Sanderling did not advance hatching date. Thus, Sanderlings increasingly hatched after their prey was maximally abundant. Surprisingly, the phenological mismatches did not affect chick growth, but the interaction of the annual width and height of the peak in food abundance did. Chicks grew especially better in years when the food peak was broad. Sanderling clutches were most likely to be depredated early in the season, which should delay reproduction. We propose that high early clutch predation may favor a later reproductive timing. Additionally, our data suggest that in most years food was still abundant after the median date of emergence, which may explain why Sanderlings did not advance breeding along with the advances in arthropod phenology.     

Estimating and Analyzing Savannah Phenology with a Lagged Time Series Model

Savannah regions are predicted to undergo changes in precipitation patterns according to current climate change projections. This change will affect leaf phenology, which controls net primary productivity. It is of importance to study this since savannahs play an important role in the global carbon cycle due to their areal coverage and can have an effect on the food security in regions that depend on subsistence farming. In this study we investigate how soil moisture, mean annual precipitation, and day length control savannah phenology by developing a lagged time series model. The model uses climate data for 15 flux tower sites across four continents, and normalized difference vegetation index from satellite to optimize a statistical phenological model. We show that all three variables can be used to estimate savannah phenology on a global scale. However, it was not possible to create a simplified savannah model that works equally well for all sites on the global scale without inclusion of more site specific parameters. The simplified model showed no bias towards tree cover or between continents and resulted in a cross-validated r² of 0.6 and root mean squared error of 0.1. We therefore expect similar average results when applying the model to other savannah areas and further expect that it could be used to estimate the productivity of savannah regions.     

Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2.

The lipopolysaccharide (LPS) of Chlamydia trachomatis L2 was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. From a total of 5 x 10(4) cm2 of infected monolayers, 22.3 mg of LPS were obtained. Compositional analysis indicated the presence of 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), GlcN, phosphorus, and fatty acids in a molar ratio of 2.8:2:2.1:4.5. Matrix-assisted laser-desorption ionization mass spectrometry performed on the de-O-acylated LPS gave a major molecular ion peak at m/z 1781.1 corresponding to a molecule of 3 Kdo, 2 GlcN, 2 phosphates, and two 3-hydroxyeicosanoic acid residues. The structure of deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide was determined by 600 MHz NMR spectroscopy as Kdoalpha2-->8Kdoalpha2-->4Kdoalpha2-->6D-GlcpNbeta1 -->6D-GlcpNalpha 1,4'-bisphosphate. These data, together with those published recently on the acylation pattern of chlamydial lipid A (Qureshi, N., Kaltashov, I., Walker, K., Doroshenko, V., Cotter, R. J., Takayama, K, Sievert, T. R., Rice, P. A., Lin, J.-S. L., and Golenbock, D. T. (1997) J. Biol. Chem. 272, 10594-10600) allow us to present for the first time the complete structure of a major molecular species of a chlamydial LPS.     

Influence of GLP-1 on Myocardial Glucose Metabolism in Healthy Men during Normo- or Hypoglycemia

Background and Aims

Glucagon-like peptide-1 (GLP-1) may provide beneficial cardiovascular effects, possibly due to enhanced myocardial energetic efficiency by increasing myocardial glucose uptake (MGU). We assessed the effects of GLP-1 on MGU in healthy subjects during normo- and hypoglycemia.

Materials and Methods

We included eighteen healthy men in two randomized, double-blinded, placebo-controlled cross-over studies. MGU was assessed with GLP-1 or saline infusion during pituitary-pancreatic normo- (plasma glucose (PG): 4.5 mM, n = 10) and hypoglycemic clamps (PG: 3.0 mM, n = 8) by positron emission tomography with ¹⁸fluoro-deoxy-glucose (¹⁸F-FDG) as tracer.

Results

In the normoglycemia study mean (± SD) age was 25±3 years, and BMI was 22.6±0.6 kg/m² and in the hypoglycemia study the mean age was 23±2 years with a mean body mass index of 23±2 kg/m². GLP-1 did not change MGU during normoglycemia (mean (+/− SD) 0.15+/−0.04 and 0.16+/−0.03 µmol/g/min, P = 0.46) or during hypoglycemia (0.16+/−0.03 and 0.13+/−0.04 µmol/g/min, P = 0.14). However, the effect of GLP-1 on MGU was negatively correlated to baseline MGU both during normo- and hypoglycemia, (P = 0.006, r² = 0.64 and P = 0.018, r² = 0.64, respectively) and changes in MGU correlated positively with the level of insulin resistance (HOMA 2IR) during hypoglycemia, P = 0.04, r² = 0.54. GLP-1 mediated an increase in circulating glucagon levels at PG levels below 3.5 mM and increased glucose infusion rates during the hypoglycemia study. No differences in other circulating hormones or metabolites were found.

Conclusions

While GLP-1 does not affect overall MGU, GLP-1 induces changes in MGU dependent on baseline MGU such that GLP-1 increases MGU in subjects with low baseline MGU and decreases MGU in subjects with high baseline MGU. GLP-1 preserves MGU during hypoglycemia in insulin resistant subjects.ClinicalTrials.gov registration numbers: {"type":"clinical-trial","attrs":{"text":"NCT00418288","term_id":"NCT00418288"}}NCT00418288: (hypoglycemia) and {"type":"clinical-trial","attrs":{"text":"NCT00256256","term_id":"NCT00256256"}}NCT00256256: (normoglycemia).     

Process monitoring of acetic acid in Escherichia coli cultivation using electrochemical detection in a flow injection system

A flow-injection system for determination of acetic acid in Escherichia coli cultivations with electrochemical detection based on immobilized acetate kinase (AK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) was developed to cope with the problems related to measurements under process conditions such as interferences from pyruvate, drift of electrode baseline and making the cultivation medium and reagents compatible. The results obtained by flow injection analysis were compared with those obtained with an enzymatic test kit.