Fluorogenic substrates for high-throughput measurements of endothelial lipase activity

Endothelial lipase (EL) has been shown to be a critical determinant for high density lipoprotein cholesterol levels in vivo; therefore, assays that measure EL activity have become important for the discovery of small molecule inhibitors that specifically target EL. Here, we describe fluorescent Bodipy-labeled substrates that can be used in homogeneous, ultra-high-throughput kinetic assays that measure EL phospholipase or triglyceride lipase activities. Triton X-100 detergent micelles and synthetic HDL particles containing Bodipy-labeled phospholipid or Bodipy-labeled triglyceride substrates were shown to be catalytic substrates for EL, LPL, and HL. More importantly, only synthetic HDL particles containing Bodipy-labeled triglyceride were ideal substrates for EL, LPL, and HL in the presence of high concentrations of human or mouse serum. These data suggest that substrate presentation is a critical factor when determining EL activity in the presence of serum.     

Environmental Statistics and Optimal Regulation

Any organism is embedded in an environment that changes over time. The timescale for and statistics of environmental change, the precision with which the organism can detect its environment, and the costs and benefits of particular protein expression levels all will affect the suitability of different strategies–such as constitutive expression or graded response–for regulating protein levels in response to environmental inputs. We propose a general framework–here specifically applied to the enzymatic regulation of metabolism in response to changing concentrations of a basic nutrient–to predict the optimal regulatory strategy given the statistics of fluctuations in the environment and measurement apparatus, respectively, and the costs associated with enzyme production. We use this framework to address three fundamental questions: (i) when a cell should prefer thresholding to a graded response; (ii) when there is a fitness advantage to implementing a Bayesian decision rule; and (iii) when retaining memory of the past provides a selective advantage. We specifically find that: (i) relative convexity of enzyme expression cost and benefit influences the fitness of thresholding or graded responses; (ii) intermediate levels of measurement uncertainty call for a sophisticated Bayesian decision rule; and (iii) in dynamic contexts, intermediate levels of uncertainty call for retaining memory of the past. Statistical properties of the environment, such as variability and correlation times, set optimal biochemical parameters, such as thresholds and decay rates in signaling pathways. Our framework provides a theoretical basis for interpreting molecular signal processing algorithms and a classification scheme that organizes known regulatory strategies and may help conceptualize heretofore unknown ones.     

Complete genome sequence of Atopobium parvulum type strain (IPP 1246)

Atopobium parvulum (Weinberg et al. 1937) Collins and Wallbanks 1993 comb. nov. is the type strain of the species and belongs to the genomically yet unstudied Atopobium/Olsenella branch of the family Coriobacteriaceae. The species A. parvulum is of interest because its members are frequently isolated from the human oral cavity and are found to be associated with halitosis (oral malodor) but not with periodontitis. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the genus Atopobium, and the 1,543,805 bp long single replicon genome with its 1369 protein-coding and 49 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Increasing elephantLoxodonta africana density is a more important driver of change in vegetation condition than rainfall

The impact of African elephantsLoxodonta africana Blumenbuch, 1797 on biodiversity is hotly debated in wildlife management circles with scientists polarised in their views. This polarisation is largely due to the individual experiences of researchers. We aimed to determine whether elephants or rainfall patterns drove changes in vegetation condition (Normalised Difference Vegetation Index; NDVI) by avoiding a site-specific approach and looking at the issue at a broader scale. We used published estimates of elephant population density from 30 sites and recorded the change in density from 1995–1999, from 1999–2002 and from 2002–2006. We also recorded the deviation of annual rainfall from the long-term mean for those periods. We modelled these variables against the change in NDVI between periods using mixed effects models. We found that elephants were more influential in driving change in vegetation condition than rainfall, and this also occurred at one of our individual test sites where long-term data were available (Kruger). Elephants and rainfall combined to drive change in vegetation condition at our other long-term test site (Amboseli). Management activities (fencing, water provision) may cause the differences between the two long-term study sites. Change in productivity driven by rainfall has ramifications for biodiversity, suggesting that elephant derived changes in vegetation productivity (NDVI) also impacts on biodiversity. Thus, this study supports previous findings from individual sites that elephants impact vegetation, however there is also a suggestion that these impacts may vary according to management actions.     

Subsidies of long‐chain polyunsaturated fatty acids from aquatic to terrestrial environments via amphibian emergence

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Characterization of a new β-glucosidase/β-xylosidase from the gut microbiota of the termite (Reticulitermes santonensis)

The gut of the termite Reticulitermes santonensis contains an interesting diversity of prokaryotic and eukaryotic microorganisms not found elsewhere. These microorganisms produce many enzyme-digesting lignocellulosic compounds, probably in cooperation with endogenous enzymes. Regarding cellulose and hemicellulose digestion in the termite gut, much remains to be learned about the relative contributions of termite enzymes and enzymes produced by different microorganisms. Here we grew bacterial colonies from termite gut suspensions, identifying 11 of them after PCR amplification of their 16S rRNA genes. After constructing in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained, we performed functional screening for α-amylase, xylanase, β-glucosidase, and endoglucanase activities. This screen revealed a clone producing β-glucosidase activity. Sequence analysis showed that the cloned genomic DNA fragment contained three complete ORFs (bglG, bglF, and bglB) organized in a putative bgl operon. The new β-glucosidase (BglB), identified with its regulators BglG and BglF, belongs to glycoside hydrolase family 1. The new β-glucosidase was expressed in E. coli and purified by affinity chromatography. The purified enzyme shows maximal activity at pH 6.0 and 40?°C. It also displays β-xylosidase activity.     

De novo lipogenesis in the differentiating human adipocyte can provide all fatty acids necessary for maturation

The primary products of de novo lipogenesis (DNL) are saturated fatty acids, which confer adverse cellular effects. Human adipocytes differentiated with no exogenous fat accumulated triacylglycerol (TG) in lipid droplets and differentiated normally. TG composition showed the products of DNL (saturated fatty acids from 12:0 to 18:0) together with unsaturated fatty acids (particularly 16:1n-7 and 18:1n-9) produced by elongation/desaturation. There was parallel upregulation of expression of genes involved in DNL and in fatty acid elongation and desaturation, suggesting coordinated control of expression. Enzyme products (desaturation ratios, elongation ratios, and total pathway flux) were also correlated with mRNA levels. We used (13)C-labeled substrates to study the pathway of DNL. Glucose (5 mM or 17.5 mM in the medium) provided less than half the carbon used for DNL (42% and 47%, respectively). Glutamine (2 mM) provided 9-10%, depending upon glucose concentration. In contrast, glucose provided most (72%) of the carbon of TG-glycerol. Pathway analysis using mass isotopomer distribution analysis (MIDA) revealed that the pathway for conversion of glucose to palmitate is complex. DNL in human fat cells is tightly coupled with further modification of fatty acids to produce a range of saturated and unsaturated fatty acids consistent with normal maturation.     

Allelopathic invasive tree (<Emphasis Type="Italic">Rhamnus cathartica</Emphasis>) alters native plant communities

Many plants release allelopathic chemicals that can inhibit germination, growth, and/or survival in neighboring plants. These impacts appear magnified with the invasion of some non-native plants which may produce allelochemicals against which native fauna have not co-evolved resistance. Our objective was to examine the potential allelopathic impact of an invasive non-native shrub/tree on multiple plant species using field observation and experimental allelopathy studies. We surveyed and collected an invasive, non-native tree/shrub (Rhamnus cathartica) at Tifft Nature Preserve (a 107-ha urban natural area near Lake Erie in Buffalo, NY). We also surveyed understory plant communities in the urban forest to examine correlations between R. cathartica abundance and local plant community abundance and richness. We then used experimental mesocosms to test if patterns observed in the field could be explained by adding increased dosages of R. cathartica to soils containing five plant species, including native and non-native woody and herbaceous species. In the highly invaded urban forest, we found that herbaceous cover, shrubs and woody seedlings negatively covaried with R. cathartica basal area and seedlings density. In the mesocosm experiments, R. cathartica resulted in significant decreases in plant community species richness, abundance, and shifted biomass allocation from roots. Our results provide evidence that R. cathartica is highly allelopathic in its invaded range, that R. cathartica roots have an allelopathic effect and that some plant species appear immune. We suggest that these effects may explain the plant’s ability to form dense monocultures and resist competitors, as well as shift community composition with species-specific impacts.