Fluorescent substrates for ADAM15 useful for assaying and high throughput screening

A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs −2, –9, and −13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M⁻¹ s⁻¹ and 4300 M⁻¹ s⁻¹, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (k_cat/K_m) of 5200 M⁻¹ s⁻¹. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 μM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.     

Phosphorylated TandeMBP: A unique protein substrate for protein phosphatase assay

To analyze a variety of protein phosphatases, we developed phosphorylated TandeMBP (P-TandeMBP), in which two different mouse myelin basic protein isoforms were fused in tandem, as a protein phosphatase substrate. P-TandeMBP was prepared efficiently in four steps: (1) phosphorylation of TandeMBP by a protein kinase mixture (Ca²⁺/calmodulin-dependent protein kinase Iδ, casein kinase 1δ, and extracellular signal-regulated kinase 2); (2) precipitation of both P-TandeMBP and protein kinases to remove ATP, Pi, and ADP; (3) acid extraction of P-TandeMBP with HCl to remove protein kinases; and (4) neutralization of the solution that contains P-TandeMBP with Tris. In combination with the malachite green assay, P-TandeMBP can be used to detect protein phosphatase activity without using radioactive materials. Moreover, P-TandeMBP served as an efficient substrate for PPM family phosphatases (PPM1A, PPM1B, PPM1D, PPM1F, PPM1G, PPM1H, PPM1K, and PPM1M) and PPP family phosphatase PP5. Various phosphatase activities were also detected with high sensitivity in gel filtration fractions from mouse brain using P-TandeMBP. These results indicate that P-TandeMBP might be a powerful tool for the detection of protein phosphatase activities.     

Mapping habitat indices across river networks using spatial statistical modelling of River Habitat Survey data

Freshwater ecosystems are declining faster than their terrestrial and marine counterparts because of physical pressures on habitats. European legislation requires member states to achieve ecological targets through the effective management of freshwater habitats. Maps of habitats across river networks would help diagnose environmental problems and plan for the delivery of improvement work. Existing habitat mapping methods are generally time consuming, require experts and are expensive to implement. Surveys based on sampling are cheaper but provide patchy representations of habitat distribution. In this study, we present a method for mapping habitat indices across networks using semi-quantitative data and a geostatistical technique called regression kriging. The method consists of the derivation of habitat indices using multivariate statistical techniques that are regressed on map-based covariates such as altitude, slope and geology. Regression kriging combines the Generalised Least Squares (GLS) regression technique with a spatial analysis of model residuals. Predictions from the GLS model are ‘corrected’ using weighted averages of model residuals following an analysis of spatial correlation. The method was applied to channel substrate data from the River Habitat Survey in Great Britain. A Channel Substrate Index (CSI) was derived using Correspondence Analysis and predicted using regression kriging. The model explained 74% of the main sample variability and 64% in a test sample. The model was applied to the English and Welsh river network and a map of CSI was produced. The proposed approach demonstrates how existing national monitoring data and geostatistical techniques can be used to produce continuous maps of habitat indices at the national scale.     

农林废弃物混配基质的理化性质及其对油茶幼苗生长效应的综合评价

以腐熟的甘蔗渣( SB)、木薯皮( CP)、花生壳( PS)和火炭灰( BA)及园土( GS)为原料,按照不同体积比配制成9种混配基质,并以等体积比泥炭和园土混配基质为对照,对各基质的理化性质及基质中油茶( Camellia oleifera Abel)幼苗的生长状况进行比较;在对基质理化指标和油茶幼苗的壮苗指数进行线性回归分析和通径分析的基础上,采用主成分分析和综合评价法对各基质的育苗效果进行综合评价。结果表明：9种农林废弃物混配基质的容重、总孔隙度和毛管孔隙度分别为0.23~0.47 g·cm-3、60.90%~67.23%和46.53%~58.27%, pH 6.72~pH 7.14,各基质的速效氮含量、速效磷含量、速效钾含量、pH值、电导率和通气孔隙度均高于或显著高于对照,容重则显著低于对照。在不同基质中油茶幼苗的茎粗、单株叶片数、叶长、叶宽、叶绿素相对含量( SPAD)、单株茎叶干质量、单株根干质量和壮苗指数均存在一定差异,而株高和根冠比却无显著差异;其中S9混配基质也V( SB)：V( CP)：V( BA)=2：1：1页中幼苗的大部分生长指标较高,表现出明显的生长优势。线性回归和通径分析结果显示：除通气孔隙度外,基质的其他8个理化指标基本涵盖了影响幼苗壮苗指数的关键因素;其中,速效钾含量是对壮苗指数直接影响最大的负相关因子,而速效磷含量、电导率和总孔隙度对壮苗指数则有较大的正向直接作用,并且三者通过速效钾含量对壮苗指数产生较大的负向间接作用;此外,基质容重对壮苗指数也有一定的负向直接作用。综合评价结果显示：S9和S7混配基质也V( SB)：V( BA)：V( GS)=4：3：3页对油茶育苗效果的综合得分较高,分别为14.363和14.337,建议将S9混配基质作为油茶育苗的首选替代基质,S7混配基质作为备选基质。     

Candida albicans β-1,2 mannosyl transferase Bmt3: Preparation and evaluation of a β (1,2), α (1,2)-tetramannosyl fluorescent substrate

We describe for the first time the chemical synthesis of a tetramannoside, containing both α (1 → 2) and β (1 → 2) linkages. Dodecylthio (lauryl) glycosides were prepared from odorless dodecyl thiol and used as donors for the glycosylation steps. This tetramannoside, was coupled to a mantyl group, and revealed to be a perfect substrate of β-mannosyltransferase Bmt3, confirming the proposed specificity and allowing the preparation of a pentamannoside sequence (β Man (1,2) β Man (1,2) α Man (1,2) α Man (1,2) α Man) usable as a novel substrate for further elongation studies.     

Mechanisms Preserving Insulin Action during High Dietary Fat Intake

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Elevational patterns of carbon,nitrogen and phosphorus in understory bryophytes on the eastern slope of Gongga Mountain,China

Aims The nutrient uptake, requirement and releasing rates of bryophytes are very different from those of tracheophytes. However, it is difficult to make a quantitative evaluation of bryophytes’ roles in nutrient cycling and their specific eco-physiological adaptations due to lack of knowledge of their concentrations and stoichiometric ratios of carbon (C), nitrogen (N) and phosphorus (P). To fill this gap, the present study aims to investigate: (i) what are the elevational trends of C, N and P concentrations and stoichiometric ratios of bryophytes? (ii) whether C, N and P concentrations and stoichiometric ratios of bryophytes differ between different bryophyte types (in terms of the growth form and living substrate)? and (iii) how do the exponent scalings of N and P of bryophytes change along the elevational gradient?     

Postglacial ecotype formation under outcrossing and self‐fertilization in Arabidopsis lyrata

The formation of ecotypes has been invoked as an important driver of postglacial biodiversity, because many species colonized heterogeneous habitats and experienced divergent selection. Ecotype formation has been predominantly studied in outcrossing taxa, while far less attention has been paid to the implications of mating system shifts. Here, we addressed whether substrate‐related ecotypes exist in selfing and outcrossing populations of Arabidopsis lyrata subsp. lyrata and whether the genomic footprint differs between mating systems. The North American subspecies colonized both rocky and sandy habitats during postglacial range expansion and shifted the mating system from predominantly outcrossing to predominantly selfing in a number of regions. We performed an association study on pooled whole‐genome sequence data of 20 selfing or outcrossing populations, which suggested genes involved in adaptation to substrate. Motivated by enriched gene ontology terms, we compared root growth between plants from the two substrates in a common environment and found that plants originating from sand grew roots faster and produced more side roots, independent of mating system. Furthermore, single nucleotide polymorphisms associated with substrate‐related ecotypes were more clustered among selfing populations. Our study provides evidence for substrate‐related ecotypes in A. lyrata and divergence in the genomic footprint between mating systems. The latter is the likely result of selfing populations having experienced divergent selection on larger genomic regions due to higher genome‐wide linkage disequilibrium.     

Binding of FAD and tryptophan to the tryptophan 6‐halogenase Thal is negatively coupled

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH₂), O₂, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH₂ by flavin reductases.