A new type of ultrasensitive bioluminogenic enzyme substrates. I. Enzyme substrates with D-luciferin as leaving group

Derivatives of D-luciferin, D-luciferin methyl ester, D-luciferin O-sulfate, D-luciferin O-phosphate, D-luciferyl-L-N alpha-arginine and D-luciferyl-L-phenylalanine were used as highly sensitive substrates for carboxylic esterase, arylsulfatase, alkaline phosphatase and carboxypeptidases A, B and N. Enzymatic cleavage of the compounds by enzymes leading to the release of D-luciferin was demonstrated. Kinetic constants have been determined for D-luciferin methyl ester and carboxylic esterase, for D-luciferin O-sulfate and arylsulfatase, for D-luciferin O-phosphate and alkaline phosphatase, for D-luciferyl-L-phenylalanine and carboxypeptidase A, and for carboxypeptidases B and N and D-luciferyl-L-N alpha-arginine. All compounds proved to be highly sensitive substrates for the respective enzymes, permitting a limit of detection for enzymes between 10 and 500 fg per assay.     

Luciferin derivatives in bioluminescence-enhanced enzyme immunoassays

Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins (illustrated with human urinary kallikrein, bradykinin and the determination of human urinary kallikrein antibody titres) have been developed. The usable ranges of the standard curves are from 5 pg to 5000 pg per litre. The relative intra-assay coefficients of variation of the tests were between 2% and 6%, and the inter-assay coefficients of variation between 4% and 12%.     

A novel bioluminogenic assay for α-chymotrypsin

The use of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin as a novel substrate for α-chymotrypsin has been demonstrated. The kinetic parameters determined are K_M = 0.38mmol/L, k_cat = 6.5 s^?1 and k_cat/k_M = 17,100 (L/mols). The test principle of the coupled assay is the release of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emission, by firefly luciferase (Photinus pyralis) and can be quantified in a luminometric assay. The detection limit for chymotrypsin was found to be 0.3 ng per assay. 6-(N-acetyl-L -phenylalanyl)-aminoluciferin has been synthesized as an example for a new class of highly sensitive substrates. By modification of the peptide residue these new substrates may be suitable for ultrasensitive detection of different proteinases.     

Further indications of the multicomponent nature of the acrosome reaction-inducing substance of human follicular fluid

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with ³H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley-Liss, Inc.     

Climate limitation at the cold edge: contrasting perspectives from species distribution modelling and a transplant experiment

The role of climate in determining range margins is often studied using species distribution models (SDMs), which are easily applied but have well-known limitations, e.g. due to their correlative nature and colonization and extinction time lags. Transplant experiments can give more direct information on environmental effects, but often cover small spatial and temporal scales. We simultaneously applied a SDM using high-resolution spatial predictors and an integral projection (demographic) model based on a transplant experiment at 58 sites to examine the effects of microclimate, light and soil conditions on the distribution and performance of a forest herb, Lathyrus vernus, at its cold range margin in central Sweden. In the SDM, occurrences were strongly associated with warmer climates. In contrast, only weak effects of climate were detected in the transplant experiment, whereas effects of soil conditions and light dominated. The higher contribution of climate in the SDM is likely a result from its correlation with soil quality, forest type and potentially historic land use, which were unaccounted for in the model. Predicted habitat suitability and population growth rate, yielded by the two approaches, were not correlated across the transplant sites. We argue that the ranking of site habitat suitability is probably more reliable in the transplant experiment than in the SDM because predictors in the former better describe understory conditions, but that ranking might vary among years, e.g. due to differences in climate. Our results suggest that L. vernus is limited by soil and light rather than directly by climate at its northern range edge, where conifers dominate forests and create suboptimal conditions of soil and canopy-penetrating light. A general implication of our study is that to better understand how climate change influences range dynamics, we should not only strive to improve existing approaches but also to use multiple approaches in concert.     

Local temperatures inferred from plant communities suggest strong spatial buffering of climate warming across Northern Europe

Recent studies from mountainous areas of small spatial extent (<2500 km²) suggest that fine‐grained thermal variability over tens or hundreds of metres exceeds much of the climate warming expected for the coming decades. Such variability in temperature provides buffering to mitigate climate‐change impacts. Is this local spatial buffering restricted to topographically complex terrains? To answer this, we here study fine‐grained thermal variability across a 2500‐km wide latitudinal gradient in Northern Europe encompassing a large array of topographic complexities. We first combined plant community data, Ellenberg temperature indicator values, locally measured temperatures (LmT) and globally interpolated temperatures (GiT) in a modelling framework to infer biologically relevant temperature conditions from plant assemblages within <1000‐m² units (community‐inferred temperatures: CiT). We then assessed: (1) CiT range (thermal variability) within 1‐km² units; (2) the relationship between CiT range and topographically and geographically derived predictors at 1‐km resolution; and (3) whether spatial turnover in CiT is greater than spatial turnover in GiT within 100‐km² units. Ellenberg temperature indicator values in combination with plant assemblages explained 46–72% of variation in LmT and 92–96% of variation in GiT during the growing season (June, July, August). Growing‐season CiT range within 1‐km² units peaked at 60–65°N and increased with terrain roughness, averaging 1.97 °C (SD = 0.84 °C) and 2.68 °C (SD = 1.26 °C) within the flattest and roughest units respectively. Complex interactions between topography‐related variables and latitude explained 35% of variation in growing‐season CiT range when accounting for sampling effort and residual spatial autocorrelation. Spatial turnover in growing‐season CiT within 100‐km² units was, on average, 1.8 times greater (0.32 °C km^?1) than spatial turnover in growing‐season GiT (0.18 °C km^?1). We conclude that thermal variability within 1‐km² units strongly increases local spatial buffering of future climate warming across Northern Europe, even in the flattest terrains.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

Remotely sensed spectral heterogeneity as a proxy of species diversity: Recent advances and open challenges

Environmental heterogeneity is considered to be one of the main factors associated with biodiversity given that areas with highly heterogeneous environments can host more species due to their higher number of available niches. In this view, spatial variability extracted from remotely sensed images has been used as a proxy of species diversity, as these data provide an inexpensive means of deriving environmental information for large areas in a consistent and regular manner. The aim of this review is to provide an overview of the state of the art in the use of spectral heterogeneity for estimating species diversity. We will examine a number of issues related to this theme, dealing with: i) the main sensors used for biodiversity monitoring, ii) scale matching problems between remotely sensed and field diversity data, iii) spectral heterogeneity measurement techniques, iv) types of species taxonomic diversity measures and how they influence the relationship between spectral and species diversity, v) spectral versus genetic diversity, and vi) modeling procedures for relating spectral and species diversity. Our review suggests that remotely sensed spectral heterogeneity information provides a crucial baseline for rapid estimation or prediction of biodiversity attributes and hotspots in space and time.