Bioluminescent assay of NADPH-dependent isocitrate dehydrogenase and its substrates and cofactors

A bioluminescent assay for NADPH-dependent isocitrate dehydrogenase and for its substrates and cofactors was developed. The method is based on continuous NADPH monitoring in the reaction. The linear range of the assay for the enzyme activity is from 0.05 U/liter to 30 U/liter. It is about 300 times more sensitive than the corresponding spectrophotometric assay at 340 nm. Good correlation exists between both assays. Isocitrate, NADP, manganese, and magnesium can be measured at picomole levels. The applicability of the assays to serum analysis is discussed.     

Distribution and development of necroticSphagnum patches in two Estonian raised bogs

Sphagnum mosses dominate the plant cover of boreal bogs and accumulate carbon as peat. However, discoloured necroticSphagnum patches are also common in bogs. NecroticSphagnum inhibits peat accumulation, and consequently these areas may sink with respect to their surroundings with healthy mosses and continuing peat accumulation. Therefore, necrotic patches in the moss carpet could have an important role in triggering the succession ofSphagnum communities and the differentiation of bog microtopography. Our main aim was to find out how necroticSphagnum patches are distributed on a microtopographic gradient and amongSphagnum species. Based on these results we discuss the development and likely role of necrotic patches. It was found that necrotic patches occur on all types of bog microforms and contain the most of commonSphagnum species. Necrotic patches were more common and larger in wet hollows. The development of necrotic patches depends on their location on the microtopographic gradient. Necrotic patches on higher microforms usually re-vegetate, whereas those in hollows can result in mud-bottom hollows.     

On the meaning of the distance-to-target weighting method and normalisation in Life Cycle Impact assessment

Distance-to-target weighting methods are widely used in life cycle impact assessment. The methods rank impacts as being more important the further away society’s activities are from achieving the desired targets for the pollutants. However, we feel that the scientific bases of the distance-to-target methods still need more clarification. This article illustrates how multiattribute value theory (MAVT) can be applied to interpret the impact category weights as well as the aggregation rule and normalisation used in the distant-to-target methods. Our comparison revealed that under certain conditions two of the three commonly used impact assessment methods (Ecoindicator 95, ET-method) applying distance-to-target weighting are consistent with the impact assessment framework derived from MAVT. This consistency holds for non-zero targets with equal importance and linear damage functions passing through the origin. We show that the MAVT framework offers a foundation for the methodological development in life cycle impact assessment.     

Species–area relationships of red-listed species in old boreal forests: a large-scale data analysis

Aim Species–area relationships are often applied, but not generally approved, to guide practical conservation planning. The specific species group analysed may affect their applicability. We asked if species–area curves constructed from extensive databases of various sectors of natural resource administration can provide insights into large‐scale conservation of boreal forest biodiversity if the analyses are restricted only to red‐listed species. Location Finland, northern Europe. Methods Our data included 12,645 records of 219 red‐listed Coleoptera and Fungi from the whole of Finland. The forest data also covered the entire country, 202,761 km². The units of species–area analyses were 224 municipalities where the red‐listed forest species have been observed. We performed a hierarchical partitioning analysis to reveal the relative importance of different potential explanatory variables. Based on the results, for all red‐listed species, species associated with coniferous trees and for Fungi, the area of economically over‐aged forests explained the best the variation in data. For species associated with deciduous trees and Coleoptera, the forest area explained better variation in data than the area of old forests. In the subsequent log–log species–area regression analyses, we used the best variables as the explanatory variable for each species group. Results There was a strong relationship between the number of all red‐listed species and the area of old forests remaining, with a z‐value of 0.45. The area explained better the number of species associated with conifer trees and Fungi than the number of species associated with deciduous trees and Coleoptera. Main conclusions The high z‐values of species–area curves indicate that the remaining old‐growth patches constitute a real archipelago for the conifer‐associated red‐listed species, since lower values had been expected if the surrounding habitat matrix were a suitable habitat for the species analysed.     

Nanovesicles released by OKT3 hybridoma express fully active antibodies

Recent findings have shown that nanovesicles preparations from either primary immune cells culture supernatants or plasma contain immunoglobulins, suggesting that a natural way of antibody production may be through exosome release. To verify this hypothesis, we used the OKT3 hybridoma clone, which produces a murine IgG2a monoclonal antibody used to reduce rejection in patients undergoing organ transplantation. We showed exosome-associated immunoglobulins in hybridoma supernatants, by Western blot, nanoscale flow cytometry and immunocapture-based ELISA. The OKT3-exo was also being able to trigger cytokines production in both CD4 and CD8 T cells. These results show that nanovesicles contain immunoglobulin and could be used for immunotherapy. These data could lead to a new approach to improve the effectiveness of therapeutic antibodies by exploiting their natural property to be expressed on nanovesicle membrane, that probably render them more stable and as a consequence more capable to interact with their specific ligand in the best way.     

Bioluminescent assay of D-3-hydroxybutyrate in serum

An enzymatic assay of D -3-hydroxybutyrate in which the hydroxybutyrate dehydrogenase reaction is coupled to the bacterial oxidoreductase—uciferase system is described. The bioluminescent assay is based on either, end-point, or on initial velocity measurements. This simple and rapid assay requires a single serum sample of 10 μl. Its linear range covers two orders of magnitude from 10^?6 mol/I upwards. This assay is suitable for the routine determination of D -3-hydroxybutyrate in human blood with good accuracy.     

Packed-bed reactor of immobilized bacterial bioluminescence enzymes: A potential high-sensitivity detector for automated analyzers

Commercial bacterial bioluminescence enzymes were immobilized on agarose with 85% recovery of coupled activity. A light-emitting continuous-flow column for NADH monitoring at picomole levels was developed. The present enzyme reactor exhibited good operational stability in continuous use, which makes the system applicable to a sensitive detector for automated analyzers.     

Bovine colostrum ultrafiltrate supplemented with adult bovine serum and transferrin: An effective fbs substitute for cultivation of vero and CHO-K1 cells

Summary A mixture containing an ultrafiltrate fraction (UF) of bovine colostrum (6.7%), adult bovine serum (BS) (1%), and human holo-transferrin (hTF) (5 mg/liter) was developed for cultivation of Chinese hamster ovary cells (CHO-K1) and African green monkey kidney cells (Vero). The growth-supporting activity of the mixture (UF/BS/hTF) was comparable to that of 1 to 10% fetal bovine serum (FBS) and considerably better than 1 to 2% BS. Cells could be directly seeded from FBS-supplemented medium to UF/BS/hTF-supplemented medium without any weaning period, even at initial plating density of 1700 cells/ml. Vero and CHO-K1 cells were cultivated in UF/BS/hTF-supplemented media for up to 43 days without any apparent reduction in growth. The UF/BS/hTF mixture could also be used as a freezing medium. Cells were passaged twice in the mixture, frozen, and stored at liquid N₂ for 11 wk. After thawing, the viability of Vero and CHO-K1 cells was reduced 13 and 7%, respectively, and both cell lines started to grow well. Additional hTF could be replaced with bovine holo-transferrin, although a high concentration (150 mg/liter) should be used for CHO-K1 cells. The results suggest that the UF/BS/hTF mixture provides a new economical alternative to FBS in cultivation of Vero and CHO-K1 cells in the presence of reduced protein amounts.