Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.     

Antisense RNA Inhibition of RbcS Gene Expression Reduces Rubisco Level and Photosynthesis in the C4 Plant Flaveria bidentis

The C4 dicot Flaveria bidentis was genetically transformed with an antisense RNA construct targeted to the nuclear-encoded gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; RbcS). RbcS mRNA levels in leaves of transformants were reduced by as much as 80% compared to wild-type levels, and extractable enzyme activity was reduced by up to 85%. There was no significant effect of transformation with the gene construct on levels of other photosynthetic enzymes. Antisense transformants with reduced Rubisco activity exhibited a stunted phenotype. Rates of photosynthesis were reduced in air at high light and over a range of CO2 concentrations but were unaffected at low light. From these results we conclude that, as is the case in C3 plants, Rubisco activity is a major determinant of photosynthetic flux in C4 plants under high light intensities and air levels of CO2.     

Prostasomes from four different species are able to produce extracellular adenosine triphosphate (ATP)

Background

Prostasomes are extracellular vesicles. Intracellularly they are enclosed by another larger vesicle, a so called “storage vesicle” equivalent to a multivesicular body of late endosomal origin. Prostasomes in their extracellular context are thought to play a crucial role in fertilization.

Methods

Prostasomes were purified according to a well worked-out schedule from seminal plasmas obtained from human, canine, equine and bovine species. The various prostasomes were subjected to SDS-PAGE separation and protein banding patterns were compared. To gain knowledge of the prostasomal protein systems pertaining to prostasomes of four different species proteins were analyzed using a proteomic approach. An in vitro assay was employed to demonstrate ATP formation by prostasomes of different species.

Results

The SDS-PAGE banding pattern of prostasomes from the four species revealed a richly faceted picture with most protein bands within the molecular weight range of 10–150 kDa. Some protein bands seemed to be concordant among species although differently expressed and the number of protein bands of dog prostasomes seemed to be distinctly fewer. Special emphasis was put on proteins involved in energy metabolic turnover. Prostasomes from all four species were able to form extracellular adenosine triphosphate (ATP). ATP formation was balanced by ATPase activity linked to the four types of prostasomes.

Conclusion

These potencies of a possession of functional ATP-forming enzymes by different prostasome types should be regarded against the knowledge of ATP having a profound effect on cell responses and now explicitly on the success of the sperm cell to fertilize the ovum.

General significance

This study unravels energy metabolic relationships of prostasomes from four different species.     

Characterization of the primary radical pair in reaction centers of Heliobacillus mobilis by 13C photo-CIDNP MAS NMR

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by (13)C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. In the (13)C photo-CIDNP MAS NMR spectra of heliobacterial membrane fragments, two sets of signals are observed, allowing characterization of the primary radical pair. One set, showing enhanced absorptive (positive) signals, arises from the BChl g donor, while the set of emissive (negative) signals is assigned to the 8(1)-hydroxy Chl a acceptor. Hence, under these sample conditions, both donor and acceptor sides are either monomeric or composed of identical cofactors. The occurrence of the differential relaxation (DR) mechanism suggests a donor triplet lifetime in the microsecond range. It appears that the occurrence of the solid-state photo-CIDNP effect is a general feature of primary radical pairs in natural photosynthesis.     

Large scale experimental effects of three levels of sheep densities on an alpine ecosystem

Plant–herbivore dynamics is a major topic in ecological research, but empirical knowledge on the ecological effects of different densities of large grazers from fully replicated experiments is rare. Previous studies have focused on grazing vs no grazing, and our understanding of the extent to which different levels of grazing alter vegetation composition, and how quickly such effects can be measured, is therefore limited. We performed a fully replicated, short‐term (four‐year) experiment using large enclosures (each ～0.3 km²) with three different sheep densities (no grazing, low grazing and high grazing, respectively) in an alpine environment with summer grazing in southern Norway to address these issues. Sheep grazing mainly affected plant species at high densities of sheep as compared to no sheep after a four‐year treatment; few effects of low sheep densities were detectable. Highly selected herbs, herbs suggested vulnerable to trampling, and woody species decreased, while most graminoids, one ruderal, one prostrate species and two bryophyte taxa increased at high sheep densities. We found contrasting responses within main functional groups highlighting that fine details of plant life histories need to be known for responses to grazing to be successfully predicted. Vascular plant cover and bare soil responded to sheep density after two years of treatment, but only for one of the species was frequency change observed at this stage. Overall, plants in low grazing plots were found to be almost unaffected. Changes in abundance were mainly found at the no grazing and high grazing treatments. Plant species that decreased at high grazing generally increased at no grazing and vice versa, suggesting a response to both cessation of grazing and enhanced grazing respectively. Our study demonstrates, beyond a simple comparison of heavily grazed and non‐grazed sites, that herbivore effects on plants are typically non‐linearly related to herbivore density, and that the speed of plant responses will depend both on the plant property examined and the grazing pressure.     

Monocomponent endoglucanase treatment increases the reactivity of softwood sulphite dissolving pulp

Softwood dissolving pulp was treated with a commercial monocomponent fungal endocellulase. The reactivity of the pulp for the production of rayon and cellulose derivatives as determined with the Fock method increased drastically with relatively low amounts of enzyme, and the yield loss and decrease of viscosity were moderate. The mechanism behind the increased reactivity is discussed.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

Age class,density and temporal effects on diet composition of sheep in an alpine ecosystem

Understanding diet selection is important since diet determines energy intake and therefore growth of ungulate populations. Yet very few studies have reported annual variation in diet. Density-dependent diet choice by large herbivores has been reported several times, but these studies are typically either short-term or they lack replication of the density treatment. In a landscape-scale experiment with 3 replicates of two densities (25 and 80 individuals/km²) of domestic sheep, we determined diet composition using microhistological analysis during 6 summer grazing seasons (2002–2007) in alpine habitats. We tested how age class, density and temporal variation (within season, annually, and over years) affected summer diet. There was marked evidence of additive effects of these factors on overall diet composition, but interactions were few. The interaction between density and annual variation was an important determinant of the proportion of the main forage component (Avenella flexuosa), but not of the proportions of herbs, Salix spp. and for “other” forage plants. Surprisingly, the density effect on this intermediate quality forage (A. flexuosa) was not consistent among years (both positive, negative and no effects), likely arising due to large variation in the proportion of the other forage plants. We discuss how foraging ecology can supplement the insight from life history theory in explaining variation in vital rates.