Changes in organic phosphorus composition in boreal forest humus soils: the role of iron and aluminium

Organic phosphorus (P) is an important component of boreal forest humus soils, and its concentration has been found to be closely related to the concentration of iron (Fe) and aluminium (Al). We used solution and solid state ³¹P NMR spectroscopy on humus soils to characterize organic P along two groundwater recharge and discharge gradients in Fennoscandian boreal forest, which are also P sorption gradients due to differences in aluminium (Al) and iron (Fe) concentration in the humus. The composition of organic P changed sharply along the gradients. Phosphate diesters and their degradation products, as well as polyphosphates, were proportionally more abundant in low Al and Fe sites, whereas phosphate monoesters such as myo-, scyllo- and unknown inositol phosphates dominated in high Al and Fe soils. The concentration of inositol phosphates, but not that of diesters, was positively related to Al and Fe concentration in the humus soil. Overall, in high Al and Fe sites the composition of organic P seemed to be closely associated with stabilization processes, whereas in low Al and Fe sites it more closely reflected inputs of organic P, given the dominance of diesters which are generally assumed to constitute the bulk of organic P inputs to the soil. These gradients encompass the broad variation in soil properties detected in the wider Fennoscandian boreal forest landscape, as such our findings provide insight into the factors controlling P biogeochemistry in the region but should be of relevance to boreal forests elsewhere.     

Chlorophyll a/b-binding proteins, pigment conversions, and early light-induced proteins in a chlorophyll b-less barley mutant.

Monospecific polyclonal antibodies have been raised against synthetic peptides derived from the primary sequences from different plant light-harvesting Chl a/b-binding (LHC) proteins. Together with other monospecific antibodies, these were used to quantify the levels of the 10 different LHC proteins in wild-type and chlorina f2 barley (Hordeum vulgare L.), grown under normal and intermittent light (ImL). Chlorina f2, grown under normal light, lacked Lhcb1 (type I LHC II) and Lhcb6 (CP24) and had reduced amounts of Lhcb2, Lhcb3 (types II and III LHC II), and Lhcb4 (CP 29). Chlorina f2 grown under ImL lacked all LHC proteins, whereas wild-type ImL plants contained Lhcb5 (CP 26) and a small amount of Lhcb2. The chlorina f2 ImL thylakoids were organized in large parallel arrays, but wild-type ImL thylakoids had appressed regions, indicating a possible role for Lhcb5 in grana stacking. Chlorina f2 grown under ImL contained considerable amounts of violaxanthin (2-3/reaction center), representing a pool of phototransformable xanthophyll cycle pigments not associated with LHC proteins. Chlorina f2 and the plants grown under ImL also contained early light-induced proteins (ELIPs) as monitored by western blotting. The levels of both ELIPs and xanthophyll cycle pigments increased during a 1 h of high light treatment, without accumulation of LHC proteins. These data are consistent with the hypothesis that ELIPs are pigment-binding proteins, and we suggest that ELIPs bind photoconvertible xanthophylls and replace "normal" LHC proteins under conditions of light stress.     

Bombesin, neurotensin and pro-gamma-melanotropin immunoreactants in human milk

The concentration of bombesin-like, neurotensin-like and pro-gamma-melanotropin-like immunoreactants in human skim milk was measured by radioimmunoassay and found to be 235 pg/ml (mean, n = 13, range 60-430 pg/ml), 63 pg/ml (mean, n = 13, range 20-105 pg/ml) and 2.4 ng/ml (mean, n = 6, range 1.2-5 ng/ml), respectively. The concentrations were 5-10 times higher than in plasma. High performance liquid chromatography showed that the neurotensin and pro-gamma-melanotropin immunoreactants co-eluted with the authentic peptides. Bombesin gave three peaks, one co-eluting with authentic bombesin and one with porcine gastrin releasing peptide 14-27, whereas another one had a shorter elution time, suggesting a less hydrophobic fragment, possibly even smaller than gastrin releasing peptide 14-27.     

Large scale geographic clines of parasite damage to Populus tremula L.

According to the geographic mosaic theory of coevolution (GMTC), clines of traits reflecting local co‐adaptation (including resistance genes) should be common between a host and its parasite and should persist across time. To test the GMTC‐assumption of persistent clinal patterns we compared the natural prevalence of two parasites on aspen Populus tremula trees: mining moths of the genus Phyllocnistis and leaf rust Melampsora spp. Damage data were collated from the Swedish National Forest Damage Inventory (2004–2006). In addition, occurrence of the parasites was scored in field conditions in two common gardens in the north and south of Sweden over five growing seasons (2004–2008), then related to biomass (stem height and diameter) and to concentrations of eleven leaf phenolics. Phyllocnistis mainly occurred in the northern garden, a distribution range which was confirmed by the countrywide inventory, although Phyllocnistis was more abundant on southern clones, providing evidence for possible local maladaptation. Melampsora occurred all over the country and in both gardens, but built up more quickly on northern clones, which suggests a centre of local clone maladaptation in the north. Stem growth also followed a clinal pattern as did the concentration of three phenolic compounds: benzoic acid, catechin and cinnamic acid. However, only benzoic acid was related to parasite presence: negatively to Phyllocnistis and positively to Melampsora and it could thus be a potential trait under selection. In conclusion, clines of Phyllocnistis were stronger and more persistent compared to Melampsora, which showed contrasting clines of varying strength. Our data thus support the assumption of the GMTC model that clines exist in the border between hot and cold spots and that they may be less persistent for parasites with an elevated gene flow, and/or for parasites which cover relatively larger hot spots surrounded by fewer cold spots.     

Thiol-directed immobilization of recombinant IgG-binding receptors

A genetic engineering approach is described for directed immobilization of IgG-binding receptors to a thiol-containing matrix using a single cysteine residue. The cysteine residue is introduced into the C-terminal part of receptors based on staphylococcal protein A. Receptors containing one, two or five IgG-binding domains were produced in Escherichia coli and subsequently immobilized on thiopropyl-Sepharose. A high amount, 5 mumol/ml gel (45 mg/ml), of recombinant receptor could be rapidly immobilized to the solid support and both the gel and the immobilized receptor could be regenerated by reduction and oxidation reactions. The gel was used for affinity purification of human IgG and analysis of IgG-binding capacity at different amounts of immobilized recombinant protein show the same maximal IgG-binding capacity (20-25 mg/ml) for all three immobilized receptors. However, at low substitution grade of receptors, the immobilized receptor molecules were shown to bind one (Z-Cys) and two (ZZ-Cys) IgG molecules, respectively. These results demonstrate that the immobilized protein molecules are in a functionally active form and that a two-domain receptor can bind two molecules of IgG without steric hindrance. Interestingly, the five-domain receptor (ZV-Cys), with a structure similar to native protein A, can only bind approximately two IgG molecules, despite the five-domain structure of the molecule.     

Lhca5 interaction with plant photosystem I

In the outer antenna (LHCI) of higher plant photosystem I (PSI) four abundantly expressed light-harvesting protein of photosystem I (Lhca)-type proteins are organized in two heterodimeric domains (Lhca1/Lhca4 and Lhca2/Lhca3). Our cross-linking studies on PSI-LHCI preparations from wildtype Arabidopsis and pea plants indicate an exclusive interaction of the rarely expressed Lhca5 light-harvesting protein with LHCI in the Lhca2/Lhca3-site. In PSI particles with an altered LHCI composition Lhca5 assembles in the Lhca1/Lhca4 site, partly as a homodimer. This flexibility indicates a binding-competitive model for the LHCI assembly in plants regulated by molecular interactions of the Lhca proteins with the PSI core.     

Direct cellular lysis/protein extraction protocol for soil metaproteomics

We present a novel direct protocol for deep proteome characterization of microorganisms in soil. The method employs thermally assisted detergent-based cellular lysis (SDS) of soil samples, followed by TCA precipitation for proteome extraction/cleanup prior to liquid chromatography-mass spectrometric characterization. This approach was developed and optimized using different soils inoculated with genome-sequenced bacteria (Gram-negative Pseudomonas putida or Gram-positive Arthrobacter chlorophenolicus). Direct soil protein extraction was compared to protein extraction from cells isolated from the soil matrix prior to lysis (indirect method). Each approach resulted in identification of greater than 500 unique proteins, with a wide range in molecular mass and functional categories. To our knowledge, this SDS-TCA approach enables the deepest proteome characterizations of microbes in soil to date, without significant biases in protein size, localization, or functional category compared to pure cultures. This protocol should provide a powerful tool for ecological studies of soil microbial communities.     

Muscle Sympathetic Nerve Activity Is Related to a Surrogate Marker of Endothelial Function in Healthy Individuals

Background

Evidence from animal studies indicates the importance of an interaction between the sympathetic nervous system and the endothelium for cardiovascular regulation. However the interaction between these two systems remains largely unexplored in humans. The aim of this study was to investigate whether directly recorded sympathetic vasoconstrictor outflow is related to a surrogate marker of endothelial function in healthy individuals.

Methods and Results

In 10 healthy normotensive subjects (3 f/7 m), (age 37±11 yrs), (BMI 24±3 kg/m²) direct recordings of sympathetic action potentials to the muscle vascular bed (MSNA) were performed and endothelial function estimated with the Reactive Hyperaemia- Peripheral Arterial Tonometry (RH-PAT) technique. Blood samples were taken and time spent on leisure-time physical activities was estimated. In all subjects the rate between resting flow and the maximum flow, the Reactive Hyperemic index (RH-PAT index), was within the normal range (1,9–3,3) and MSNA was as expected for age and gender (13–44 burst/minute). RH-PAT index was inversely related to MSNA (r = −0.8, p = 0.005). RH-PAT index and MSNA were reciprocally related to time (h/week) spent on physical activity (p = 0.005 and p = 0.006 respectively) and platelet concentration (PLT) (p = 0.02 and p = 0.004 respectively).

Conclusions

Our results show that sympathetic nerve activity is related to a surrogate marker of endothelial function in healthy normotensive individuals, indicating that sympathetic outflow may be modulated by changes in endothelial function. In this study time spent on physical activity is identified as a predictor of sympathetic nerve activity and endothelial function in a group of healthy individuals. The results are of importance in understanding mechanisms underlying sympathetic activation in conditions associated with endothelial dysfunction and emphasise the importance of a daily exercise routine for maintenance of cardiovascular health.     

Rapid turn-over of plasma membrane sphingomyelin and cholesterol in baby hamster kidney cells after exposure to sphingomyelinase

Plasma membrane sphingomyelin in baby hamster kidney (BHK-21) cells was hydrolyzed with sphingomyelinase (Staphylococcus aureus) and the effects on membrane cholesterol translocation and the properties of membrane bound adenylate cyclase and Na+/K(+)-ATPase were determined. Exposure of confluent BHK-21 cells to 0.1 U/ml of sphingomyelinase led to the degradation (at 37 degrees C) of about 60% of cell sphingomyelin. No simultaneous hydrolysis of phosphatidylcholine occurred. The hydrolysis of sphingomyelin subsequently led to the translocation (within 40 min) of about 50-60% of cell [3H]cholesterol from a cholesterol oxidase susceptible pool to an oxidase resistant compartment. The translocation of [3H]cholesterol from the cell surface to intracellular membranes was accompanied by a paralleled increase in [3H]cholesterol ester formation. When cells were first exposed to sphingomyelinase (to degrade sphingomyelin) and then incubated without the enzyme in serum-free media, the mass of cell sphingomyelin decreased initially (by 60%), but then began to increase and reached control levels within 3-4 h. The rapid re-synthesis of sphingomyelin was accompanied by an equally rapid normalization of cell [3H]cholesterol distribution. The re-formation of cell sphingomyelin also led to a decreased content of cellular [3H]cholesterol esters, indicating that unesterified [3H]cholesterol was pulled out of the cholesterol ester cycle and transported to the cell surface. Exposure of BHK-21 cells to sphingomyelinase further led to a dramatically decreased activity of ouabain-sensitive Na+/K(+)-ATPase, whereas forskolin-stimulated adenylate cyclase activity was not affected. The activity of Na+/K(+)-ATPase returned to normal in parallel with the normalization of cell sphingomyelin mass and cholesterol distribution. We conclude that sphingomyelin has profound effects on the steady-state distribution of cell cholesterol, and that manipulations of cell sphingomyelin levels directly and reversibly affects the apparent distribution of cholesterol. Changes in the lipid composition of the plasma membrane also appears to selectively affect important metabolic reactions in that compartment.