The impact of simulated chronic nitrogen deposition on the biomass and N2-fixation activity of two boreal feather moss–cyanobacteria associations

Bryophytes achieve substantial biomass and play several key functional roles in boreal forests that can influence how carbon (C) and nitrogen (N) cycling respond to atmospheric deposition of reactive nitrogen (N_r). They associate with cyanobacteria that fix atmospheric N₂, and downregulation of this process may offset anthropogenic N_r inputs to boreal systems. Bryophytes also promote soil C accumulation by thermally insulating soils, and changes in their biomass influence soil C dynamics. Using a unique large-scale (0.1 ha forested plots), long-term experiment (16 years) in northern Sweden where we simulated anthropogenic N_r deposition, we measured the biomass and N₂-fixation response of two bryophyte species, the feather mosses Hylocomium splendens and Pleurozium schreberi. Our data show that the biomass declined for both species; however, N₂-fixation rates per unit mass and per unit area declined only for H. splendens. The low and high treatments resulted in a 29% and 54% reduction in total feather moss biomass, and a 58% and 97% reduction in total N₂-fixation rate per unit area, respectively. These results help to quantify the sensitivity of feather moss biomass and N₂ fixation to chronic N_r deposition, which is relevant for modelling ecosystem C and N balances in boreal ecosystems.     

Untreated silicone breast implant rupture

Implant rupture is a well-known complication of breast implant surgery that can pass unnoticed by both patient and physician. To date, no prospective study has addressed the possible health implications of silicone breast implant rupture. The aim of the present study was to evaluate whether untreated ruptures are associated with changes over time in magnetic resonance imaging findings, serologic markers, or self-reported breast symptoms. A baseline magnetic resonance imaging examination was performed in 1999 on 271 women who were randomly chosen from a larger cohort of women having cosmetic breast implants for a median period of 12 years (range, 3 to 25 years). A follow-up magnetic resonance imaging examination was carried out in 2001, excluding women who underwent explantation in the period between the two magnetic resonance imaging examinations (n = 44). On the basis of these examinations, the authors identified 64 women who had at least one ruptured implant at the first magnetic resonance imaging examination and, for comparison, all women who had intact implants at both examinations (n = 98). Magnetic resonance images from the two examinations were compared and changes in rupture configuration were evaluated. Comparisons were also made for self-reported breast symptoms occurring during the study period and for changes in serum values of antinuclear antibodies, rheumatoid factor, and cardiolipin antibodies immunoglobulin G and immunoglobulin M. The majority of the women with implant rupture had no visible magnetic resonance imaging changes of their ruptured implants. For 11 implants (11 percent) in 10 women, the authors observed progression of silicone seepage, either as a conversion from intracapsular into extracapsular rupture (n = 7), as progression of extra-capsular silicone (n = 3), or as increasing herniation of the silicone within the fibrous capsule (n = 1); however, in most cases, these changes were minor. Some changes could be ascribed to trauma, but others seemed spontaneous. There was no increase in levels of autoantibodies during the study period in either study group. Women with untreated implant ruptures reported a significant increase in nonspecific breast changes (odds ratio, 2.1; 95 percent confidence interval, 1.2 to 3.8) compared with women without ruptures. On the basis of this first study of women with untreated silicone breast implant rupture, the authors conclude that implant rupture is a relatively harmless condition, which only rarely progresses and gives rise to notable symptoms. Even so, because of a small risk of silicone spread, the authors suggest that women with implant ruptures be followed clinically, if not operated on. Because implant ruptures often occur asymptomatically, any woman with silicone implants, regardless of rupture status, should be evaluated at regular intervals.     

The Danish Registry for Plastic Surgery of the Breast: establishment of a nationwide registry for prospective follow-up,quality assessment,and investigation of breast surgery

Although numerous epidemiologic studies have examined the long-term safety of silicone breast implants during the past decade, there is a relative lack of surveillance data on short-term health effects and complications following cosmetic surgery of the breast. The Danish Registry for Plastic Surgery of the Breast, established in May of 1999, provides plastic surgeons with a nationwide system for the collection of preoperative, perioperative, and postoperative data on women undergoing breast implantation, breast reduction, or mastopexy. The purpose of the Registry is to examine short-term and, eventually, long-term local complications and possible health effects, and to contribute to an ongoing evaluation of surgical results and surveillance of the products. Furthermore, the Registry will allow the identification of new areas for research into cosmetic and reconstructive breast surgery. Women accepting registration in the Danish Registry for Plastic Surgery of the Breast complete a self-administered questionnaire focusing on medical history and demographic and behavioral factors. Preoperative blood samples are drawn for storage. Surgical data, postoperative results, and complications are registered following surgery and at postoperative visits. Currently, registration has been initiated at 24 private and public clinics, representing more than 80 percent of the plastic surgery clinics in Denmark. As of November of 2001, a total of 1472 women with breast implants and 560 women with breast reduction were included in the Registry. These figures are expected to increase annually by 1000 women undergoing breast implantation and 500 women undergoing breast reduction or mastopexy. The authors present their experience of establishing the first nationwide comprehensive clinical-epidemiologic database and biological bank for cosmetic and reconstructive surgery procedures.     

Characterization of a recombinant granzyme B derivative as a "restriction" protease

Blood coagulation factor Xa (FXa) and Thrombin are well-known serine proteases often used for processing of recombinant fusion proteins, but because they are purified from bovine blood or other animal sources, there is a risk of pathogenic contaminants in the preparation of the proteases. We report here the characterization of a recombinant serine protease produced in Escherichia coli, which can be used as a specific and efficient alternative to FXa and Thrombin as processing protease. This recombinant protease is derived from human granzyme B (GrB). The protease is found to be very stable in general, and it performs very well in the cleavage of several different fusion proteins tested and was even found superior to processing by FXa in two cases.     

The Tyrosine Sulfate-rich Domains of the LRR Proteins Fibromodulin and Osteoadherin Bind Motifs of Basic Clusters in a Variety of Heparin-binding Proteins, Including Bioactive Factors

The small leucine-rich repeat proteins, fibromodulin and osteoadherin, have N-terminal extensions with a variable number of O-sulfated tyrosine residues. This modification combined with a number of aspartic and glutamic acid residues results in a highly negatively charged domain of less than 30 amino acids. We hypothesized that this domain shares functional properties with heparin regarding binding to proteins and polypeptides containing clusters of basic amino acids. Two other family members, PRELP and chondroadherin, have distinctly different clusters of basic amino acids in their N and C termini, respectively, and PRELP is known to bind to heparin via this domain. Another heparin-binding protein is the cytokine Oncostatin M, with a different cluster of basic amino acids in its C terminus. We used polypeptides representing these basic domains in solid phase assays and demonstrate interactions with the negatively charged N-terminal domain of fibromodulin and full-length osteoadherin. The tyrosine sulfate domains also bound heparin-binding proteins such as basic fibroblast growth factor-2, thrombospondin I, MMP13, the NC4 domain of collagen IX, and interleukin-10. Fibronectin with large heparin-binding domains did not bind, neither did CILP containing a heparin-binding thrombospondin type I motif without clustered basic amino acids. Affinity depends on the number and position of the sulfated tyrosine residues shown by different binding properties of 10-kDa fragments subfractionated by ion-exchange chromatography. These interactions may sequester growth factors, cytokines, and matrix metalloproteinases in the extracellular matrix as well as contribute to its organization.The integrity of the extracellular matrix depends on a multitude of interactions between molecular constituents leading to the formation of major macromolecular assemblies important for tissue functions. A major component in most types of extracellular matrix is the network of fibrillar structures primarily composed of collagen I in fibrous tissues and bone, whereas cartilage contains the similar collagen II.These collagen fibrils contain a number of associated molecules, often bound to their surface. One such molecule is the distinct collagen IX, containing three triple helical domains each surrounded by non-triple helical domains. Another set of molecules binding to triple helical collagen is the members of the small leucine-rich repeat protein family, such as fibromodulin (1), lumican (2), decorin (3), biglycan (4), PRELP (5), chondroadherin (6), and possibly osteoadherin. The typical LRR³ protein contains 10–11 repeats of some 25 amino acids with leucine residues at conserved locations. This domain represents a common denominator for the family and contains structures providing for interaction with, e.g. triple helical collagen (7–9). The LRR proteins contain an extension at either the N- or C-terminal end or, in a few cases, at both ends. These extensions may contribute to a second function exemplified by PRELP, where the N-terminal with a stretch of clustered arginine residues provides binding to heparin/heparan sulfate containing optimally five or more disaccharides with three sulfate groups each (10). In decorin and biglycan, the N-terminal extension have substituents of glycosaminoglycan chains of dermatan/chondroitin sulfate that can contribute to collagen binding (11) as well as provide putative self interactions with a similar chain on another molecule. In particular, it has been shown that decorin and biglycan will bind via their protein core to the N-terminal globular domain of collagen VI (4) and direct the formation of the collagen VI-beaded filament network, provided that the glycosaminoglycan chains are intact.There are a number of proteins known to interact with heparin. Whereas heparin is not present in the extracellular matrix, these proteins may bind to stretches within the heparan sulfate chains enriched in disaccharides having high sulfate content. The heparan sulfate is found particularly as a component of cell surface proteoglycans such as glypicans (12) and syndecans (13) and of the extracellular matrix proteoglycans perlecan (14) and agrin (15). Important ligands for these chains are growth factors exemplified by members of the FGF family. Other molecules that bind to heparan sulfate include fibronectin, having two such domains with molecular weights of around 20,000 (16). Also several members of the metalloproteinase family contain heparin-binding motifs as do many cytokines.The most common heparin-binding sequence contains clusters of basic amino acids, often with additional proline residues. PRELP and chondroadherin as well as the other proteins mentioned represent examples having such sequences. A different type of motif, first found in thrombospondin I, contains consecutive repeats of a WXZ sequence, where the tryptophan may be mannosylated (17, 18). This is referred to as the thrombospondin type I motif heparin-binding structure. Thrombospondin I in addition contains a heparin-binding basic cluster of amino acids (19). CILP on the other hand only contains the thrombospondin type 1 motif. These domains can bind to heparin with high affinity, an interaction that can be disrupted by high salt.A very different type of extension is found in the N-terminal part of fibromodulin and osteoadherin. These proteins contain a number of tyrosine residues, which may and often do, carry a sulfate group. Thus, fibromodulin contains up to nine such residues and osteoadherin as many as eight, where six are located in the N-terminal and two in the C-terminal extension (20). Any given preparation contains molecules within the same species with a range of levels of O-sulfate-substituted tyrosine. The functional significances of these domains have been unknown. We now show that these domains can mimic heparin in several interactions.     

Identification and characterization of the integrin alpha2beta1 binding motif in chondroadherin mediating cell attachment

Chondroadherin is a leucine-rich repeat protein known to mediate adhesion of isolated cells via the integrin α(2)β(1) and to interact with collagen. In this work, we show that cell adhesion to chondroadherin leads to activation of MAPKs but does not result in cell spreading and division. This is in contrast to the spreading and dividing of cells grown on collagen, although the binding is mediated via the same α(2)β(1) receptor. We identified a cell binding motif, CQLRGLRRWLEAK(318) by mass spectrometry after protease digestion of chondroadherin. Cells adhering to the synthetic peptide CQLRGLRRWLEAK(318) remained round, as was observed when they bound to the intact protein. The peptide added in solution was able to inhibit cell adhesion to the intact protein in a dose-dependent manner and was also verified to bind to the α(2)β(1) integrin. A cyclic peptide, CQLRGLRRWLEAKASRPDATC(326), mimicking the structural constraints of this sequence in the intact protein, showed similar efficiency in inhibiting binding to chondroadherin. The unique peptide motif responsible for cellular binding is primarily located in the octamer sequence LRRWLEAK(318). Binding of cells to the active peptide or to chondroadherin immobilized on cell culture plates rapidly induces intracellular signaling (i.e. ERK phosphorylation). Thus, chondroadherin interaction with cells may be central for maintaining the adult chondrocyte phenotype and cartilage homeostasis. The peptides, particularly the more stable cyclic peptide, open new opportunities to modulate cell behavior in situations of tissue pathology.     

Effects of High Doses of Cholecalciferol in Normal Subjects: A Randomized Double-Blinded,Placebo-Controlled Trial

Background

Vitamin D repletion with high doses of vitamin D is often recommended to patients and healthy subjects. The safety, especially concerning changes in urinary calcium excretion is of great importance.

Methods

In a double-blinded, placebo-controlled study in 40 healthy volunteers, we examined the changes in mineral metabolism during supplementation with 3000 IU of oral cholecalciferol daily during 4 months.

Results

Both 25(OH)vitamin D and 1,25(OH)₂vitamin D increased significantly in the active treated group as compared to the placebo group (186% versus 14% (P<0.001) and 28% versus – 8% (P<0.001)). No change was observed in urinary calcium excretion in the active group compared to the placebo group (P = 0.891). Fibroblast growth factor 23 increased significantly by 10% (P<0.018) in the active group. However, there was no difference in changes in FGF23 between treatment groups (P = 0.457).

Conclusion

High dose cholecalciferol significantly increases 25(OH)vitamin D and 1,25(OH)₂vitamin D levels compared to placebo. No changes in urinary calcium excretion or other measured components of the mineral metabolism were found between groups.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00952562","term_id":"NCT00952562"}}NCT00952562.     

The return of an experimentally N-saturated boreal forest to an N-limited state: observations on the soil microbial community structure,biotic N retention capacity and gross N mineralisation

Background and aims

To find out how N-saturated forests can return to an N-limited state, we examined the recovery of biotic N sinks under decreasing N supply.

Methods

. We studied a 40-year-old experiment in Pinus sylvestris forest, with control plots, N0, three N treatments, N1-N3, of which N3 was stopped after 20 years, allowing observation of recovery.

Results

In N3, the N concentration in foliage was still slightly elevated, but the N uptake capacity of ectomycorrhizal (ECM) roots in N3 was no longer lower than in N0. Per area the amount of a biomarker for fungi, here mainly attributed ECM, was higher in N3 and N0 than in N1 and N2. Retention of labeled ¹⁵NH₄ ⁺ by the soil was greater in the control (99 %) and N3 (86 %), than in N1 (45 %) and N2 (29 %); we ascribe these differences to biotic retention because cation exchange capacity did not vary. Gross N mineralisation and retention of N correlated, negatively and positively, respectively, with abundance of ECM fungal biomarker.

Conclusions

. The results suggest a key role for ECM fungi in regulating the N cycle. We propose, in accordance with plant C allocation theory, that recovery is driven by increased tree below-ground C allocation to ECM roots and fungi.     

Mass spectrometric characterisation of post-translational modification and genetic variation in human tetranectin

Tetranectin, a plasminogen-binding trimeric C-type lectin-like protein primarily involved in tissue remodeling and development, was scanned for covalent modifications and sequence heterogeneity, using a combination of mass spectrometric and classical protein chemical analytical methods. Electrospray ionisation mass spectrometry showed the presence of eight components of different mass and abundance in plasma tetranectin, all of higher mass than that calculated from the cDNA sequence. To identify and locate residues accounting for the heterogeneity, samples of tetranectin were subjected to proteolytic cleavage. Peptide fragments, in mixtures or in purified form, were analysed by matrix-assisted-laser-desorption-ionisation mass spectrometry and, where required, by Edman sequencing and compared to the cDNA sequence. Our results show that the mass heterogeneity in plasma tetranectin is due to sequence heterogeneity at position 85 and the presence of a partially sialylated oligosaccharide prosthetic group attached to Thr-4. Residue 85 is encoded in the cDNA as a Ser residue, but plasma tetranectin is a 1:1 mixture of Ser85 and Gly-85 sequence variants. Mass spectrometric analysis of enzymatic and mild acid hydrolysates of an N-terminal glycopeptide showed that the composition and partial covalent structure of the O-linked oligosaccharide prosthetic group is < or =N-acetylhexosamine < or =[hexose, (sialic acid)0-3].