Amphitrite ornata, a Marine Worm, Contains Two Dehaloperoxidase Genes

Amphitrite ornata, a terebellid polychaete, inhabits marine environments that are contaminated by biogenically produced halometabolites. These halogenated organic compounds are toxic and quite diverse. To survive in this environment, A. ornata produces a novel dehaloperoxidase (DHP I) that detoxifies haloaromatic compounds. In this study we identified and characterized two dehaloperoxidase genes, designated dhpA and dhpB, from an A. ornata complementary DNA library. The deduced amino acid sequences (DHP A and DHP B) of the two dhp genes both contain 137 amino acid residues, but they differ at 5 amino acid positions. Allelic variation was observed for both genes as well. Polymerase chain reaction–restriction fragment length polymorphism assays of genomic DNA from 19 in individuals showed that each individual contains both the dhpA and the dhpB genes. Therefore, the two types of DHP are encoded by separate genes and are not alleles of a single gene. Furthermore, DHP A and DHP B may have different substrate specificities since they have amino acid differences in the active site. Accepted January 16, 2001.     

The effects of symbiotic crabs on the pumping activity and growth rates of Chaetopterus variopedatus

This study investigates how the presence of symbiotic crabs (Pinnixa chaetopterana or Polyonyx gibbesi) in the tubes of the polychaete Chaetopterus variopedatus affects the worms' pumping activity and growth rates under laboratory and field conditions. In the field, worms whose tubes are inhabited by Pinnixa beat their fan segments significantly more frequently than do worms hosting Polyonyx, but other measures of pumping activity do not differ according to symbiont species. In the lab, worms tend to move water through their tubes at higher rates when crabs are present. In 7-month laboratory experiments, growth rates of worms hosting either species of crab did not differ from growth rates of worms without crab symbionts. Although worms hosting Polyonyx are, on average, significantly larger than worms hosting Pinnixa, this appears to be due to competition between the crab species for hosts and not due to differential effects on host growth. Unlike the crabs in this study, pea crab species inhabiting bivalves are known to have strong deleterious effects on host growth and reproduction, suggesting that the evolution of virulence in symbiotic interactions is dependent upon specific ecological context.     

Proteomic analysis of human osteoarthritis synovial fluid

Background

Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography.

Results

We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples.

Conclusions

We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

The modification of the cytotoxic effect of leucocidin by N-ethylmaleimide, flavine mononucleotide and menadione

1. The movement of the cytoplasmic granules in the leucocidin-treated leucocyte is prevented in the presence of N-ethylmaleimide or menadione. This effect follows a change of state in the cytoplasm. It may not be due to reaction with SH groups. When granule movement is prevented in this way the subsequent addition of Ca(2+) and ATP does not induce the secretion of the proteins of the granules. 2. Menadione or iodoacetate stimulates some effects of suboptimum amounts of leucocidin. This effect probably follows a reaction with SH groups. 3. Flavine mononucleotide inhibits some effects of suboptimum amounts of leucocidin. 4. Leucocidin decreases the stimulation of glucose oxidation due to menadione but increases that due to flavine mononucleotide. Leucocidin decreases the adsorption of menadione by leucocytes but increases that of flavine mononucleotide. 5. The redox state of the nicotinamide-adenine nucleotide coenzymes is not altered during leucocidin action and flavine mononucleotide and menadione do not undergo significant continuous oxidation and reduction when added to the leucocyte.     

Interplay between nitrogen deposition and grazing causes habitat degradation

Abstract Increased atmospheric nitrogen (N) deposition has been held responsible for the large‐scale invasion of graminoids (grasses, sedges and rushes) in a wide range of habitats from forests to upland heaths, causing dramatic changes in plant species composition. Concurrently with an increase in N deposition over the last century, livestock grazing has intensified in many parts of the world following policy reform, leading to large‐scale degradation of natural and seminatural ecosystems. On the basis of a series of experiments conducted in a Scottish montane ecosystem, we discovered that grazing and N deposition do not operate independently, and the interplay between them is leading to the replacement of valuable moss‐dominated habitat by grasses and sedges. Our study indicates that in setting ‘critical loads’ of N, widely used to minimize habitat degradation, it is necessary to account for substantial amplification of N‐deposition effects by grazing.     

Polychaete chaetae: Function, fossils, and phylogeny

In this article we review the phylogenetic distribution of majorchaetal types within the Polychaeta, discuss what has been demonstratedabout chaetal function in modern worms, and examine what isknown about the evolution of chaete through the fossil record.We conclude with specific cautions about how chaetae are treatedin phylogenetic analyses and make suggestions about how theycould be used to provide a stronger phylogenetic signal.     

Cytochrome P4501A induced differentially in endothelial cells cultured from different organs of Anguilia rostrata

Summary Endothelial cells are a structural barrier and an active regulator of many bodily processes. Cytochrome P4501A (CYPIA) activity is induced in the endothelium of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney, and rete mirabile of the eel, Anguilla rostrata, to aryl hydrocarbon receptor (AhR) agonists. In heart endothelial cells, the maximum response (based on O-deethylation of 7-ethoxyresorufin to resorufin [EROD] activity) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 113 pmol/mg/min, was at 1 nM TCDD and the peak response to β-napthoflavone (βNF), 135 pmol/mg/min, was at 3 μM βNF. The maximum response to TCDD in the kidney endothelial cells is 12 pmol/mg/min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and βNF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg/min, respectively) in untreated or dimethylsulfoxide-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vasculature.     

Cytochrome P450 1 genes in early deuterostomes (tunicates and sea urchins) and vertebrates (chicken and frog): origin and diversification of the CYP1 gene family

Cytochrome P450 family 1 (CYP1) proteins are important in a large number of toxicological processes. CYP1A and CYP1B genes are well known in mammals, but the evolutionary history of the CYP1 family as a whole is obscure; that history may provide insight into endogenous functions of CYP1 enzymes. Here, we identify CYP1-like genes in early deuterostomes (tunicates and echinoderms), and several new CYP1 genes in vertebrates (chicken, Gallus gallus and frog, Xenopus tropicalis). Profile hidden Markov models (HMMs) generated from vertebrate CYP1A and CYP1B protein sequences were used to identify 5 potential CYP1 homologs in the tunicate Ciona intestinalis genome. The C. intestinalis genes were cloned and sequenced, confirming the predicted sequences. Orthologs of 4 of these genes were found in the Ciona savignyi genome. Bayesian phylogenetic analyses group the tunicate genes in the CYP1 family, provisionally in 2 new subfamilies, CYP1E and CYP1F, which fall in the CYP1A and CYP1B/1C clades. Bayesian and maximum likelihood analyses predict functional divergence between the tunicate and vertebrate CYP1s, and regions within CYP substrate recognition sites were found to differ significantly in position-specific substitution rates between tunicates and vertebrates. Subsequently, 10 CYP1-like genes were found in the echinoderm Strongylocentrotus purpuratus (sea urchin) genome. Several of the tunicate and echinoderm CYP1-like genes are expressed during development. Canonical xenobiotic response elements are present in the upstream genomic sequences of most tunicate and sea urchin CYP1s, and both groups are predicted to possess an aryl hydrocarbon receptor (AHR), suggesting possible regulatory linkage of AHR and these CYPs. The CYP1 family has undergone multiple rounds of gene duplication followed by functional divergence, with at least one gene lost in mammals. This study provides new insight into the origin and evolution of CYP1 genes.     

Arctic mosses govern below-ground environment and ecosystem processes

Mosses dominate many northern ecosystems and their presence is integral to soil thermal and hydrological regimes which, in turn, dictate important ecological processes. Drivers, such as climate change and increasing herbivore pressure, affect the moss layer thus, assessment of the functional role of mosses in determining soil characteristics is essential. Field manipulations conducted in high arctic Spitsbergen (78° N), creating shallow (3 cm), intermediate (6 cm) and deep (12 cm) moss layers over the soil surface, had an immediate impact on soil temperature in terms of both average temperatures and amplitude of fluctuations. In soil under deep moss, temperature was substantially lower and organic layer thaw occurred 4 weeks later than in other treatment plots; the growing season for vascular plants was thereby reduced by 40%. Soil moisture was also reduced under deep moss, reflecting the influence of local heterogeneity in moss depth, over and above the landscape-scale topographic control of soil moisture. Data from field and laboratory experiments show that moss-mediated effects on the soil environment influenced microbial biomass and activity, resulting in warmer and wetter soil under thinner moss layers containing more plant-available nitrogen. In arctic ecosystems, which are limited by soil temperature, growing season length and nutrient availability, spatial and temporal variation in the depth of the moss layer has significant repercussions for ecosystem function. Evidence from our mesic tundra site shows that any disturbance causing reduction in the depth of the moss layer will alleviate temperature and moisture constraints and therefore profoundly influence a wide range of ecosystem processes, including nutrient cycling and energy transfer.