A hemodialysis cohort study of protocol-based anticoagulation management.

BACKGROUND: Chronic hemodialysis patients frequently require anticoagulation treatment with warfarin for a variety of co-morbidities. The optimal method for monitoring and dose adjustment of warfarin-based anticoagulation in this population, however, remains unclear. To examine this more closely, we reviewed all hemodialysis patients at a single institution on chronic warfarin therapy for a 10-month period prior to and after the institution of a standardized protocol for warfarin dose adjustment and monitoring. Anticoagulation efficacy was assessed by time within the therapeutic INR range (TTR), and resource utilization was assessed by the number of weekly INR measurements required for monitoring. RESULTS: We retrospectively analyzed 4481 patient-days of warfarin therapy data (from 25 hemodialysis patients) in the pre-protocol timeframe, and 3308 patient-days of warfarin therapy data (from 21 hemodialysis patients) in the on-protocol timeframe. Time within the therapeutic INR range (TTR) did not improve with institution of the dosing protocol-51.18% using non protocol-based management, and 51.57% using protocol-based management (p 0.73). However, overall resource utilization was reduced with institution of protocolized warfarin monitoring-from 1.71 INR measurements per patient-week pre-protocol, to 1.20 INR measurements per patient-week (p < 0.0001) post-protocol. CONCLUSIONS: In this single-center study, institution of a standardized dosing protocol in a hemodialysis population on chronic warfarin therapy did not improve the rate of on-target anticoagulation, but did result in significantly lower resource utilization. We support protocol-based warfarin management in the hemodialysis population, but future work should examine the rate of on-target anticoagulation typically achieved in this group.

Submarine groundwater discharge from the South Australian Limestone Coast region estimated using radium and salinity.

The Tertiary Limestone Aquifer (TLA) is one of the major regional hydrogeological systems of southern Australia. Submarine groundwater discharge (SGD) of freshwater from the TLA occurs through spring creeks, beach springs and diffusively through beach sands, but the magnitude of the total flux is not known. Here, a range of potential environmental tracers (including temperature, salinity, (222)Rn, (223)Ra, (224)Ra, (226)Ra, (228)Ra, and (4)He) were measured in potential sources of SGD and in seawater along a 45 km transect off the coastline to evaluate SGD from the TLA. Whilst most tracers had a distinct signature in the sources of water to the coastline, salinity and the radium quartet had the most distinct SGD signal in seawater. A one-dimensional advection-dispersion model was used to estimate the terrestrial freshwater component of SGD (Qfw) using salinity and the recirculated seawater component (Qrsw) using radium activity in seawater. Qfw was estimated at 1.2-4.6 m(3) s(-1), similar in magnitude to previously measured spring creek discharge (∼3 m(3) s(-1)) for the area. This suggests that other terrestrial groundwater discharge processes (beach springs and diffuse discharge through beach sands) were no more than 50% of spring creek discharge. The largest component of total SGD was Qrsw, estimated at 500-1000 m(3) s(-1) and possibly greater. The potential for wave, storm, or buoyancy-driven porewater displacement from the seafloor could explain the large recirculation flux for this section of the Southern Ocean Continental Shelf.

Sinks for nitrogen inputs in terrestrial ecosystems: a meta-analysis of 15N tracer field studies.

Effects of anthropogenic nitrogen (N) deposition and the ability of terrestrial ecosystems to store carbon (C) depend in part on the amount of N retained in the system and its partitioning among plant and soil pools. We conducted a meta-analysis of studies at 48 sites across four continents that used enriched 15N isotope tracers in order to synthesize information about total ecosystem N retention (i.e., total ecosystem 15N recovery in plant and soil pools) across natural systems and N partitioning among ecosystem pools. The greatest recoveries of ecosystem 15N tracer occurred in shrublands (mean, 89.5%) and wetlands (84.8%) followed by forests (74.9%) and grasslands (51.8%). In the short term (< 1 week after 15N tracer application), total ecosystem 15N recovery was negatively correlated with fine-root and soil 15N natural abundance, and organic soil C and N concentration but was positively correlated with mean annual temperature and mineral soil C:N. In the longer term (3-18 months after 15N tracer application), total ecosystem 15N retention was negatively correlated with foliar natural-abundance 15N but was positively correlated with mineral soil C and N concentration and C:N, showing that plant and soil natural-abundance 15N and soil C:N are good indicators of total ecosystem N retention. Foliar N concentration was not significantly related to ecosystem 15N tracer recovery, suggesting that plant N status is not a good predictor of total ecosystem N retention. Because the largest ecosystem sinks for 15N tracer were below ground in forests, shrublands, and grasslands, we conclude that growth enhancement and potential for increased C storage in aboveground biomass from atmospheric N deposition is likely to be modest in these ecosystems. Total ecosystem 15N recovery decreased with N fertilization, with an apparent threshold fertilization rate of 46 kg N x ha(-1) x yr(-1) above which most ecosystems showed net losses of applied 15N tracer in response to N fertilizer addition.

Comparative water use by the riparian trees Melaleuca argentea and Corymbia bella in the wet-dry tropics of northern Australia.

We examined sources of water and daily and seasonal water use patterns in two riparian tree species occupying contrasting niches within riparian zones throughout the wet-dry tropics of northern Australia: Corymbia bella Hill and Johnson is found along the top of the levee banks and Melaleuca argentea W. Fitzg. is restricted to riversides. Patterns of tree water use (sap flow) and leaf water potential were examined in four trees of each species at three locations along the Daly River in the Northern Territory. Predawn leaf water potential was higher than -0.5 MPa throughout the dry season in both species, but was lower at the end of the dry season than at the beginning of the dry season. Contrary to expectations, predawn leaf water potential was lower in M. argentea trees along the river than in C. bella trees along the levees. In contrast, midday leaf water potential was lower in the C. bella trees than in M. argentea trees. There were no seasonal differences in tree water use in either species. Daily water use was lower in M. argentea trees than in C. bella trees. Whole-tree hydraulic conductance, estimated from the slope of the relationship between leaf water potential and sap flow, did not differ between species. Xylem deuterium concentrations indicated that M. argentea trees along the riverbank were principally reliant on river water or shallow groundwater, whereas C. bella trees along the levee were reliant solely on soil water reserves. This study demonstrated strong gradients of tree water use within tropical riparian communities, with implications for estimating riparian water use requirements and for the management of groundwater resources.

Structure-activity relationship of biaryl acylsulfonamide analogues on the human EP(3) prostanoid receptor.

Potent and selective ligands for the human EP3 prostanoid receptor are described. Biaryl compounds bearing a tethered ortho substituted acidic moiety were identified as potent EP3 antagonists based on the SAR described herein. The binding affinity of key compounds on all eight human prostanoid receptors is reported.

Localization of phosphodiesterase-4 isoforms in the medulla and nodose ganglion of the squirrel monkey.

Pre-clinical and clinical studies are currently underway to evaluate the potential of phosphodiesterase-4 (PDE4) inhibitors for the treatment of chronic obstructive pulmonary disease and other inflammatory conditions of the airways. The most common side effect associated with this class of compounds is emesis. The squirrel monkey provides a model for evaluating the efficacy of PDE4 inhibitors and their emetic potential. The distribution of three PDE4 isoforms (A, C and D) has been investigated in the squirrel monkey medulla and nodose ganglion to determine which isoform(s) could be responsible for the emetic adverse effects. The distribution of PDE4 isoforms was delineated using immunohistochemistry with antibodies specific for PDE4A, PDE4C and PDE4D and by in situ hybridization with isoform-selective riboprobes. PDE4A was present in the medulla where expression was mostly restricted to glial cells and the vasculature. PDE4C was not detected in either the medulla or nodose ganglion. Finally, the PDE4D isoform was localized to neurons in the nodose ganglion and found through many structures of medulla including the area postrema, neurons of the nucleus tractus solitarius and locus coeruleus. These data are consistent with a role for PDE4D in the emetic response.

Structure-activity relationship of cinnamic acylsulfonamide analogues on the human EP3 prostanoid receptor.

Potent and selective antagonists of the human EP3 receptor have been identified. The structure-activity relationship of the chemical series was conducted and we found several analogues displaying sub-nanomolar K(i) values at the EP3 receptor and micromolar activities at the EP1, EP2 and EP4 receptors. The effect of added human serum albumin (HSA) on the binding affinity at the EP3 receptor was also investigated.

Key structural features of prostaglandin E(2) and prostanoid analogs involved in binding and activation of the human EP(1) prostanoid receptor.

The structure-activity relationship (SAR) of prostaglandin (PG) E(2) at the human EP(1) prostanoid receptor (designated hEP(1)) was examined via the binding and activation of this receptor by a series of 55 prostanoids and analogs. Using clonal human embryonic kidney 293 cell lines expressing recombinant hEP(1), affinity (K(i)), potency (EC(50)), and efficacy data were obtained using a radioligand competitive binding assay and an aequorin-based calcium functional assay. All compounds behaved as full agonists (90-100% of the response elicited by PGE(2)) in this assay, and the correlation between the K(i) and EC(50) values was highly significant (R(2) = 0.86). The results from the SAR analysis can be summarized as follows: 1) the existence and configuration of hydroxyl groups at the 11 and 15 positions of PGE(2) and prostanoid analog structures play a critical role in agonist activity; 2) the carboxyl group is also important for activity and modification of the carboxylic acid to various esters results in greatly reduced affinity and potency; 3) the activity of structures with moderate or weak potency can be enhanced by modification of the omega-tail; and 4) modifications to the ketone at the 9-position are better tolerated, with 9-deoxy-9-methylene-PGE(2) being the most potent agonist tested in the functional assay. The impact of other modifications on agonist potency is also discussed. The results from this study have identified, for the first time, the key structural features of PGE(2) and related prostanoids and prostanoid analogs necessary for activation of hEP(1).

Structure-activity relationship on the human EP3 prostanoid receptor by use of solid-support chemistry.

Potent and selective EP3 receptor ligands were found by making a library using solid-support chemistry. These compounds can be obtained by a Suzuki coupling reaction of a solid-supported benzyl bromide using various boronic acids. The yields obtained for this reaction were in the range of 24-95% of arylmethyl cinnamic acid 1 after cleavage from the Wang resin.

The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs.

Stable cell lines that individually express the eight known human prostanoid receptors (EP(1), EP(2), EP(3), EP(4), DP, FP, IP and TP) have been established using human embryonic kidney (HEK) 293(EBNA) cells. These recombinant cell lines have been employed in radioligand binding assays to determine the equilibrium inhibitor constants of known prostanoid receptor ligands at these eight receptors. This has allowed, for the first time, an assessment of the affinity and selectivity of several novel compounds at the individual human prostanoid receptors. This information should facilitate interpretation of pharmacological studies that employ these ligands as tools to study human tissues and cell lines and should, therefore, result in a greater understanding of prostanoid receptor biology.

New class of biphenylene dibenzazocinones as potent ligands for the human EP1 prostanoid receptor.

A new class of potent and selective ligands for the human EP1 prostanoid receptor is described. SAR studies reported herein allowed the identification of several potent dibenzazocinones bearing an acylsulfonamide side chain. The binding affinity of these compounds on all eight human prostanoid receptors is reported.

A gene-type-specific enhancer regulates the carbamyl phosphate synthetase I promoter by cooperating with the proximal GAG activating element.

The rat carbamyl phosphate synthetase I gene is expressed in two cell types: hepatocytes and epithelial cells of the intestinal mucosa. The proximal promoter contains a single activating element, GAG, two repressor elements (sites I and III) and an anti-repressor element (site II). Although these elements together exhibit the potential for complex regulation, they are unable to confer tissue-specific promoter activity. Here we have identified a cell-type-specific enhancer that lies 10 kilobases upstream of the promoter. Unexpectedly, the enhancer also functioned in a gene-type-specific manner. The enhancer stimulated promoter activity exclusively through the proximal GAG element. Abrogation of GAG, either directly by mutation of GAG or indirectly by sites I and III repressors, abolished enhancer activation. Conversely, activation of the heterologous thymidine kinase promoter by the enhancer required the introduction of GAG. The requirement for GAG, therefore, functions to constrain the enhancer to a specific target promoter.

Binding of GM1 ganglioside to a synthetic peptide derived from the lysosomal sphingolipid activator protein saposin B.

Saposin B is a lysosomal sphingolipid activator protein which activates GM1 ganglioside hydrolysis by lysosomal beta-galactosidase. To identify the structural elements of saposin B implicated in sphingolipid binding, we studied a synthetic peptide corresponding to a predicted alpha-helix, sapB-18, spanning residues 52-69 of saposin B. The circular dichroism spectrum of sapB-18 at pH 4.4 was consistent with a 44% alpha-helix content. As shown by intrinsic Tyr fluorescence studies of sapB-18, this peptide binds the GM1 ganglioside with a Kd of about 7 microM. Thus, we suggest that a putative amphipathic alpha-helix between residues 52 and 69 of saposin B plays a major role in the recognition and binding of GM1 ganglioside by saposin B.

Modulation of human saposin B sphingolipid-binding specificity by alternative splicing. A study with saposin B-derived synthetic peptides.

The saposins A, B, C, and D, produced by proteolytic maturation of the same precursor protein, prosaposin, are sphingolipid-binding proteins which function as activators for lysosomal enzymes involved in sphingolipid hydrolysis. The alternative splicing of the prosaposin gene results in the inclusion or exclusion of exon 8 into transcribed prosaposin mRNA through the use of alternative acceptor sites. The relative abundance of each alternatively spliced mRNA was determined by reverse transcription-polymerase chain reaction in various human tissues and cell lines. Exon 8 codes for only three amino acid residues, Gln-Asp-Gln, in the saposin B domain of prosaposin. The prosaposin mRNA containing exon 8 is the major species in cultured skin fibroblasts, brain, and pituitary glands together with a smaller amount of mRNA devoid of exon 8, whereas the prosaposin mRNA detected in liver and lymphoblasts was devoid of exon 8 insertion. Previous structural modeling studies on saposin B have suggested that the Gln-Asp-Gln insertion occurs in an amphipathic alpha-helix region of the protein which is implicated in the binding of GM1-ganglioside. We report that synthetic peptides containing the alpha-helix, with and without the Gln-Asp-Gln insertion, have different binding affinities for GM1-ganglioside, sulfatide, and sphingomyelin. The insertion of the Gln-Asp-Gln sequence completely abolishes the capacity of the peptide to bind GM1-ganglioside, whereas its affinity for sulfatide and sphingomyelin is increased about 4-fold and almost 2-fold, respectively. No significant binding of glucosylceramide was observed with both peptides. These results suggest that alternative splicing of prosaposin mRNA may change binding specificity of saposin B presumably to adapt to the variable sphingolipid composition of tissues.

Binding of GM1-ganglioside to a synthetic peptide derived from the lysosomal sphingolipid-activator-protein saposin B.

Saposin B is a lysosomal sphingolipid-activator-protein which activates GM1-ganglioside hydrolysis by lysosomal beta-galactosidase. To identify the structural elements of saposin B implicated in sphingolipid binding, we studied a synthetic peptide corresponding to a predicted alpha-helix, sapB-18, spanning residues 52 to 69 of saposin B. The circular dichroism spectrum of sapB-18 at pH 4.4 was consistent with a 44% alpha-helix content. As shown by intrinsic Tyr fluorescence studies of sapB-18, this peptide binds the GM1-ganglioside with a Kd of about 7 microM. Thus, we suggest that a putative amphipathic alpha-helix between residues 52 and 69 of saposin B plays a major role in the recognition and binding of GM1-ganglioside by saposin B.

Human neuraminidase is a 60 kDa-processing product of prosaposin.

Human neuraminidase was purified from placenta as part of a large molecular weight complex with lysosomal beta-galactosidase and carboxypeptidase. Passage of this purified complex through a sialic acid-affinity column (fetuin-agarose) retained a minor 60 kDa protein which was eluted with 100 mM N-acetylneuraminic acid. This 60 kDa protein is recognized in Western blots of the purified complex by an anti-prosaposin antibody which at the same time was able to inhibit neuraminidase activity in the preparation. Furthermore, probing of cultured skin fibroblasts of patients affected with neuraminidase deficiency using the antiprosaposin antibody revealed an abnormal 57 kDa protein. These results indicate that the 60 kDa protein is derived from prosaposin and has the characteristics of a neuraminidase.

2,3-Diphosphoglycerate is a nonselective inhibitor of inositol 1,4,5-trisphosphate action and metabolism.

Inositol 1,4,5-trisphosphate (InsP3) is an important second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C in response to Ca2(+)-mobilizing stimuli. InsP3 interacts with specific intracellular receptors and triggers the release of sequestered Ca2+ from an intracellular store. We have looked at the influence of 2,3-diphosphoglycerate on the action and metabolism of InsP3 in the bovine adrenal cortex. 2,3-Diphosphoglycerate blocked InsP3 binding to adrenal cortex microsomes with a half-maximal efficiency of 0.5 mM. Scatchard analyses revealed that 2,3-diphosphoglycerate did not change the maximal capacity of the microsomes, but decreased their binding affinity for InsP3. The Ca2(+)-releasing activity of InsP3 on the same microsomal preparation was monitored with the fluorescent indicator, Fura-2. 2,3-Diphosphoglycerate blocked this activity with a half-maximal efficiency of 2 mM. The effect of 2,3-diphosphoglycerate could be overcome by supramaximal doses of InsP3, indicating a competitive inhibitory effect. The activity of InsP3 phosphatase from bovine adrenal cortex microsomes was also studied. 2,3-Diphosphoglycerate inhibited the activity of the phosphatase with a half-maximal efficiency of 0.3 mM. Lineweaver-Burke plots revealed that this effect was competitive. Finally, 2,3-diphosphoglycerate was also able to inhibit the activity of a partially purified preparation of InsP3 kinase from bovine adrenal cortex cytosol. The half-maximal dose was around 10 mM and the Lineweaver-Burke plot showed that the inhibition was competitive. These results show that 2,3-diphosphoglycerate can be considered as a structural analog of InsP3. Its inhibitory effects, however, are not selective enough to use it as an InsP3 protective agent in Ca2(+)-mobilization studies.

Differential effects of heparin on inositol 1,4,5-trisphosphate binding, metabolism, and calcium release activity in the bovine adrenal cortex.

In a wide variety of cells, inositol-1,4,5-triphosphate is a second messenger that interacts with specific intracellular receptors and triggers the release of sequestered Ca2+ from an intracellular store. We have looked at the influence of heparin on the action and metabolism of inositol-1,4,5-triphosphate in the bovine adrenal cortex. Heparin blocked inositol-1,4,5-trisphosphate binding with half-maximal efficiency around 10 micrograms/ml. Scatchard analyses revealed that heparin did not change the affinity but decreased the number of available binding sites. The Ca2+-releasing activity of inositol-1,4,5-trisphosphate was monitored with the fluorescent indicator, fura-2. Heparin blocked this activity with half-maximal effeciency around 10 micrograms/ml. The effect of heparin could be overcome by a supramaximal dose of inositol-1,4,5-trisphosphate (25 microM). The activity of inositol-1,4,5-trisphosphate-3-kinase from bovine adrenal cortex cytosol was also studied. Heparin inhibited the activity of the kinase with a half-maximal effeciency around 0.4 microgram/ml. Lineweaver-Burk plots revealed that this potent effect was noncompetitive. Finally, we observed that heparin is without effect on inositol-1,4,5-trisphosphate-5-phosphatase (at concentrations as high as 2 mg/ml). These results are consistent with the suggestion that the binding sites for inositol-1,4,5-trisphosphate are the intracellular receptors responsible for the Ca2+-mobilizing effects of inositol-1,4,5-trisphosphate. These results also show that the kinase, the phosphatase, and the receptor are three different molecular entities, which are affected in a different manner by heparin.