Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins: OPPORTUNITIES FOR POTENT, SELECTIVE MODULATION OF ANTITHROMBIN FUNCTION*

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891–31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80–87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.Antithrombin (AT),³ a plasma glycoprotein and a member of the serpin superfamily of proteins, is a major regulator of the coagulation cascade. Its primary targets are thrombin, factor Xa (fXa), and factor IXa (fIXa) (1). It has also been suggested to inhibit several other coagulation enzymes (2–6), albeit with much weaker inhibitory efficiency. Antithrombin alone is a rather poor inhibitor of factors IIa, Xa, and IXa and requires the presence of heparin to exhibit its full anticoagulant potential.Heparin is a highly sulfated polysaccharide that greatly enhances the rate of AT inhibition of these enzymes under physiological conditions (1). This acceleration forms the basis for heparin''s use as an anticoagulant for the past several decades. Yet heparin is associated with bleeding complications and suffers from a number of other limitations. In addition, the animal origin of the drug is also a cause for concern as suggested by recent incidences of oversulfated chondroitin sulfate contaminating unfractionated heparin (UFH) preparations and resulting in numerous deaths (7–9). Although low molecular weight heparins (LMWHs) are superior to UFH with respect to therapeutic complications, the iatrogenic bleeding risk is not completely eliminated. Likewise, fondaparinux, or the minimal antithrombin binding pentasaccharide sequence (H5), is also associated with bleeding (10, 11) and lacks an effective antidote to reverse excessive anticoagulation.The major reason for the limitations of UFH and LMWH therapies is the presence of numerous negative charges on each polymeric chain. UFH and LMWH are linear co-polymers of glucosamine and uronic acid residues that are decorated with numerous sulfate groups generating a massive polyanion (12, 13). This polyanion is capable of interacting with a large number of plasma proteins and proteins present on cells lining the vasculature, which likely induce many of the UFH and LMWH complications (14, 15). Fondaparinux displays a much better pharmacological profile primarily because of its limited number of sulfate and carboxylate groups.To design better anticoagulants that are less polyanionic and more hydrophobic than UFHs and LMWHs, we recently prepared low molecular weight variants of lignin, called sulfated dehydropolymers (DHPs) (Fig. 1), as functional mimetics of heparin. These designed molecules were prepared in a simple two-step chemo-enzymatic process involving enzymatic coupling of 4-hydroxycinnamic acids followed by the chemical sulfation of the resulting DHPs (16). In terms of structural polydispersity and heterogeneity, sulfated DHPs are similar to LMWHs. Sulfated DHPs are composed of many oligomeric chains of varying lengths and contain different inter-monomeric linkages such as β-O-4 and β-5. Yet sulfated DHPs are completely unlike LMWHs with respect to the nature of their backbone. In contrast to the highly anionic scaffold of the heparins, sulfated DHPs possess a highly aromatic scaffold with fewer anionic groups (Fig. 1). In fact, in terms of structure, sulfated DHPs are unlike any other class of anticoagulant investigated to date, including the heparins, the coumarins, the hirudins, the peptidomimetics, and the small molecule direct inhibitors. Functionally, the sulfated DHPs display plasma and blood anticoagulation similar to that of LMWHs (17, 18). Yet mechanistically, the sulfated DHPs were found to exhibit a novel mechanism of anticoagulation involving an exosite II-mediated allosteric inhibition of thrombin (17). The DHPs represent the first example of an exclusive exosite II-dependent inactivation of the catalytic function of thrombin.Open in a separate windowFIGURE 1.A representative structure of sulfated DHPs. CDSO3, FDSO3, and SDSO3 were chemo-enzymatically synthesized in two steps from the corresponding starting 4-hydroxycinnamic acid monomers, caffeic acid (CA), ferulic acid (FA), or sinapic acid (SA). The average molecular mass of CDs, FDs, and SDs was in the range of 3,000–4,000 Da. Two types of common linkages present in sulfated DHPs include the β-O-4 and β-5 linkages (shown as shaded ovals).In this work we study the interaction of sulfated DHPs with AT at a molecular level. This study reveals that the indirect antithrombin-mediated pathway may contribute to the inhibition efficiency of sulfated DHPs, thus realizing molecules that can utilize both the direct and indirect inhibition pathways. These studies uncover the ability of a specific sulfated DHP to induce potent antithrombin inhibition of free fVIIa. The antithrombin-mediated effects originate from the sulfated DHPs binding to the heparin-binding site (HBS) of the serpin through non-ionic forces that contribute more than 80% of the total binding energy. Our work supports the idea that aromatic scaffolds, which exhibit hydrophobic and hydrophilic nature, can be designed to target the HBS of antithrombin for modulation of its inhibitory functions.     

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

Background

All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.

Methods

Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.

Results

Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (π_s) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (π_a) was 7.0 × 10^-4 in the M segment and 17.3 × 10^-4 in the S segment sequences. The low π_a relative to π_s suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10^-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10^-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.

Conclusion

Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.     

Biochemical characterization of arabidopsis developmentally regulated G-proteins (DRGs)

Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as several medical conditions. Individual subfamilies within the G-protein superfamily possess unique nucleotide binding and hydrolysis rates that are intrinsic to their cellular function, and so characterization of these rates for a particular G-protein may provide insight into its cellular activity. We have produced recombinant active DRG protein using a bacterial expression system and refolding, and performed biochemical characterization of their GTP binding and hydrolysis. We show that recombinant Arabidopsis thaliana atDRG1 and atDRG2a are able to bind GDP and GTP. We also show that DRGs can hydrolyze GTP in vitro without the assistance of GTPase-activating proteins and guanine exchange factors. The atDRG proteins hydrolyze GTP at a relatively slow rate (0.94 × 10⁻³ min⁻¹ for DRG1 and 1.36 × 10⁻³ min⁻¹ for DRG2) that is consistent with their nearest characterized relatives, the Obg subfamily. The ability of DRGs to bind nucleotide substrates without assistance, their slow rate of GTP hydrolysis, heat stress activation and domain conservation suggest a possible role as a chaperone in ribosome assembly in response to stress as it has been suggested for the Obg proteins, a different but related G-protein subfamily.     

The water balance and water sources of a Eucalyptus plantation over shallow saline groundwater

Eucalypt plantations have been trialled in recent years as a control measure for shallow groundwater associated with secondary salinity. Uncertainty still remains as to the potential growth and water use of these plantations; these relate mainly to the problems associated with drought stress and accumulated solutes in the root zone resulting from saline groundwater uptake. This study investigates the water balance and identifies water sources of a 21 year old unirrigated Eucalyptus grandis W. Hill ex Maiden (flooded or rose gum) and E. camaldulensis Dehnh. (river red gum) plantation over shallow saline groundwater in the Shepparton Irrigation Region of northern Victoria, Australia. Water sources used by the plantation were identified using a monthly water balance approach, together with investigations of stable isotopes of water (deuterium and oxygen-18), soil water and chloride. We found these trees to be heavily reliant on rainwater, and derive approximately 15% of their transpiration requirements from saline (10,000 mg l^?1) groundwater at the capillary fringe. Rainfall at the site is relatively low (465 mm year^?1 on average) and groundwater uptake provides a stable water source that leads to a slight extension of the growing period of these trees. There is little potential for recharge, with subsurface water moving into the groundwater depression created by tree water uptake.     

Linkage and sequence analysis indicate that CCBE1 is mutated in recessively inherited generalised lymphatic dysplasia

Generalised lymphatic dysplasia (GLD) is characterised by extensive peripheral lymphoedema with visceral involvement. In some cases, it presents in utero with hydrops fetalis. Autosomal dominant and recessive inheritance has been reported. A large, non-consanguineous family with three affected siblings with generalised lymphatic dysplasia is presented. One child died aged 5 months, one spontaneously miscarried at 17 weeks gestation, and the third has survived with extensive lymphoedema. All three presented with hydrops fetalis. There are seven other siblings who are clinically unaffected. Linkage analysis produced two loci on chromosome 18, covering 22 Mb and containing 150 genes, one of which is CCBE1. A homozygous cysteine to serine change in CCBE1 has been identified in the proband, in a residue that is conserved across species. High density SNP analysis revealed homozygosity (a region of 900 kb) around the locus for CCBE1 in all three affected cases. This indicates a likely ancestral mutation that is common to both parents; an example of a homozygous mutation representing Identity by Descent (IBD) in this pedigree. Recent studies in zebrafish have shown this gene to be required for lymphangiogenesis and venous sprouting and are therefore supportive of our findings. In view of the conserved nature of the cysteine, the nature of the amino acid change, the occurrence of a homozygous region around the locus, the segregation within the family, and the evidence from zebrafish, we propose that this mutation is causative for the generalised lymphatic dysplasia in this family, and may be of relevance in cases of non-immune hydrops fetalis.     

Small molecule inhibitors of HIV RT Ribonuclease H

Two classes of compounds, thiocarbamates 1 and triazoles 2, have been identified as HIV RT RNase H inhibitors using a novel FRET-based HTS assay. The potent analogs in each series exhibited selectivity and were active in cell-based assays. In addition, saturable, 1:1 stoichiometric binding to target was established and time of addition studies were consistent with inhibition of RT-mediated HIV replication.     

Diet of two large sympatric teleosts, the ling (Genypterus blacodes) and hake (Merluccius australis)

Ling and hake are tertiary consumers, and as a result both may have an important structuring role in marine communities. The diets of 2064 ling and 913 hake from Chatham Rise, New Zealand, were determined from examination of stomach contents. Ling was a benthic generalist, and hake a demersal piscivore. The diet of ling was characterised by benthic crustaceans, mainly Munida gracilis and Metanephrops challengeri, and demersal fishes, mainly Macrourids and scavenged offal from fishing vessels. The diet of hake was characterised by teleost fishes, mainly macrourids and merlucciids. Multivariate analyses using distance-based linear models found the most important predictors of diet variability were depth, fish length, and vessel type (whether the sample was collected from a commercial or research vessel) for ling, and fish length and vessel type for hake. There was no interspecific predation between ling and hake, and resource competition was largely restricted to macrourid prey, although the dominant macrourid species predated by ling and hake were different. Cluster analysis of average diet of intraspecific groups of ling and hake confirmed the persistent diet separation. Although size is a central factor in determining ecological processes, similar sized ling and hake had distinctly different foraging ecology, and therefore could influence the ecosystem in different ways, and be unequally affected by ecosystem fluctuations.     

A comparison of univariate and multivariate gene selection techniques for classification of cancer datasets

Background  

Gene selection is an important step when building predictors of disease state based on gene expression data. Gene selection generally improves performance and identifies a relevant subset of genes. Many univariate and multivariate gene selection approaches have been proposed. Frequently the claim is made that genes are co-regulated (due to pathway dependencies) and that multivariate approaches are therefore per definition more desirable than univariate selection approaches. Based on the published performances of all these approaches a fair comparison of the available results can not be made. This mainly stems from two factors. First, the results are often biased, since the validation set is in one way or another involved in training the predictor, resulting in optimistically biased performance estimates. Second, the published results are often based on a small number of relatively simple datasets. Consequently no generally applicable conclusions can be drawn.