Quantification of heparin's antimetastatic effect by single‐cell force spectroscopy

In circulation, cancer cells induce platelet activation, leading to the formation of a cancer cell‐encircling platelet cloak which facilitates each step of the metastatic cascade. Since cancer patients treated with the anticoagulant heparin showed reduced metastasis rates and improved survival, it is supposed that heparin suppresses the cloak's formation by inhibiting the interaction between platelet's adhesion molecule P‐selectin with its ligands on cancer cells. To quantify this heparin effect, we developed a single‐cell force spectroscopy approach and quantified the adhesion (maximum adhesion force [F_A] and detachment work [W_D]) between platelets and human non‐small cell lung cancer cells (A549). A configuration was used in which A549 cells were glued to tipless cantilevers and force‐distance (F‐D) curves were recorded on a layer of activated platelets. The concentration‐response relationship was determined for heparin at concentrations between 1 and 100 U/mL. Sigmoid dose‐response fit revealed half‐maximal inhibitory concentration (IC₅₀) values of 8.01 U/mL (F_A) and 6.46 U/mL (W_D) and a maximum decrease of the adhesion by 37.5% (F_A) and 38.42% (W_D). The effect of heparin on P‐selectin was tested using anti‐P‐selectin antibodies alone and in combination with heparin. Adding heparin after antibody treatment resulted in an additional reduction of 9.52% (F_A) and 7.12% (W_D). Together, we quantified heparin's antimetastatic effect and proved that it predominantly is related to the blockage of P‐selectin. Our approach represents a valuable method to investigate the adhesion of platelets to cancer cells and the efficiency of substances to block this interaction.     

Identification and characterization of a novel heparin‐binding peptide for promoting osteoblast adhesion and proliferation by screening an Escherichia coli cell surface display peptide library

Heparin/heparan sulfate (HS) plays a key role in cellular adhesion. In this study, we utilized a 12‐mer random Escherichia coli cell surface display library to identify the sequence, which binds to heparin. Isolated insert analysis revealed a novel heparin‐binding peptide sequence, VRRSKHGARKDR, designated as HBP12. Our analysis of the sequence alignment of heparin‐binding motifs known as the Cardin–Weintraub consensus (BBXB, where B is a basic residue) indicates that the HBP12 peptide sequence contains two consecutive heparin‐binding motifs (i.e. RRSK and RKDR). SPR‐based BIAcore technology demonstrated that the HBP12 peptide binds to heparin with high affinity (K_D = 191 nM ). The HBP12 peptide is found to bind the cell surface HS expressed by osteoblastic MC3T3 cells and promote HS‐dependent cell adhesion. Moreover, the surface‐immobilized HBP12 peptide on titanium substrates shows significant increases in the osteoblastic MC3T3‐E1 cell adhesion and proliferation. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)₄K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)₄K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)₄K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)₄K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity

Heparin and heparin‐like molecules are known to modulate the cellular responses to vascular endothelial growth factor‐A (VEGF‐A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF‐A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF‐A are many and varied, in part due to the endogenous cell‐surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell‐surface heparan sulfates. We showed that VEGF‐induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross‐bridging role between VEGF‐A₁₆₅ and putative heparin‐binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross‐bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF‐A₁₆₅ binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far‐UV circular dichroism spectra of VEGF‐A₁₆₅ showed that heparin can also modulate the conformation and secondary structure of the protein. J. Cell. Biochem. 111: 461–468, 2010. © 2010 Wiley‐Liss, Inc.     

Kinetic and functional characterisation of the heparin‐binding peptides from human transglutaminase 2

Transglutaminase 2 (TG2) is an autoantigen in celiac disease (CD) and it has multiple biologic functions including involvement in cell adhesion through interactions with integrins, fibronectin (FN), and heparan sulfate proteoglycans. We aimed to delineate the heparin‐binding regions of human TG2 by studying binding kinetics of the predicted heparin‐binding peptides using surface plasmon resonance method. In addition, we characterized immunogenicity of the TG2 peptides and their effect on cell adhesion. The high‐affinity binding of human TG2 to the immobilized heparin was observed, and two TG2 peptides, P1 (amino acids 202–215) and P2 (261–274), were found to bind heparin. The amino acid sequences corresponding to the heparin‐binding peptides were located close to each other on the surface of the TG2 molecule as part of the α‐helical structures. The heparin‐binding peptides displayed increased immunoreactivity against serum IgA of CD patients compared with other TG2 peptides. The cell adhesion reducing effect of the peptide P2 was revealed in Caco‐2 intestinal epithelial cell attachment to the FN and FN‐TG2 coated surfaces. We propose that TG2 amino acid sequences 202–215 and 261–274 could be involved in binding of TG2 to cell surface heparan sulfates. High immunoreactivity of the corresponding heparin‐binding peptides of TG2 with CD patient's IgA supports the previously described role of anti‐TG2 autoantibodies interfering with this interaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Direct cell–cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis

The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell–cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell–cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co‐culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule‐1 (ICAM‐1) and of osteoclastogenesis‐related genes (RANKL, RANK, TNF‐α, and IL‐1β) was highly up‐regulated in the co‐cultures compared to mono‐cultures and the 5–10‐fold up‐regulation reflected a synergistic increase due to direct cell–cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)₂VitD₃ and dexamethasone. In case of indirect cell–cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast‐like cells that were formed in co‐culture with periodontal ligament fibroblasts was significantly augmented compared to mono‐cultures. Our data indicate that cell–cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts. J. Cell. Physiol. 222: 565–573, 2010. © 2009 Wiley‐Liss, Inc.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

Matrilysin (MMP‐7) cleaves C‐type lectin domain family 3 member A (CLEC3A) on tumor cell surface and modulates its cell adhesion activity

Matrilysin (MMP‐7) plays important roles in tumor progression. Previous studies have suggested that MMP‐7 binds to tumor cell surface and promotes their metastatic potential. In this study, we identified C‐type lectin domain family 3 member A (CLEC3A) as a membrane‐bound substrate of MMP‐7. Although this protein is known to be expressed specifically in cartilage, its message was found in normal breast and breast cancer tissues as well as breast and colon cancer cell lines. Because few studies have been done on CLEC3A, we overexpressed its recombinant protein in human cancer cells. CLEC3A was found in the cell membrane, extracellular matrix (ECM), and culture medium of the CLEC3A‐expressing cells. CLEC3A has a basic sequence in the NH₂‐terminal domain and showed a strong heparin‐binding activity. MMP‐7 cleaved the 20‐kDa CLEC3A protein, dividing it to a 15‐kDa COOH‐terminal fragment and an NH₂‐terminal fragment with the basic sequence. The 15‐kDa fragment no longer had heparin‐binding activity. Treatment of the CLEC3A‐expressing cells with MMP‐7 released the 15‐kDa CLEC3A into the culture supernatant. Furthermore, the 20‐kDa CLEC3A promoted cell adhesion to laminin‐332 and fibronectin substrates, but this activity was abrogated by the cleavage by MMP‐7. These results suggest that CLEC3A binds to heparan sulfate proteoglycans on cell surface, leading to the enhancement of cell adhesion to integrin ligands on ECM. It can be speculated that the cleavage of CLEC3A by MMP‐7 weakens the stable adhesion of tumor cells to the matrix and promotes their migration in tumor microenvironments. J. Cell. Biochem. 106: 693–702, 2009. © 2009 Wiley‐Liss, Inc.     

Mechanism of interaction of thrombospondin with human endothelium and inhibition of sickle erythrocyte adhesion to human endothelial cells by heparin

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [¹²⁵I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28–40%, (ii) the RGD peptide or MoAbs against integrin α_vβ₃ or the calcium binding region of TSP inhibited binding by 18–28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.     

Partial Nitridation‐Induced Electrochemistry Enhancement of Ternary Oxide Nanosheets for Fiber Energy Storage Device

Fiber‐based power sources are receiving interest in terms of application in wearable electronic devices. Herein, fiber‐shaped all‐solid‐state asymmetric energy storage devices are fabricated based on a partially nitridized NiCo₂O₄ hybrid nanostructures on graphite fibers (GFs). The surface nitridation leads to a 3D “pearled‐veil” network structure, in which Ni–Co–N nanospheres are mounted on NiCo₂O₄ nanosheets' electrode. It is demonstrated that the hybrid materials are more potent than the pure NiCo₂O₄ in energy storage applications due to a cooperative effect between the constituents. The Ni–Co–N segments augment the pristine oxide nanosheets by enhancing both capacity and rate performance (a specific capacity of 384.75 mAh g⁻¹ at 4 A g⁻¹, and a capacity retention of 86.5% as the current is increased to 20 A g⁻¹). The whole material system has a metallic conductivity that renders high‐rate charge and discharge, and an extremely soft feature, so that it can wrap around arbitrary‐shaped holders. All‐solid‐state asymmetric device is fabricated using Ni–Co–N/NiCo₂O₄/GFs and carbon nanotubes/GFs as the electrodes. The flexible device delivers outstanding performance compared to most oxide‐based full devices. These structured hybrid materials may find applications in miniaturized foldable energy devices.     

Basic residues in the 37-loop of activated protein C modulate inhibition by protein C inhibitor but not by α1-antitrypsin

The role of lysines 37–39 (chymotrypsin numbering) in the 37-loop of the serine protease activated protein C (APC) was studied by expressing acidic and neutral recombinant APC (rAPC) mutants. Activity of the APC mutants was assessed using human plasma and plasma-purified and recombinant derivatives of protein C inhibitor (PCI; also known as plasminogen activator inhibitor-3) and α₁-antitrypsin, with and without heparin. The catalytic properties of the mutants to small peptidyl substrates were essentially the same as wild-type rAPC (wt-rAPC), yet their plasma anticoagulant activities were diminished. Analysis of the rAPC-protease inhibitor complexes formed after addition of wt-rAPC and mutants to plasma revealed no change in the inhibition pattern by α₁-antitrypsin but a reduction in mutant complex formation by PCI in the presence of heparin. Using purified serpins, we found that inhibition rates of the mutants were the same as wt-rAPC with α₁-antitrypsin; however, PCI (plasma-derived and recombinant forms) inhibition rates of the acidic mutants were slightly faster than that of wt-rAPC without heparin. By contrast, PCI–heparin inhibition rates of the mutants were not substantially accelerated compared to wt-rAPC. The mutants had reduced heparin-binding properties compared to wt-rAPC. Molecular modeling of the PCI–APC complex with heparin suggests that heparin may function not only to bridge PCI to APC, but also to alleviate putative non-optimal intermolecular interactions. Our results suggest that the basic residues of the 37-loop of APC are involved in macromolecular substrate interactions and in heparin binding, and they influence inhibition by PCI (with or without heparin) but not by α₁-antitrypsin, two important blood plasma serpins.     

From Pb to Bi: A Promising Family of Pb‐Free Optoelectronic Materials and Devices

Lead‐based organic–inorganic hybrid perovskite materials are widely used in optoelectronic devices due to their excellent photophysical properties. However, the main issues which hinder its commercialization are the toxicity caused by lead and the intrinsic instability of the material. Recently, many lead‐free halide materials with good intrinsic stability have been reported, among which bismuth‐based halide materials have attracted extensive research due to their structure and promising optoelectronic properties. In this review, bismuth‐based materials are divided into binary BiX₃ (X = I, Br, Cl), ternary A_aBi_bX_a+3b (A = Cs, Rb, MA, Ag, etc.), and quaternary A₂AgBiX₆ (A = Cs, Rb, MA, etc.) according to its elemental composition. The structure and optoelectronic properties of bismuth‐based halide materials, which may be helpful for the development of bismuth‐based halide materials and lead‐free perovskites in the future, are summarized and highlighted.     

Analysis and Optimum Design of Hybrid Plasmonic Slab Waveguides

Propagation properties of hybrid plasmonic slab waveguides are studied in detail using transfer matrix method considering structural and material aspects. Hybrid metal–insulator, hybrid metal–insulator–metal, and hybrid insulator–metal–insulator waveguides are considered. Propagation length (L _p), spatial length (L _s), and mode length (L _m) are utilized as three common figures of merit to compare and optimize the waveguides according to the layer thicknesses and metal/dielectric materials. The effect of constituting materials including metals (such as silver, gold, copper, and aluminum) and dielectrics (common dielectric materials used in photonic integrated circuit technologies such as silicon and silicon compounds, III–V compounds, and polymers) are discussed. It is found that hybrid waveguides are partially to completely superior to conventional plasmonic waveguides, providing a better balance between confinement and loss.     

Rhamnogalacturonan‐I is a determinant of cell–cell adhesion in poplar wood

The molecular basis of cell–cell adhesion in woody tissues is not known. Xylem cells in wood particles of hybrid poplar (Populus tremula × P. alba cv. INRA 717‐1B4) were separated by oxidation of lignin with acidic sodium chlorite when combined with extraction of xylan and rhamnogalacturonan‐I (RG‐I) using either dilute alkali or a combination of xylanase and RG‐lyase. Acidic chlorite followed by dilute alkali treatment enables cell–cell separation by removing material from the compound middle lamellae between the primary walls. Although lignin is known to contribute to adhesion between wood cells, we found that removing lignin is a necessary but not sufficient condition to effect complete cell–cell separation in poplar lines with various ratios of syringyl:guaiacyl lignin. Transgenic poplar lines expressing an Arabidopsis thaliana gene encoding an RG‐lyase (AtRGIL6) showed enhanced cell–cell separation, increased accessibility of cellulose and xylan to hydrolytic enzyme activities, and increased fragmentation of intact wood particles into small cell clusters and single cells under mechanical stress. Our results indicate a novel function for RG‐I, and also for xylan, as determinants of cell–cell adhesion in poplar wood cell walls. Genetic control of RG‐I content provides a new strategy to increase catalyst accessibility and saccharification yields from woody biomass for biofuels and industrial chemicals.     

Heparin Modulates Integrin-Mediated Cellular Adhesion: Specificity of Interactions with α and β Integrin Subunits

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin α_IIbβ₃, and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (α_vβ₃, α_vβ₅, and α_IIbβ₃). By comparing K562 cells expressing a common α subunit (Kα_vβ₃, Kα_vβ₅) with cells expressing a common β subunit (Kα_vβ₃, Kα_IIbβ₃), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5–7.5 μg/ml consistently enhanced the adhesion of β₃expressing cells (Kα_vβ₃,Kα_IIbβ₃). In contrast, UH at 0.5–7.5 μg/ml inhibited Kα_vβ₅adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kα_vβ₃cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the β subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.     

Resveratrol induces cell‐cycle disruption and apoptosis in chemoresistant B16 melanoma

Resveratrol, a naturally occurring polyphenol, has been shown to possess chemopreventive activities. In this study, we show that resveratrol (0–500 µM) inhibits the growth of a doxorubicin‐resistant B16 melanoma cell subline (B16/DOX) (IC₅₀ = 25 µM after 72 h, P < 0.05). This was accomplished by imposing an artificial checkpoint at the G₁–S phase transition, as demonstrated by cell‐cycle analysis and down‐regulation of cyclin D1/cdk4 and increased of p53 expression level. The G₁‐phase arrest of cell cycle in resveratrol‐treated (10–100 µM) B16/DOX cells was followed by the induction of apoptosis, which was revealed by pyknotic nuclei and fragmented DNA. Resveratrol also potentiated at subtoxic dose (25 µM for 24 h) doxorubicin cytotoxicity in the chemoresistant B16 melanoma (P < 0.01). When administered to mice, resveratrol (12.5 mg/kg) reduced the growth of an established B16/DOX melanoma and prolonged survival (32% compared to untreated mice). All these data support a potential use of resveratrol alone or in combination with other chemotherapeutic agents in the management of chemoresistant tumors. J. Cell. Biochem. 110: 893–902, 2010. © 2010 Wiley‐Liss, Inc.     

Heparin‐integrin interaction in endothelial cells: Downstream signaling and heparan sulfate expression

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up‐regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [³⁵S]‐sulfate metabolically labeling of the cells. We observed that the up‐regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine‐phosphorylation of focal adhesion‐associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin‐dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca²⁺ release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca²⁺ chelator, Ca²⁺ signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin‐induced up‐regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca²⁺/NO pathways. J. Cell. Physiol. 227: 2740–2749, 2012. © 2011 Wiley Periodicals, Inc.     

Preparation and properties of biologically active fluorescent heparins

Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl 0.12 mol; anticoagulant activity, 117 USP units/ mg) was reacted with 5-isothiocyanatofluorescein in 0.5M carbonate buffer (pH 8.5) at 35°C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (λ_em 516 nm, λ_ex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively).The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting the assay were virtually identical.     

Mercury(II) promotes the in vitro aggregation of tau fragment corresponding to the second repeat of microtubule‐binding domain: Coordination and conformational transition

The loss of metal homeostasis and the toxic effect of metal ion are important events in neurodegenerative and age‐related diseases, such as Alzheimer's disease (AD). For the first time, we investigated the impacts of mercury(II) ions on the folding and aggregation of Alzheimer's tau fragment R2 (residues 275‐305: VQIIN KKLDL SNVQS KCGSK DNIKH VPGGGS), corresponding to the second repeat unit of the microtubule‐binding domain, which was believed to be pivotal to the biochemical properties of full tau protein. By ThS fluorescence assay and electron microscopy, we found that mercury(II) dramatically promoted heparin‐induced aggregation of R2 at an optimum molar ratio of 1: 2 (metal: protein), and the resulting R2 filaments became smaller. Isothermal titration calorimetry (ITC) experiment revealed that the strong coordination of mercury(II) with R2 was an enthalpy‐controlled, entropy‐decreased thermodynamic process. The exceptionally large magnitude of heat release (ΔH₁ = ?34.8 Kcal mol^?1) suggested that the most possible coordinating site on the R2 peptide chain was the thiol group of cysteine residue (Cys291), and this was further confirmed by a control experiment using Cys291 mutated R2. Circular dichroism spectrum demonstrated that this peptide underwent a significant conformational change from random coil to β‐turn structure upon its binding to mercury(II) ion. This study was undertaken to better understand the mechanism of tau aggregation, and evaluate the possible role of mercury(II) in the pathogenesis of AD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1100–1107, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com