Heparin in molluscs: chemical, enzymatic degradation and C and H n.m.r. spectroscopical evidence for the maintenance of the structure through evolution

The structural features and anticoagulant activity of heparins isolated from three species of molluscs (Anomalocardia brasilian, Donnax striatus and Tivela mactroides) are reported. It is shown by chemical analyse, type of products formed by action of heparinase and heparitinase II, anticoagulant activity, ¹³C and ¹H n.m.r. spectroscopy, that the mollusc heparins are virtually indistinguishable from heparins present in mammalian tissues. These data, taken as a whole, suggest that heparin has maintained its main structural features through evolution. The implications of these findings are discussed.     

Mast cells are present in epithelial layers of different tissues of the mollusc Anomalocardia brasiliana. In situ characterization of heparin and a correlation of heparin and histamine concentration

Heparin, other sulphated glycosaminoglycans and histamine were extracted from various dissected organs of Anomalocardia brasiliana, a mollusc from the South Atlantic, and quantified. A good correlation between heparin and histamine content was found in the labial palp, intestine, ctenidium, mantle and foot tissues. The tissue location of metachromatic cells, putatively containing heparin, was identified histologically with Alcian Blue, Toluidine Blue, Masson trichrome, Haematoxylin-Eosin and PAS. Except for the foot, cells containing metachromatic granules were found in the epithelium surfaces of all the organs analysed. An in situ identification of heparin using nitrous acid and heparinase degradation has established unequivocally the presence of this compound in the metachromatic cells. The location of 'mast-like' cells at the epithelium surface of mollusc tissues exposed to the environment are very similar to the distribution of mammalian and other vertebrate mast cells and gives support to the suggestion for a role of mast cells in defense mechanisms.     

Mast cells are present in epithelial layers of different tissues of the mollusc <Emphasis Type="Italic">Anomalocardia brasiliana</Emphasis>. <Emphasis Type="Italic">In situ</Emphasis> characterization of heparin and a correlation of heparin and histamine concentration

Heparin, other sulphated glycosaminoglycans and histamine were extracted from various dissected organs of Anomalocardia brasiliana, a mollusc from the South Atlantic, and quantified. A good correlation between heparin and histamine content was found in the labial palp, intestine, ctenidium, mantle and foot tissues. The tissue location of metachromatic cells, putatively containing heparin, was identified histologically with Alcian Blue, Toluidine Blue, Masson trichrome, Haematoxylin–Eosin and PAS. Except for the foot, cells containing metachromatic granules were found in the epithelium surfaces of all the organs analysed. An in situ identification of heparin using nitrous acid and heparinase degradation has established unequivocally the presence of this compound in the metachromatic cells. The location of 'mast-like' cells at the epithelium surface of mollusc tissues exposed to the environment are very similar to the distribution of mammalian and other vertebrate mast cells and gives support to the suggestion for a role of mast cells in defense mechanisms.     

Protein-protein interaction conferring stability to an extracellular acetyl (xylan) esterase produced by Termitomyces clypeatus

Acetyl esterase (AE) activity present in the culture filtrate of Termitomyces clypeatus was separated into lower molar mass (LMM) and higher molar mass (HMM) protein fractions during BioGel P-200 gel chromatography. AE was purified as a 30 kDa nonglycosylated protein from LMM fractions by CM-Sepharose ion exchange chromatography and HPGPLC. Although the HMM fraction had a number of enzyme activities (sucrase, beta-xylosidase, beta-glucosidase, and alpha-L-arabinofuranosidase) other than AE, protein present in the fraction was eluted as a single protein peak in HPGPLC and gave a single band in native PAGE. The fraction, subsequently purified by DEAE-Sephadex chromatography, was a SDS-PAGE homogeneous 80 kDa glycoprotein, but with both AE and cellobiase activities. The aggregate dissociated during ConA-Sepharose chromatography and 30 kDa AE and 56 kDa glycosylated cellobiase were purified separately. The dissociation caused significant loss of cellobiase activity but not that of AE. AE purified from both HMM and LMM fractions was characterized to be the same enzyme in terms of molar masses, pI (7.3), and other physicochemical properties. AE as an aggregate with cellobiase showed higher thermostability, temperature optimum, and resistance toward chemical denaturants than those of purified AE. Compared to cellobiase purified earlier from the same fungus, the enzyme present with AE in the aggregate also showed higher catalytic activity, thermostability, and temperature optimum. The study indicated that the formation of such SDS-resistant enzyme aggregate was associated with significant changes in the physicochemical properties of the enzymes, mainly toward improvement of rigidity of enzymes, and sometimes with the improvement of catalytic activity.     

Existence of a polar digitalis-like factor in mammalian hypothalamus

We were able to partially purify a polar digitalis-like factor from rat and bovine hypothalami based on the capacity to inhibit [3H]ouabain binding to intact human erythrocytes. This factor was characterized in reference to the digitalis-like factor that we have isolated and reported on. Hypothalamic factor shared digitalis-like activities and physicochemical properties with the one derived from human urine and mammalian plasma. These findings strongly suggest that a polar digitalis-like factor identical to the circulatory factor does exist in mammalian hypothalamus.     

Enhancing the pharmacodynamic profile of a class of selective COX-2 inhibiting nitric oxide donors

We report herein the development, synthesis, physicochemical and pharmacological characterization of a novel class of pharmacodynamic hybrids that selectively inhibit cyclooxygenase-2 (COX-2) isoform and present suitable nitric oxide releasing properties. The replacement of the ester moiety with the amide group gave access to in vivo more stable and active derivatives that highlighted outstanding pharmacological properties. In particular, the glycine derivative proved to be extremely active in suppressing hyperalgesia and edema.     

Pimprinine, an extracellular alkaloid produced by Streptomyces CDRIL-312: fermentation, isolation and pharmacological activity

Pimprinine, an extracellular alkaloid has been isolated from the culture filtrate of Streptomyces CDRIL-312. Pimprinine was subsequently purified using silica gel column chromatography and also by preparatory thin layer chromatography. Some physicochemical properties, antimicrobial activities (in-vitro) and pharmacological activities of pimprinine were studied. Pimprinine showed promising anticonvulsant activity in both minimum and maximum electric seizure threshold test in mice. Its anticonvulsant activity is very much comparable to that of phenyl hydantion sodium. Pimprinine also inhibited effectively tremorine-induced tremors and analgesia in mice.     

Anti-inflammatory properties of a heparin-like glycosaminoglycan with reduced anti-coagulant activity isolated from a marine shrimp

The anti-inflammatory properties of a heparin-like compound from the shrimp Litopenaeus vannamei are related. Besides reducing significantly (p<0.001) the influx of inflammatory cells to injury site in a model of acute inflammation, shrimp heparin-like compound was able to reduce the matrix metalloproteinase (MMPs) activity in the peritoneal lavage of inflamed animals. Moreover, this compound also reduced almost 90% the activity of MMP-9 secreted by human activated leukocytes. Negligible anti-coagulant activities in aPPT assay and a poor bleeding potential make this compound a better alternative than mammalian heparin as a possible anti-inflammatory drug.     

Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6

Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.     

The pharmacological profile of the low molecular weight heparin 21-23 in man: anticoagulant, lipolytic and protamine reversible effects

The anticoagulant, lipolytic and protamine reversible effects of high doses of low molecular weight (LMW) heparin 21-23 and unfractionated heparin were compared in man. 7,500 units of each heparin were applied, which corresponds to 90 mg LMW heparin and 48 mg unfractionated heparin. The anticoagulant properties of the LMW heparin are characterized by a doubled half life of factor Xa activity, smaller influence on aPTT and thrombin after intravenous (i.v.) and subcutaneous (s.c.) injection, and higher bioavailability of factor Xa activity after s.c. administration (90% versus 15%). Protamine chloride completely neutralizes the effect on aPTT and thrombin and reduces the anti factor Xa activity by 60%. The bleeding time is prolonged by both normal and LMW heparin by 20%. This effect is normalized by protamine chloride, too. Thrombelastography with recalcified whole blood demonstrates that protamine chloride shortens but not completely normalizes the coagulation time in presence of either unfractionated or LMW heparin. The half life of lipoprotein lipase (LPL) activity is 60 min after i.v. administration of unfractionated heparin and 120 min with LMW heparin. Although the release of lipases (LPL and HTGL) is higher after i.v. and s.c. administration of the LMW heparin they do not induce higher releases of free fatty acids. This indicates that the lipolytic activity of this LMW heparin and unfractionated heparin is similar. The results show an improved anticoagulant pharmacological profile of this LMW heparin as compared to unfractionated heparin. Protamine normalizes the anticoagulant effects of LMW heparin with exception of a residual anti factor Xa activity and normalizes the changes of bleeding time and thrombelastography.     

The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0–102.8% and 101.6–102.8%, respectively), good intermediate precision (1.9–2.1% and 2.4–4.2%, respectively) and good reproducibility (4.0–4.8% and 3.6–6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.     

Enhanced chemical and biological activities of a newly biosynthesized eugenol glycoconjugate, eugenol α-d-glucopyranoside

Eugenol, the essential component (over 90 %) of clove oil from Eugenia caryophyllata Thunb. (Myrtaceae), is a phenolic compound well known for its versatile pharmacological actions, including analgesic, local anesthetic, anti-inflammatory, antimicrobial, antitumor, and hair-growing effects. However, the application of eugenol is greatly limited mainly because of its unwanted physicochemical properties, such as low solubility, liability to sublimation, and pungent odor. Since glycosylation has been suggested to improve the physicochemical and biological properties of the parental compound, we have previously developed a novel and efficient way to biosynthesize highly purified eugenol α-D-glucopyranoside (α-EG). In light of the widely acknowledged importance of pure eugenol and the potential superiority of the glycosylation, it is crucial to further explore and compare the physicochemical and biological properties of these two phenolic compounds. In this study, we demonstrate that glucosylation is a promising method for modification of phenolic compound, and that α-EG is superior over its parent eugenol, in all of the tested aspects, including physicochemical properties, antioxidation activity, and antimicrobial and antitumor activities. These results strongly suggest that α-EG, as a novel prodrug, may serve as a useful probe and potential therapeutic drug in both fundamental research and clinical application in the coming future.     

Identification of protein phosphatases-1 and 2A and inhibitor-2 in oocytes of the starfish Asterias rubens and Marthasterias glacialis

Protein phosphatases present in the particulate and soluble fractions of oocytes of the starfish Asterias rubens and Marthasterias glacialis have been classified according to the criteria used for these enzymes from mammalian cells. The major protein phosphatase activity in the particulate fraction had very similar properties to protein phosphatase-1 from mammalian tissues, including preferential dephosphorylation of the beta subunit of phosphorylase kinase, sensitivity to inhibitor-1 and inhibitor-2, inhibition of phosphorylase phosphatase activity by protamine and heparin, and retention by heparin-Sepharose. The major protein phosphatase in the soluble fraction had very similar properties to mammalian protein phosphatase-2A, including preferential dephosphorylation of the alpha subunit of phosphorylase kinase, insensitivity to inhibitors-1 and 2, activation by protamine and heparin, and exclusion from heparin-Sepharose. An acid-stable and heat-stable protein was detected in the soluble fraction of starfish oocytes, whose properties were indistinguishable from those of inhibitor-2 from mammalian tissues. It inhibited protein phosphatase-1 specifically, and its apparent molecular mass on SDS polyacrylamide gels was 31 kDa. Furthermore, an inactive hybrid formed between the starfish oocyte inhibitor and the catalytic subunit of mammalian protein phosphatase-1 could be reactivated by preincubation with MgATP and mammalian glycogen synthase kinase-3. The remarkable similarities between starfish oocyte protein phosphatases and their mammalian counterparts are indicative of strict phylogenetic conservation of these enzymes. The results will facilitate further analysis of the role of protein phosphorylation in the control of starfish oocyte maturation by the hormone 1-methyladenine.     

Comparison of Physical and Chemical Properties of Green and Brown Brazilian Propolis Collected in Minas Gerais,Brazil

Among the 13 types of propolis classified in Brazil according to their physicochemical properties, green propolis and brown propolis are the most commonly found and used. In this work, a comparison of the physicochemical properties of green and brown propolis produced in Minas Gerais, Brazil was performed according to the methodology established by the Brazilian legislation. And, the content of 9 bioactive compounds in the samples was determined by RP-HPLC. GrProp showed a higher content of pinocembrin, artepillin C and baccharin, and a higher quantity of total flavonoids, in comparison with BrwProp. The mechanical mass content in both types of propolis was above the limit established by legislation. However, the other physicochemical parameters were within the limits. The chemical composition, especially the flavonoid content and the free radical (DPPH) scavenger property confer to both types of propolis a promising pharmacological activity.     

Glycosaminoglycan composition of the large freshwater mollusc bivalve Anodonta anodonta

In this paper, glycosaminoglycans from the body of the large freshwater mollusc bivalve Anodonta anodonta were recovered at about 0.6 mg/g of dry tissue, composed of chondroitin sulfate (approximately 38%), nonsulfated chondroitin (about 21%), and heparin (41%). This last polysaccharide was found to consist of a large percentage (approximately 88%) of a fast-moving species possessing a lower molecular mass and sulfate group amount and about 12% of a more sulfated, slow-moving component having a greater molecular mass. The chondroitin sulfate was composed of approximately 28% of the 6-sulfated disaccharide, 46% of the 4-sulfated disaccharide, and about 26% of the nonsulfated disaccharide, with a charge density value of 0.74. Heparin was subjected to the oligosaccharide mapping after treatment with heparinase and then separation of the resulting unsaturated oligosaccharides by SAX-HPLC. A heparin sample from Anodonta anodonta showed a degree of sulfation similar to that of bovine mucosal heparin because of the presence of approximately the same mol % of the trisulfated disaccharide (DeltaUA2S(1-->4)-alpha-D-GlcN2S6S), a slight modification of the other oligosaccharides, and a significant increase of the disaccharide bearing the sulfate group in position 3 of the N-sulfoglucosamine 6-sulfate (-->4)-beta-D-GlcA(1-->4)-alpha-D-GlcN2S3S6S(1-->) part of the ATIII-binding region. However, the anticoagulant activity of mollusc heparin was quite similar to that of pharmaceutical grade heparin. The data obtained again emphasize the heterogeneity of GAGs from molluscs.     

Adaptation of the Lyme disease spirochaete to the mammalian host environment results in enhanced glycosaminoglycan and host cell binding

The Lyme disease spirochaete, Borrelia burgdorferi, is transmitted to mammals by Ixodes ticks and can infect multiple tissues. Host cell attachment may be critical for tissue colonization, and B. burgdorferi cultivated in vitro recognizes heparin- and dermatan sulphate-related glycosaminoglycans (GAGs) on the surface of mammalian cells. To determine whether growth of the spirochaete in the mammalian host alters GAG binding, we assessed the cell attachment activities of B. burgdorferi grown in vitro or in dialysis membrane chambers implanted intraperitoneally in rats. Host-adapted B. burgdorferi exhibited approximately threefold better binding to purified heparin and dermatan sulphate and to GAGs expressed on the surface of cultured endothelial cells. Three B. burgdorferi surface proteins, Bgp, DbpA and DbpB, have been demonstrated previously to bind to GAGs or to GAG-containing molecules, and we show here that recombinant derivatives of each of these proteins were able to bind to purified heparin and dermatan sulphate. Immunofluorescent staining of in vitro-cultivated or host-adapted spirochaetes revealed that DbpA and DbpB were present on the bacterial surface at higher levels after host adaptation. Recombinant Bgp, DbpA and DbpB each partially inhibited attachment of host-adapted B. burgdorferi to cultured mammalian cells, consistent with the hypothesis that these proteins may promote attachment of B. burgdorferi during growth in the mammalian host. Nevertheless, the partial nature of this inhibition suggests that multiple pathways promote mammalian cell attachment by B. burgdorferi in vivo. Given the observed increase in cell attachment activity upon growth in the mammalian host, analysis of host-adapted bacteria will facilitate identification of the cell binding pathways used in vivo.     

A C-type lectin from the tunicate, Styela plicata, that modulates cellular activity

Previous studies have identified proteins from tunicates (invertebrate members of the Phylum Chordata) that have physicochemical and functional properties similar to those of the inflammatory cytokine, interleukin 1 (IL-1). Here we characterize one of those proteins from the tunicate, Styela plicata, that can stimulate tunicate and mammalian cell proliferation, activate phagocytosis, increase interleukin 2 (IL-2) secretion by mammalian peripheral blood mononuclear cells and enhance IL-2 receptor (IL-2R) expression by mammalian EL-4.IL-2 cells. Partial amino acid sequence data showed that the S. plicata protein resembles three C-type lectins (TC14, TC14-1 and TC14-2) from a closely related tunicate species, Polyandrocarpa misakiensis. Its similarity to carbohydrate recognition domains (CRDs) from P. misakiensis lectins suggests that the S. plicata protein modulates the activities of mammalian immunocompetent cells by interacting with carbohydrate moieties of glycosylated cell surface receptors.     

Mechanism of inhibition of mammalian DNA topoisomerase I by heparin.

We have previously shown that heparin is a potent inhibitor of a mammalian DNA topoisomerase I. We have now investigated the mechanism of its inhibition. This was carried out first by scrutinizing the structural features of heparin molecules responsible for the inhibition. Commercial heparin preparation was fractionated by antithrombin III-Sepharose into non-adsorbed, low-affinity and high-affinity fractions, of which only the high-affinity fraction of heparin is known to contain a specific oligosaccharide sequence responsible for the binding to antithrombin III. These fractions all exhibited essentially similar inhibitory activities. Furthermore, when chemically sulphated to an extent comparable with or higher than heparin, otherwise inactive glycosaminoglycans such as heparan sulphate, chondroitin 4-sulphate, dermatan sulphate and neutral polysaccharides such as dextran and amylose were converted into potent inhibitors. Sulphated dermatan sulphate, one of the model compounds, was further shown to bind competitively to the same sites on the enzyme as heparin. These observations strongly suggested that topoisomerase inhibition by heparin is attributable primarily, if not entirely, to the highly sulphated polyanionic nature of the molecules. In a second series of experiments we examined whether heparin inhibits only one or both of the topoisomerase reactions, i.e. nicking and re-joining. It was demonstrated that both reactions were inhibited by heparin, but the nicking reaction was more severely affected than was the re-joining reaction.     

Molecular mechanism for ligand stabilization in the mollusc myoglobin possessing the distal Val residue.

Myoglobin extracted from the triturative stomach of Dolabella auricularia, a common mollusc found on the Japanese coast, possesses naturally occurring substitution at the distal E7 position (Val-E7) and its oxygen affinity is only slightly lower than those of the common mammalian myoglobins possessing the usual His-E7. 1H nuclear magnetic resonance studies of Dolabella met-cyano myoglobin have revealed that a guanidino NH proton of Arg-E10 is hydrogen-bonded to the Fe-bound CN-. The role of Arg-E10 as a hydrogen-bond donor for Fe-bound ligand in the present myoglobin appears to be responsible for its relatively high ligand affinity.