Preparation, characterization and anticoagulant activity in vitro of heparin-like 6-carboxylchitin derivative

A series of heparin-like 6-carboxylchitin derivatives with different N-acetyl group and sulfate group contents were prepared. Their structures were characterized by element analysis, FT-IR, (13)C NMR, and gel permeation chromatography. Their anticoagulant activity in vitro was investigated for human plasma with respect to activated partial thromboplastin time (APTT). The results showed all 6-carboxylchitin derivatives prolonged APTT within the scope of studied degree of sulfation (0.28-1.03) and Mws (4.3-13.7 kDa). Their anticoagulant activity strongly depended on their structures. 3,6-O-sulfated group promoted the anticoagulant activity. Only incorporation of N-sulfated group into deacetylated 6-carboxylchitin could not improve the anticoagulant activity. But, N-sulfated group and O-sulfated group had the synergistic action, and N-sulfated group could promote the anticoagulant activity for the N,O-sulfated chitin derivatives. In addition, acetyl group took a role in the anticoagulant activity, too.     

Chemical modification,characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides

A natural lacquer polysaccharide with complex branches was separated into two fractions, LPH (MW 16.9x10(4)) and LPL (MW 6.85x10(4)). Results of 13C NMR and FT-IR indicated they had the same structure. The treatment of LPL with sodium periodate led to a partial cut-off of side chains with 4-O-methyl-D-glucuronic acid in the terminal. These polysaccharides were sulfated in the presence of Py*SO3/DMSO. Depending on the reaction conditions, the products showed a different degree of sulfation (DS) ranging from 0.57 to 1.57 and different molecular weights ranging from 1.71x10(4) to 3.49x10(4). FT-IR analysis showed the equatorial primary OH at O-6 and the axial secondary OH at O-4 were sulfated. Activated partial thromboplastin time (APTT), prothrombin time and thrombin time (TT) assays showed the sulfated polysaccharides could prolong APTT and TT, but not TP. These activities strongly depended on the DS, the molecular weights (MW) and the branching structure of polysaccharides. DS of above 0.8 was essential for anticoagulant activity. The anticoagulant activity increased with the DS and the molecular weights. The molecular weights played a more important role. The branching structure of polysaccharides increased the activities. In our studies, the sulfated polysaccharides with the DS of 1.15 and the highest MW of 3.49x10(4) had the best blood anticoagulant activities.     

Chemical modification of erythropoietin: an increase in in vitro activity by guanidination

Human recombinant erythropoietin (rHuEPO) was chemically modified with several group-specific reagents in order to study the role of each kind of amino-acid residue in its biological activity. Guanidination of the amino groups of the lysine residues yielded derivatives that showed higher activities in vitro than native rHuEPO, whereas amidination had no effect on the activity. By contrast, modification of the positive charges of the lysine residues to neutral or negative charges, such as in carbamylation, trinitrophenylation, acetylation or succinylation, caused a significant loss of rHuEPO activity. Chemical modification of other amino-acid residues, such as arginine and tyrosine residues or carboxyl groups, also led to loss of activity.     

Polyphenolic-polysaccharide compounds from selected medicinal plants of Asteraceae and Rosaceae families: Chemical characterization and blood anticoagulant activity

The preparations from selected traditional medicinal plants in Poland (Asteraceae and Rosaceae families), were prepared in the multi-step process of isolation and their anticoagulant activity was measured by APTT and PT tests. The most promising effect was observed for the substances extracted from Fragaria vesca (Rosaceae) and Echinacea purpurea (Asteraceae). They showed interesting activity with respect to the activity of 5th International Standard for Unfractionated Heparin in APTT method. The structure characterization by IR, HPLC, GLC-MS and colorimetric methods revealed that these preparations are macromolecular polysaccharide–polyphenolic conjugates, similar to cell wall acidic macromolecular fragments which are common in the higher plants. The high content of hexuronic acids, as well as phenolic glycoconjugates seems to be responsible for the observed anticoagulant activity.     

Chemical modification of chloroperoxidase for enhanced stability and activity

Chloroperoxidase from Caldariomyces fumago (CPO, EC 1.11.1.10) is one of the most interesting enzymes from the group of heme peroxidases and has been extensively applied in synthetic processes. Nevertheless, the practical application of CPO is limited due to its very low operational stability, especially in the presence of peroxidative compounds. For this reason, effect of chemical modifications of CPO in the stability of the enzyme was studied. Side-chain selective modifications of amino groups of Lys residues, and carboxyl groups of Asp and Glu residues, as well as crosslinking and periodate oxidation of sugar moiety were carried out. The stability of modified CPOs was evaluated at elevated pH and temperature, and in the presence of tert-butyl hydroperoxide. Effect of modification of CPO on the performance of the reaction of Cbz-ethanolamine oxidation was studied as well. Those modifications that involved carboxyl groups via carbodiimide coupled method and the periodate oxidation of the sugar moiety produced better catalysts than native CPO in terms of stability and activity at elevated pH values and temperatures.     

Chemical modification of reveromycin A and its biological activities

Various derivatives of reveromycin A, a novel inhibitor of eukaryotic cell growth, were prepared and their inhibitory effects on both isoleucyl-tRNA synthetase activity and in vitro protein synthesis, and activities on the morphological reversion of src(ts)-NRK cells were assayed. The C5 hydroxyl group and C24 carboxyl group are particularly important for these activities.     

Sulfonation of papain-treated chitosan and its mechanism for anticoagulant activity

The novel low-molecular-weight chitosan polysulfate (MW 5120-26,200 Da) was prepared using the depolymerization of chitosan with papain (EC. 3.4.22.2). The sulfonation of depolymerized products was performed using chlorosulfonic acid in N,N-dimethylformamide under semi-heterogeneous conditions. The structures of the products were characterized by FTIR, ¹³C NMR, and ¹H NMR (1D, 2D NMR) spectroscopy. The present study sheds light on the mechanism of anticoagulant activity of chitosan polysulfate. Anticoagulant activity was investigated by an activated partial thromboplastin assay, a thrombin time assay, a prothrombin time assay, and thrombelastography. Surface plasmon resonance also provided valuable data for understanding the relationship between the molecular binding of sulfated chitosan to two important blood clotting regulators, antithrombin III and heparin cofactor II. These results show that the principal mechanism by which this chitosan polysulfate exhibits anticoagulant activity is mediated through heparin cofactor II and is dependent on polysaccharide molecular weight.     

Identification of a Vibrio furnissii oligopeptide permease and characterization of its in vitro hemolytic activity

We describe purification and characterization of an oligopeptide permease protein (Hly-OppA) from Vibrio furnissii that has multifaceted functions in solute binding, in in vitro hemolysis, in antibiotic resistance, and as a virulence factor in bacterial pathogenesis. The solute-binding function was revealed by N-terminal and internal peptide sequences of the purified protein and was confirmed by discernible effects on oligopeptide binding, by accumulation of fluorescent substrates, and by fluorescent substrate-antibiotic competition assay experiments. The purified protein exhibited host-specific in vitro hemolytic activity against various mammalian erythrocytes and apparent cytotoxicity in CHO-K1 cells. Recombinant Hly-OppA protein and an anti-Hly-OppA monoclonal antibody exhibited and neutralized the in vitro hemolytic activity, respectively, which further confirmed the hemolytic activity of the gene product. In addition, a V. furnissii hly-oppA knockout mutant caused less mortality than the wild-type strain when it was inoculated into BALB/c mice, indicating the virulence function of this protein. Finally, the in vitro hemolytic activity was also confirmed with homologous ATP-binding cassette-type transporter proteins from other Vibrio species.     

Phosphorylated modification and in vitro antioxidant activity of Radix Hedysari polysaccharide

Phosphorylated modification of a polysaccharide obtained from Radix Hedysari (RHP) was studied. Three phosphorylated polysaccharides (RHPP) with variable degrees of substitution (DS(p)) were obtained with 4-dimethylaminopyridine (DMAP) and N, N' Dicyclocarbodiimide (DCC) as catalyst. The structures of RHPP were characterized by FT-IR spectra and (13)C NMR spectra. Depending on different reaction time, RHPP showed different DS(p) ranging from 0.30 to 0.66, and different Mw ranging from 86.6 to 89.7?KDa. Compared with RHP, RHPP exhibited superior antioxidant activities in vitro, which indicated that phosphorylated modification could enhance antioxidant activities of RHP. Furthermore, it was obvious that the DS(p) had a significant effect on the antioxidant activity.     

Modulation of diacylglycerol kinase theta activity by alpha-thrombin and phospholipids

Diacylglycerol kinase modulates the levels of diacylglycerol and phosphatidic acid, two critical lipid second messengers, yet little is known about the effects of cellular stimulation on the kinetic behavior of this enzyme. We examined the effects of alpha-thrombin and activating phospholipids on the activity and substrate affinity of a soluble diacylglycerol kinase, DGKtheta. Our data demonstrate that the apparent binding parameters of DGKtheta increase following thrombin stimulation, suggesting that alpha-thrombin antagonizes DGKtheta activity. Interestingly, this effect is obscured in the presence of high bulk substrate concentrations. Given the known stimulatory effects of phosphatidylserine on many diacylglycerol kinases, we examined the effects of various phospholipids on DGKtheta and found that phosphatidic acid is a more effective activator than phosphatidylserine. Phosphatidic acid decreased the apparent surface K(M) (K(M(surf))app) of DGKtheta for dioleoylglycerol (DOG) and promoted binding to vesicles in a dose-dependent manner. Phosphatidylserine also lowered the K(M(surf))app of DGKtheta, though higher concentrations were required to achieve the same effect. Interestingly, PS promoted binding to vesicles only when present at levels beyond that required to saturate enzyme activity, suggesting that PS and PA activate DGKtheta through different mechanisms. The potential physiological implications of these findings are discussed.     

Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana

The isolation, some structural features, physicochemical properties and pharmacological activities of a heparin from Anomalocardia brasiliana are reported. It is shown that the mollusc heparin is very similar to those present in mammalian tissues with regard to chemical composition, physicochemical properties, pharmacological activities and susceptibility to heparinase and heparitinase II from Flavobacterium heparinum, as well as to the types of products formed by the action of these enzymes. Three significant quantitative differences were observed for the mollusc heparin when compared with the ones from mammalian origin, namely, a higher degree of binding with antithrombin III (45%), higher molecular weight (27-43 kDa) and higher anticoagulant activity (320 I.U./mg). The possible biological role of heparin is discussed in view of the present findings.     

Chemical composition and in vitro antibacterial activity of Seseli libanotis

Summary The antibacterial efficacy of the methanol extract of the aerial parts of Seseli libanotis by using disc diffusion assay against 107 strains from 52 bacterial species and the chemical composition of the essential oil using GC/MS analysis were studied for the first time. The minimum inhibitory concentrations (MIC) of single compounds were determined by the microbroth dilution method. Gas chromatography–mass spectrophotometry analyses allowed 13 compounds to be determined; the main constituents of the essential oil of aerial parts of Seseli libanotis were trans-caryophyllene (20.39%), spathulenol (11.89%), (−)-caryophyllene oxide (11.47%), euasarone (10.66%) and delta-cadinene (9.16%). The methanol extract of Seseli libanotis had a broad-spectrum antibacterial activity (>14 mm inhibition zone in diameter) in particular against Bacillus cereus, Bacillus dipsauri, Bacillus lentimorbus, Bacillus sphaericus, Bacillus subtilis, Corynebacterium ammoniagenes, Kocuria rosea, Neisseria subflava and Micrococcus lylae. These inhibitory effects are interesting in relation to the prevention of microbial contamination in foods.     

Influence of functional groups on the in vitro anticoagulant activity of chitosan sulfate

A new method for the chemical modification of chitosan sulfate was used to prepare N-propanoyl-, N-hexanoyl- and N,O-quaternary substituted chitosan sulfate. Structural analysis by elemental analysis, FTIR, 13C NMR, and 1H NMR spectroscopy, and gel-permeation chromatography showed that these methods could conveniently be used for the introduction of functional groups. The influences of the acyl or quaternary groups on the anticoagulant activity of the polysaccharides were studied with respect to activated partial thromboplastin time (APTT) thrombin time (TT), and prothrombin time (PT). The propanoyl and hexanoyl groups increased the APTT activity, and the propanoyl groups also increased the TT anticoagulant activity slightly, while the N,O-quaternary chitosan sulfate showed only a slight TT coagulant activity.     

Chemical modification of recombinant human granulocyte colony-stimulating factor by polyethylene glycol increases its biological activity in vivo.

Recombinant human granulocyte colony-stimulating factor (rHuG-CSF) produced in Escherichia coli was chemically modified by polyethylene glycol (PEG) of molecular weights 4,500 or 10,000. The neutrophils observed at 32 hours after intravenous injection of the rHuG-CSF modified with PEG (4,500) or PEG (10,000) to mice were, respectively, 2.5 times and 5 times more than that observed after the injection of the unmodified rHuG-CSF. These results show that the covalent attachment of PEG to rHuG-CSF enhanced its pharmacological activity in vivo and that the modification with the larger PEG molecule is more effective to enhance the in vivo activity of rHuG-CSF.     

Chemical modification of the carbohydrate moiety in N-acetylmuramoyl-l-alanyl-d-isoglutamine,and the immunoadjuvant activity

Five carbohydrate analogs of N-acetylmuramoyl-l-alanyl-d-isoglutamine have been synthesized from benzyl 2-acetamido-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-glucopyranoside (1) and the corresponding 6-O-benzoyl derivative (2). Chlorination of 1 and 2 with triphenylphosphine in carbon tetrachloride gave the 4,6-dichloro compound 3 and the 6-O-benzoyl-4-chloro compound (4), which were treated with tributyltin hydride, to yield benzyl 2-acetamido-2,4,6-trideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-xylo-hexopyranoside (6) and benzyl 2-acetamido-6-O-benzoyl-2,4-dideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-xylo-hexopyranoside (7), respectively. Methanesulfonylation of 8, derived from 7 by debenzoylation, gave the 6-methanesulfonate, which underwent displacement with azide ion to afford benzyl 2-acetamido-6-azido-2,4,6-trideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-xylo-hexopyranoside (10). Hydrolysis of the methyl ester group in compounds 3, 5 (debenzoylated 4), 6, 8, and 10 gave the corresponding free acids, which were coupled with l-alanyl-d-isoglutamine benzyl ester, to yield the dipeptide derivatives in excellent yields. Hydrogenation of the dipeptide derivatives thus obtained gave the five carbohydrate analogs of N-acetylmuramoyl-l-alanyl-d-isoglutamine, respectively, in good yields. The immunoadjuvant activity of the N-acetylmuramoyl-dipeptide analogs was examined.