Fucosylated Chondroitin Sulfate 9-18 Oligomers Exhibit Molecular Size-Independent Antithrombotic Activity while Circulating in the Blood.

Fucosylated chondroitin sulfate (FCS) oligosaccharides extracted from sea cucumber and depolymerized exhibit potent anticoagulant activity. Knowledge of the antithrombotic activity of different size oligosaccharides and their fucose (Fuc) branch sulfation pattern should promote their development for clinical applications. We prepared highly purified FCS trisaccharide repeating units from hexasaccharide (6-mer) to octadecasaccharide (18-mer), including those with 2,4-disulfated and 3,4-disulfated Fuc branches. All 10 oligosaccharides were identified by their nuclear magnetic resonance structures and ESI-FTMS spectroscopy. In vitro anticoagulant activities and surface plasmon resonance binding tests indicated those of larger molecular sizes and 2,4-disulfated Fuc branches showed stronger anticoagulant effects with respect to anti-FXase activity, as well as stronger binding to FIXa among various clotting proteins. However, both types of FCS 9-mer to 18-mer exhibited molecular size-independent potent antithrombotic activity in vivo at the same dose. In addition, both types of the FCS 6-mer exhibited favorable antithrombotic activity in vivo, although they showed weak anticoagulant activity in vitro. Combining absorption and metabolism studies, we conclude that FCS 9-18 oligomers could remain in the circulation to interact with various clotting proteins to prevent thrombus formation, and appreciable quantities of these oligomers could be excreted through the kidneys. All FCS 9-18 oligomers also resulted in no bleeding, hypotension, or platelet aggregation risk during blood circulation. Thus, FCS 9-18 oligomers with 2,4-disulfated or 3,4-disulfated Fuc branches exhibit potent and safe antithrombotic activity needed for clinical applications.

Insulin polymers in the plasma of obese subjects are associated with elevated levels of carbonyl groups and are decreased by (-)-epicatechin.

We investigated whether oxidative damage and insulin polymerization at a systemic level are associated with the insulin resistance (IR) observed in obese subjects. We evaluated 3 groups (n=16/each) divided according to body mass index (BMI): Normal weight (NW) with a BMI of 18.5-24.9, obese 1 (O1) 30-34.9, and obese 3 (O3)>40 kg/m(2). IR and oxidative damage status of the groups were established by HOMA value and the analysis of biomarkers of oxidative stress in plasma. Insulin polymers in systemic circulation were detected using an antibody specific coupled to magnetic beads, which were incubated in plasma from the study groups. Analysis of magnetic beads by electrophoresis on polyacrylamide gel and silver stain assessed the presence of insulin polymers. The inhibition of polymers formation was studied by the presence of (-)-epicatechin. We demonstrated that O1 and O3 subjects with IR showed higher oxidative damage to their plasma lipids and proteins than NW subjects. This oxidative damage was associated with the presence of insulin polymers in the plasma of the O1 and O3 subjects. This polymer showed a high concentration of carbonyl groups by Western blot, suggesting the participation of oxidative damage in the generation of the polymer. The antioxidant (-)-epicatechin decreased the formation of the insulin polymer, indicating that the prevention of oxidative damage can inhibit insulin polymerization. Our study revealed an association between the presence of carbonyl stress, IR, and insulin polymer formation in obese subjects. This study also demonstrates that the antioxidant (-)-epicatechin inhibits insulin polymerization.

Interactions between solubilized polymer molecules and blood components.

In biomedical field, a variety of natural or synthetic polymers are being exponentially developed and used in vivo to improve human health. In practical applications, these biopolymers would enter blood circulation directly or indirectly, positively or passively, rapidly or slowly. Blood is a special tissue of human body, which fulfills many important missions to sustain normal metabolism. The contact with blood of the biopolymers, which are seen as foreign substances by the body, would be harmful or even instantaneously lethal, depending on the nature and the dose of the biopolymers administered. Therefore, it is critical to clearly understand the potential influences of the foreign polymers on blood before the polymers are applied from the lab to bedside. In this review, we discuss the recent studies on the interactions of foreign, solubilized polymer molecules (excluding formed polymer materials) with blood constituents (red blood cells, white blood cells, platelets, plasma proteins, etc.), to gain insight into the potential in vivo applications of the biopolymers in the biomedical field.

Purification and partial elucidation of the structure of an antioxidant carbohydrate biopolymer from the probiotic bacterium Bacillus coagulans RK-02.

An exopolysaccharide (EPS) was isolated from Bacillus coagulans RK-02 and purified by size exclusion chromatography. The purified, homogeneous EPS had an average molecular weight of ∼3 × 104 Da by comparison with FITC-labeled dextran standards. In vivo evaluations showed that, like other reported polysaccharides, this EPS displayed significant antioxidant activity. FTIR spectroscopy analysis showed the presence of hydroxy, carboxy, and α-glycosidic linkages and a mannose residue. GC analysis indicated that the EPS was a heteropolymer composed of glucose, mannose, galactose, glucosamine, and fucose as monomeric constituent units. Partial elucidation of the structure of the carbohydrate biopolymer based on GC-MS and NMR analysis showed the presence of two unique sets of tetrasaccharide repeating units that have 1→3 and 1→6 glycosidic linkages. This is also the first report of a Gram-positive bacterial polysaccharide with both fucose as a sugar monomer and 1→3 and 1→6 glycosidic linkages in the molecular backbone.

High molecular weight multimer form of adiponectin as a useful marker to evaluate insulin resistance and metabolic syndrome in Japanese men.

Adiponectin is an adipocyte-specific secretory protein that possesses antidiabetic and antiatherosclerotic properties. Recent studies have demonstrated that the high molecular weight (HMW) multimer form is the active form of this protein. In patients with type 2 diabetes mellitus, HMW-total adiponectin ratio was reported to be a more useful marker than total adiponectin in the prediction of insulin resistance and metabolic syndrome. In the present study of healthy Japanese male subjects without any medication, we investigated the hypothesis that measuring only HMW adiponectin may be as effective as HMW-total ratio to predict insulin resistance and/or metabolic syndrome. This was a working community-based cross-sectional study of 637 male subjects aged 30 to 65 years. Total and HMW adiponectin concentrations in serum were measured by enzyme-linked immunosorbent assay using commercially available kits. Serum HMW adiponectin level was inversely correlated with homeostasis model assessment of insulin resistance (HOMA-IR) (r = -0.375, P < .0001) even after adjustment for age and body mass index (r' = -0.245, P < .0001). When we divided the study subjects into quartile groups with equal numbers of subjects, HOMA-IR in the 4 groups based on serum HMW adiponectin level was significantly different (P < .01). Metabolic syndrome score in the 4 groups based on serum HMW adiponectin level was also significantly different (P < .01). Area under the curve of receiver operator characteristic curves of HMW adiponectin (0.73) to evaluate the presence of insulin resistance (HOMA-IR >2.5) was larger than that of total adiponectin (0.68) or HMW-total ratio (0.70). Area under the curve of receiver operator characteristic curves of HMW adiponectin (0.70) to evaluate the presence of metabolic syndrome (body mass index-based modified criteria) was also larger than that of total adiponectin (0.65), but equal to that of HMW-total ratio (0.70). These results suggest that simply measuring HMW adiponectin may be as effective as HMW-total ratio to evaluate the presence of insulin resistance and metabolic syndrome, at least in nondiabetic subjects who are not receiving any medication.

An increased polymeric IgA level is not a prognostic marker for progressive IgA nephropathy.

BACKGROUND: Elution of IgA from renal biopsies of patients with primary IgA nephropathy (IgAN) has suggested that mesangial IgA deposits are mainly multimeric in nature. This macromolecular IgA consists of dimeric and polymeric IgA and may be derived from the circulation. In children with IgAN, circulating macromolecular IgA levels correlate with bouts of macroscopic haematuria, but in adults a correlation with disease activity is less clear. Therefore, we have designed a novel method to assess the levels of polymeric IgA (pIgA) in sera from patients and controls. METHODS: A novel precipitation assay using recombinant CD89 was developed to measure pIgA. Polymeric IgA levels were measured in serum samples obtained from healthy volunteers (n = 21) and patients with IgAN (n = 51). Subsequently, serum pIgA levels were correlated with clinical parameters of disease. RESULTS: Serum pIgA levels were significantly increased in patients with IgAN. However, pIgA concentrations relative to total IgA were significantly lower in sera of patients with IgAN. No correlation was found between serum pIgA levels and clinical parameters of IgAN, such as decline of glomerular filtration rate, haematuria or proteinuria. CONCLUSIONS: Although absolute levels of serum pIgA are increased in patients with IgAN as compared with controls, levels of pIgA relative to total serum IgA are lower. No significant correlation was found between serum concentrations of pIgA and clinical parameters of disease. These data support the notion that it is not the size alone, but the physicochemical composition of the macromolecular IgA that is the key factor leading to mesangial deposition.

[Clinical significance of assessment of the metabolism of connective-tissue biopolymers in patients with type 1 diabetes mellitus and with diabetic nephropathy]. / Klinicheskoe znachenie otsenki obmena biopolimerov soedinitel'noi tkani u bol'nykh sakharnym diabetom tipa 1 s diabeticheskoi nefropatiei.

The purpose of the case study was to define a degree of destructive changes in the connective tissue at different stages of diabetic nephropathy (DN) progression. One hundred and twenty eight DN patients with type 1 diabetes mellitus, disease duration-5 to 20 years (classification according C. Mogensen, 1983) were examined. Special biochemical tools were used to evaluate the connective-tissue condition. The parameters of metabolism of the connective-tissue biopolymers were investigated in blood, urea and saliva, i.e. general and sulphated glucosamine glycine (GAG), sialic acid (SA) and oxyproline fractions. The results showed an increasing value of total and sulphated GAG and SA with a worsening DN severity, which is indicative of a destruction degree of glycoprotein-complexes in the microvascular basal membranes. A high level of bound oxyproline fractions denotes the fibril-genesis processes, which start to activate yet at the preclinical DN stage. The changes detected previously in the parameters of metabolism of the connective-tissue biopolymers, as observed in DN patients, ensure a timely choice of a therapy and a proper monitoring of its efficiency.

Hydrodynamic properties of rigid macromolecules composed of ellipsoidal and cylindrical subunits.

A procedure is devised for the calculation of hydrodynamic properties of rigid macromolecules composed subunits that are modeled as ellipsoids of revolution and cylinders. Owing to the axial symmetry of these shapes, smooth shell models can be constructured for the subunit structure. The bead shell model so constructed is employed for the calculation of the properties. A computer program, HYDROSUB, has been written implementing both the model building and the hydrodynamic calculation. A detailed example of the use of this methodology is presented for the case of the solution properties of the human antibody molecule immunoglobulin G3 (IgG3). Finally, hints are given on other uses and applications of the procedure.

Fc alpha RI/CD89 circulates in human serum covalently linked to IgA in a polymeric state.

The FcR for IgA CD89/FcalphaRI, is a type I receptor glycoprotein, expressed on myeloid cells, with important immune effector functions. In vitro CD89 can be released from CD89-expressing cells upon activation. Little information is available on the existence of this soluble molecule in vivo. Using specific and sensitive ELISA techniques (detection limit 50 pg/ml), we were not able to detect circulating CD89 in human sera. However, using Western blotting, a 30-kDa soluble CD89 molecule was demonstrated in both serum and plasma. Moreover, using a specific semiquantitative dot-blot system, we found CD89 in all human sera tested (mean concentration 1900 ng/ml). Size fractionation of human serum using gel filtration chromatography showed that the CD89 molecule was predominantly present in larger molecular mass fractions. Direct complexes between IgA and CD89 were demonstrated by anti-IgA affinity purification, and when analyzed under nonreducing conditions appeared to be covalently linked. Size fractionation of affinity-purified IgA showed the presence of soluble CD89 only in the high molecular mass fractions of IgA, but not in monomeric IgA. High molecular mass complexes of CD89-IgA could be distinguished from J chain containing dimeric IgA. These data show that CD89 circulates in complex with IgA, and suggest that CD89 might contribute to the formation of polymeric serum IgA.

Cutting edge: complement-activating complex of ficolin and mannose-binding lectin-associated serine protease.

Both ficolins and mannose-binding lectin (MBL) are lectins characterized by the presence of collagen-like and carbohydrate-binding domains in a subunit, although their carbohydrate-binding moieties are quite different. A fibrinogen-like domain is in ficolins, and a carbohydrate recognition domain is in MBL. On binding to pathogens, human MBL activates the complement system via the lectin pathway in association with two types of MBL-associated serine proteases (MASP), MASP-1 and MASP-2 and its truncated form, small MBL-associated protein (sMAP, also called MAp19). We report here that ficolin/P35, a human serum ficolin, was found to copurify with MASPs and sMAP. MASPs that were complexed with ficolin/P35 exhibited proteolytic activities against complement components C4, C2, and C3. The ficolin/P35-MASPs-sMAP complex that was bound to Salmonella typhimurium activated complement. These findings indicate that ficolin/P35 is a second collagenous lectin capable of activating the lectin pathway and thus plays a role in innate immunity.

Actin polymerisation in neutrophils of rheumatoid arthritis patients in relation to treatment with non-steroidal anti-inflammatory drugs.

There is evidence that neutrophil functions such as chemotaxis and oxygen radical formation are disturbed in rheumatoid arthritis (RA). Medication might also influence these functions. Cyclic formation and depolymerisation of actin microfilaments is crucial in cell motility, but this phenomenon has not been studied in RA. The aim of this study was to investigate basal and dynamic (formyl-methionyl-leucyl-phenylalanine (fMLP)-induced) neutrophil actin polymerisation in ten RA patients (a) during therapy with non-steroidal anti-inflammatory drugs (NSAIDS) and (b) after stopping NSAIDS> The results were compared with those of ten age-matched controls. Basal F-actin content in RA patients with NSAIDS was significantly lower than in RA patients without NSAIDS and controls: 35.5 (25.0-49.0), 50.5 (27.0-75.0) and 52.5 (32.0-85.0), respectively. Conversely, upon stimulation with fMLP, the actin polymerisation curve of RA patients with NSAIDS was higher than for RA patients without NSAIDS and controls. These results suggest that, in RA, the effects orf NSAIDS on neutrophil functions might be related to changes in the actin polymerisation-depolymerisation cycle.

Impaired actin polymerization and depolymerization in neutrophils from patients with thermal injury.

Acquired neutrophil dysfunction is considered an important cause of increased susceptibility to infection in patients with burns. In the early postinjury phase, large amounts of circulating chemo-attractants, cytokines and endotoxins induce strong systemic activation of neutrophils which may impair their motile functions. Actin is the most prevalent component of the microfilament lattice that generates force for the neutrophil motile responses, and in the present study we examined the dynamics of actin polymerization and depolymerization in neutrophils from 11 patients with large burns. At admission, the amount of polymerized actin in unstimulated neutrophils was 39.9 per cent higher than that of parallel controls. In addition, there was a positive correlation between the amount of polymerized actin and the total body surface area (TBSA) burn. The time course of patient neutrophil actin polymerization in response to FMLP, C5a, (Ser-IL-8)72, (Ala-IL-8)77 and crosslinking of surface Fc gamma RII was similar to that of controls, and the maximal amount of neutrophil F-actin was demonstrated after 30 s stimulation. At the peak of actin polymerization, however, patient neutrophils contained 27.3, 24.0, 24.7 and 25.6 per cent more polymerized actin than control cells stimulated with FMLP, (Ser-IL,-8)77, (Ala-8)77 and Fc gamma RII crosslinking, respectively. However, the relative increase of neutrophil F-actin following stimulation was significantly lower in patients than in controls. Moreover, the rate of patient neutrophil actin depolymerization was 39.0, 23.5, 63.3 and 51.7 per cent lower than that of controls after stimulation with FMLP, C5a (Ser-IL-8)72 and Fc gamma RII crosslinking, respectively. At discharge, the dynamics of neutrophil actin polymerization and depolymerization were similar to that of controls. The results demonstrate that in neutrophils during the early postburn phase, there are increased basal levels of polymerized actin, a lower responsiveness to stimulation and a reduced rate of actin depolymerization. As periodic polymerization and depolymerization of actin is essential for all neutrophil motile responses, it is probable that the alterations observed may contribute significantly to the overall neutrophil dysfunction following thermal injury.

Increased binding of polymeric lambda-IgA to cultured human mesangial cells in IgA nephropathy.

IgA nephropathy (IgAN) is characterized by raised plasma lambda-IgA1 and mesangial polymeric lambda-IgA1 deposits. It remains uncertain whether the predominant glomerular lambda-IgA1 deposits represent a selective uptake of polymeric IgA or a non-specific uptake due to elevated circulating lambda-IgA1 levels in response to an unidentified antigen. In this study, we explored whether there is an increased binding of monomeric IgA1 (mIgA1) or polymeric IgA1 (pIgA1) from patients with IgAN to cultured human mesangial cells (HMC). Total IgA1 in plasma from patients or healthy controls was isolated by jacalin-agarose column as jacalin-bound proteins (JBP). Monomeric IgA1 and pIgA1 were distinctly separated by FPLC. HMC were incubated with IgA preparations and IgA bound to HMC was determined by flow cytometry analysis using standard curves constructed by known concentrations of kappa-IgA1 or lambda-IgA1. In order to avoid any increased binding of IgA to HMC due to elevated kappa- or lambda-IgA concentrations in JBP samples from patients, JBP samples from patients or controls were appropriately diluted to achieve comparable levels of total IgA1. No differences in the total mIgA1 or pIgA1 concentration, percentage of mIgA1 or pIgA1, or the kappa/lambda ratio of mIgA1 or pIgA1 were found between adjusted JBP samples from patients or healthy controls. We found a sharp rise in percentage of pIgA1 among IgA1 bound to HMC (70%), despite the fact that only 3% of the IgA1 in the adjusted JBP samples were polymeric, suggesting that pIgA1 had a higher affinity to HMC than mIgA1. Furthermore, the kappa/lambda ratios of pIgA1 bound to HMC were significantly lower than the kappa/lambda ratios of pIgA1 in adjusted JBP only with IgAN patients but not healthy controls (P = 0.0026). Our data suggest a preferential mesangial binding of polymeric lambda-IgA1 from patients with IgAN. These polymeric lambda-IgA immune complexes are likely to be "pathogenic" and are important in the pathogenesis of IgAN.

The involvement of Kupffer cells and liver endothelial cells in the clearance of large sized soluble IgA aggregates in rats.

These findings suggests that AIgA is bound both by KC and EC in normal situations. Because of the great phagocytic capacity of KC, the contribution of EC in handling AIgA in normal rats is minimal. However, when KC are defect or absent (as after Cl2MDP treatment), the handling of AIgA by EC may become of mayor importance, i.e. it will take over the phagocytic function of KC. Studies concerning a possible receptor on EC involved in binding of AIgA are in progress.

Specific desensitization of actin polymerization of bovine platelets.

Polymerization of actin induced by activation of platelets was investigated using deoxyribonuclease I inhibition assay. When platelets were activated with ADP or 5-hydroxytryptamine, actin was polymerized quickly followed by rapid depolymerization to the initial level. Reactivation with the same agonist, however, did not cause the polymerization of actin, though with different agonists actin polymerized quite normally. The mechanism for this agonist-specific desensitization of actin polymerization was investigated by the use of a calcium ionophore A23187. It was suggested that the cause for the desensitization is the inability of platelets to mobilize Ca2+ in response to specific agonist.