Binding and biological properties of lipopolysaccharide Proteus vulgaris O25 (48/57)–chitosan complexes

In this study the negatively charged Proteus vulgaris O25 LPS was chosen for studying interaction with polycationic chitosan. The complex formation of LPS with chitosan was demonstrated using gradient centrifugation and laser interferometry method. The presented results have shown that laser interferometry method is sensitive enough for LPS–chitosan interaction studies. The changing in the ultra structure of LPS during binding with chitosan was observed by electronic microscope. The interaction of P. vulgaris O25 LPS with chitosan was shown to modulate significantly the biological activities of LPS. The toxicity of P. vulgaris O25 LPS decreased 10-fold after forming complexes with chitosan at injection to mice in the similar concentration of endotoxin. The complex LPS–chitosan was less effective than LPS alone in Limulus amabocyte lysate assay. Induction of TNF biosynthesis by LPS–chitosan complex was found to be 65% lower than that by parent LPS at concentration of 100 ng/ml.     

Comparative study of electrokinetic potentials and binding affinity of lipopolysaccharides-chitosan complexes

Electrokinetic properties of complexes of chitosan (Ch) with lipopolysaccharides (LPSs) from Escherichia coli O55:B5, Yersinia pseudotuberculosis 1B 598, and Proteus vulgaris O25 (48/57) and their size distribution were investigated using zeta-potential distribution assay and quasi-elastic light scattering. The interaction of LPS from different microorganisms with chitosan at the same w/w ratio of components (1:1) resulted in the formation of complexes in which the negative charge of LPS was neutralized (LPS from E. coli) or overcompensated (Y. pseudotuberculosis and P. vulgaris). The changing in size of the endotoxin aggregates during binding with chitosan was observed. The binding constants of chitosan with LPSs were determined by a method with using the anionic dye Orange II. The LPS from E. coli possess higher affinity to chitosan in comparison with the two others samples of endotoxin.     

Fas-deficient mice have impaired alveolar neutrophil recruitment and decreased expression of anti-KC autoantibody:KC complexes in a model of acute lung injury

Background

Chronic obstructive pulmonary disease (COPD) is characterized by progressive worsening of airflow limitation associated with abnormally inflamed airways in older smokers. Despite correlative evidence for a role for tumor necrosis factor-alpha in the pathogenesis of COPD, the anti-tumor necrosis factor-alpha, infliximab did not show clinical efficacy in a double-blind, placebo-controlled, phase II clinical trial. This study sought to evaluate the systemic inflammatory profile associated with COPD and to assess the impact of tumor necrosis factor neutralization on systemic inflammation.

Methods

Serum samples (n = 234) from the phase II trial were collected at baseline and after 24 weeks of placebo or infliximab. Additionally, baseline serum samples were obtained from an independent COPD cohort (n = 160) and 2 healthy control cohorts (n = 50; n = 109). Serum concentrations of a broad panel of inflammation-associated analytes were measured using a 92-analyte multiplex assay.

Results

Twenty-five proteins were significantly elevated and 2 were decreased in COPD, including highly elevated CD40 ligand, brain-derived neurotrophic factor, epidermal growth factor, acute-phase proteins, and neutrophil-associated proteins. This profile was largely independent of smoking status, age, and clinical phenotype. The majority of these associations of serum analytes with COPD are novel findings. Increased serum creatine kinase-muscle/brain and myoglobin correlated modestly with decreased forced expiratory volume at 1 second, suggesting cardiac involvement. Infliximab did not affect this systemic inflammatory profile.

Conclusions

A robust systemic inflammatory profile was associated with COPD. This profile was generally independent of disease severity. Because anti-tumor necrosis factor-alpha did not influence systemic inflammation, how to control the underlying pathology beyond symptom suppression remains unclear.

Trial Registration

ClinicalTrials.gov, No.: NCT00056264.     

Antioxidant properties of some different molecular weight chitosans

Chitosan, a cationic polysaccharide, is widely employed as dietary supplement and in pharmacological and biomedical applications. Although numerous studies have focused on its applications as pharmaceutical excipients or bioactive reagents, relationships between molecular weight (Mr) and biological properties remain unclear. The focus of this study was on the antioxidant properties of several Mr chitosans. We measured the ability of seven Mr chitosans (CT1; 2.8 kDa, CT2; 17.0 kDa, CT3; 33.5 kDa, CT4; 62.6 kDa, CT5; 87.7 kDa, CT6; 604 kDa, CT7; 931 kDa) to protect plasma protein from oxidation by peroxyl radicals derived from 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH). A comparison of the antioxidant action of high Mr chitosans (CT6–CT7) with that of low Mr chitosans (CT1–CT5) showed that low Mr chitosans (CT1–CT5) were more effective in preventing the formation of carbonyl groups in plasma protein exposed to peroxyl radicals. AAPH substantially increases plasma protein carbonyl content via the oxidation of human serum albumin (HSA). We also measured the ability of these chitosans to protect HSA against oxidation by AAPH. Low Mr chitosans (CT1–CT5) were found to effectively prevent the formation of carbonyl groups in HSA, when exposed to peroxyl radicals. Low Mr chitosans were also good scavengers of N-centered radicals, but high Mr chitosans were much less effective. We also found a strong correlation between antioxidant activity and the Mr of chitosans in vitro. These activities were also determined by using the ‘TPAC’ test. These results suggest that low Mr chitosans (CT1–CT3) may be absorbed well from the gastrointestinal tract and inhibit neutrophil activation and oxidation of serum albumin that is frequently observed in patients plasma undergoing hemodialysis, resulting in a reduction in oxidative stress associated with uremia.     

壳寡糖抑制脂多糖诱导的PIECs表达IL-8和VCAM-1作用的初步研究

目的：观察壳寡糖对脂多糖（LPS）诱导的猪髋动脉内皮细胞（PIECs）炎症损伤的影响以及潜在的分子机制。方法：以脂多糖（1g／mE）＊《激PIECs细胞,建立炎症损伤模型,以RT—PCR和Westernblot的方法观察壳寡糖（COS）预保护PIECs细胞24h,对白介素－8（IL－8）和血管细胞粘附分子．1（VCAM－1）表达水平的影响,以及对JNK信号蛋白磷酸化和c-Fos转录因子表达的影响。结果：壳寡糖可抑制脂多糖刺激的PIECs表达IL－8和VCAM－1,并抑制JNK信号通路的磷酸化和转录因子c-Fos的表达。结论：壳寡糖对脂多糖刺激的PIECs细胞中IL－8和VCAM—1表达的抑制作用是通过抑制上游的JNK信号通路磷酸化和转录因子c-Fos的表达实现的,从而缓解脂多糖对细胞造成的炎症损伤。     

Formation and properties of polyelectrolyte complexes of chitosan hydrochloride and sodium dextransulfate

Methods of potentiometry, turbidimetry, colorimetry, IR spectroscopy, and element analysis were used to investigate the conditions of formation and the properties of non-stoichiometric polyelectrolyte complexes of chitosan hydrochloride (CHC) and sodium dextransulfate (the molecules of both polysaccharides appear as semirigid chains, but their charges are opposite). It was determined that the complexes' formation of polyelectrolytes studied is predominantly electrostatic in the presence of urea. As was also found turbidity and stability of the polycomplexes solutions depended markedly on pH value of CHC and a nature of the low-molecular-weight salts added. The complexes obtained were soluble in water, aqueous urea, and water-organic mixtures. The extent of solubility depended on the composition of the complexes and could be influenced by addition of appropriate concentrations of certain low-molecular-weight salts.     

The immunological effects of taxanes

In the past decade, taxanes have gained notoriety as promising chemotherapeutic agents against different forms of cancer. These molecules were initially characterized as mitotic inhibitors, and their anti-neoplastic actions were attributed to their ability to suppress cellular division via microtubule stabilization. Less appreciated is the observation that taxanes induce other biological effects, especially in the immune system. For example, taxanes are immunostimulatory against neoplasms, supporting the idea that these agents suppress cancer through several mechanisms and not solely through inhibiting cell division. In addition, these drugs potentially regulate other aspects of immune function, such as lymphocyte activation, giving further support to their immunomodulatory capacity. In summary, taxanes effect multiple actions and potentially have greater therapeutic application beyond cancer chemotherapy. Received: 29 February 2000 / Accepted: 29 March 2000     

Rheological properties of chitosan-tripolyphosphate complexes: From suspensions to microgels

Complex fluids formed by crosslinking of chitosan (CS, 330 kDa) with sodium tripolyphosphate (TPP) have been studied by dynamic light scattering (DLS), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), and rheology. The effects of chitosan/TPP ratios, initial chitosan or TPP concentrations, and ultrasonication time on the chitosan-TPP complex formation have been investigated. It was found that the optimum condition for CS-TPP nanoparticle formation occurred at CS/TPP mass ratio of 3.75 and with 9 min sonication treatment (energy output 3.75 W/mL). At the same initial chitosan concentration, small particle sizes (i.e., particle size < 300 nm) resulted in the formation of CS-TPP nanoparticle suspensions, which showed a lower viscosity than pure chitosan solutions, and their viscosities increased as the CS-TPP nanoparticles sizes increased. Centrifugation of CS-TPP particles of larger particle sizes (i.e., 360-870 nm) at 11,000 × g caused the formation of CS-TPP microgels. Dynamic rheological studies indicated that both storage modulus (G′) and loss modulus (G″) increased with particle sizes. During centrifugation processing, strong centrifugal force surmounted the electrostatic repulsion between CS-TPP particles and caused particles to stick with each other to form CS-TPP microgels. The water contents of microgels negligibly depended on particle size, suggesting that the free volumes of microgels were not affected by particle size, therefore supporting our pseudo-hard sphere assumption for CS-TPP nanoparticles.     

Purification and immunological properties of proton-ATPase complexes from yeast and rat liver mitochondria

Proton-ATPase complexes from yeast and rat liver mitochondria were isolated by a simple method previously employed for the purification of the proton-ATPase complex from chloroplasts. After reconstitution into liposomes, the purified complexes were active in the ATP-Pi exchange reaction, the rate of which was 120 and at least 200 nmol/mg of protein/min for the rat liver and yeast mitochondria ATPases, respectively. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, each complex exhibited 11 to 12 different polypeptides. The isolated ATPase complexes from rat liver and yeast mitochondria, from Swiss chard chloroplasts, and Escherichia coli membranes were reacted with antibodies prepared against the various subunits of ATPase complexes. From all the combinations of antigen-antibody examined, only the antibodies against beta subunit cross-reacted with the corresponding subunit of all the ATPase complexes tested. These results indicate that certain amino acid sequences in the beta subunit have been preserved in all of the proton-ATPase complexes.     

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation

Abstract  Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.     

Simvastatin inhibits lipopolysaccharide-induced tumor necrosis factor-alpha expression in neonatal rat cardiomyocytes: The role of reactive oxygen species

Tumor necrosis factor-alpha (TNF-alpha) is implicated in heart failure and cardiomyocytes themselves can express TNF-alpha. Nevertheless, the mechanisms and regulations of TNF-alpha expression in cardiomyocytes remain poorly understood. The present study was to investigate the effects of simvastatin on TNF-alpha expression in cardiomyocytes and the underlying molecular mechanisms. In neonatal rat cardiomyocytes, RT-PCR and ELISA showed lipopolysaccharide (LPS)-induced TNF-alpha expression was attenuated by simvastatin pretreatment in a dose-dependent manner. The reactive oxygen species (ROS) scavenger N-acetylcysteine and the NADPH oxidase inhibitor diphenyleneiodonium also inhibited the LPS-induced expression of TNF-alpha. Dichlorofluorescein-fluorescence and cytochrome c reduction assay indicated LPS increased ROS generation and NADPH oxidase activity in cardiomyocytes, which were abrogated by simvastatin. Furthermore, similar to LPS, exogenous hydrogen peroxide also increased TNF-alpha secretion, but simvastatin did not significantly affect the hydrogen peroxide-induced TNF-alpha secretion. All the effects of simvastatin as mentioned above were completely reversed by concomitant pretreatment with mevalonate, a key intermediate during cholesterol synthesis. These results suggest that simvastatin attenuates LPS-induced TNF-alpha expression in cardiomyocytes via inhibition of activation of NADPH oxidase and subsequent ROS generation.     

Immobilized isocrite dehydrogenase: some aspects of its catalytic, chemical and immunological properties

Isocitrate dehydrogenase from Azotobacter vinelandii has been immobilized on Sepharose 4B with an efficiency of between 60 and 75%. The immobilized enzyme is assayed by a flow technique which monitors a final steady state level of product formation. By the assay system described it is estimated that the immobilized enzyme retains between 30 and 40% of the catalytic activity of the free enzyme. Studies have been carried out on the substrate dependence of the enzyme. The enzyme requires magnesium ions with optimal concentrations of 10⁻³m and above. The dependence on isocitrate and TPN⁺ concentrations was determined and analyzed by double-reciprocal plots. The immobilized enzyme is inactivated by DTNB [5,5′-dithiobis(2-nitrobenzoic acid)] and reactivated by DTT (dithiothreitol). The DTNB-modified enzyme can be reactivated by potassium cyanide. Comparison of these reactions with those of the free enzyme suggest that the steric environment of the active site was not grossly altered by immobilization. Some supporting evidence is derived from the identity of the energies of activation, 16,600 cal/mole, of free and immobilized enzyme catalyzed oxidation of isocitrate. Furthermore, the immobilized enzyme is inactivated by antibody prepared against the free enzyme. The covalently attached enzyme is resistant to tryptic digestion except in the presence of 2 m urea. This suggests that exposed lysyl residues which may be the primary site of attack by trypsin are utilized in immobilization. Treatment of the enzyme with 2 m urea unfolds the enzyme to a conformation which has very little activity but which recovers full activity upon removal of the urea. Interaction of the enzyme with antibody suggest that the antibody reacts univalently. The second valence can be satisfied by addition of free enzyme. The free enzyme bound to the immobilized enzyme-antibody complex is active. Preliminary attempts to dissociate the enzyme-antibody complexes have been unsuccessful.     

Interactions of myelin basic protein with palmitoyllysophosphatidylcholine: characterization of the complexes and conformations of the protein

The stoichiometry of palmitoyllysophosphatidylcholine/myelin basic protein (PLPC/MBP) complexes, the location of the protein in the lysolipid micelles, and the conformational changes occurring in the basic protein and peptides derived from it upon interaction with lysolecithin micelles were investigated by circular dichroic spectropolarimetry, ultracentrifugation, electron paramagnetic resonance (EPR) and ³¹P, ¹³C, and ¹H nuclear magnetic resonance spectroscopy (NMR), and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes were formed by the association of one protein molecule with approximately 141 lysolipid molecules. Small-angle X-ray scattering data indicated that the PLPC/MBP complexes form particles with a radius of gyration of 3.8 nm. EPR spectral parameters of the spin labels 5–, and 16-doxylstearate incorporated into lysolecithin/basic protein aggregates, and ¹³C- and ¹H-NMR relaxation times of PLPC indicated that the addition of the protein did not affect the environment and location of the labels and the organization of the lysolipid micelles. The data suggested that MBP lies primarily near the surface of the micelles, with segments penetrating beyond the interfacial region into the hydrophobic interior, but without any part of the protein being protected against rapid exchange of its amide groups with the aqueous environment. The basic protein acquired about 20% -helix when bound to lysolipid micelles. Circular dichroic spectra of sequential peptides derived by cleavage of the protein revealed the formation of -helical regions in the association with lysolecithin. Specific residues in myelin basic protein that participated in binding to the micelles were identified from magnetic resonance data on changes in the chemical shifts and intensities of assigned resonances, and line broadening of peaks by fatty acid spin-labels incorporated into the micelles. Correspondence to: G. L. Mendz     

Hydrodynamic and molecular characteristics of polyelectrolyte complexes between sodium dextransulfate and chitosan hydrochloride

Hydrodynamic and molecular characteristics of particles of polyelectrolyte complexes (PEC) between sodium dextransulfate and chitosan hydrochloride were studied by various physicochemical methods (high-rate sedimentation, viscosimetry, turbidimetry, and diffusion). As was shown, the complex formation is accompanied by increase in the average sizes with simultaneous changes in the shape of the particles of the investigated PEC. According to the proposed polycomplexes model, side by side aggregation of the taken macromolecules could cause disordering of adjacent helical parts of polyanion-matrix that facilitates the formation of sphere-like polycomplexes particles.     

The P2 protein of bovine root myelin: isolation and some clinical and immunological properties

Abstract— A small basic protein (mol.wt. 12,000), referred to as the P₂ protein, was extracted with dilute acid from delipidated bovine root myelin and purified by ion exchange chromatography on cellulose phosphate. It appeared homogeneous on polyacrylamide gel electrophoresis. The P₂ protein had a distinctly different amino acid composition than the larger basic protein (mol.wt. 18,000), referred to as the P₁ protein, that is also present in peripheral nerve myelin. It contained relatively more hydrophobic residues and much less histidine and proline. The P₂ protein conjugated with peroxidase was bound by lymph node cells and infiltrates in rabbits sensitized with whole bovine root myelin. No binding was evident with the bovine central nervous system myelin basic protein. Chemically and immunologically, the P₂ protein appears to be specific to peripheral nervous system myelin. The isolated P₂ protein produced mild clinical symptoms of experimental allergic neuritis, but no histological evidence of disease. It was suggested that the P₂ protein is an important antigen for experimental allergic neuritis, and that its antigenic determinants are likely to be conformation-dependent.     

Physico-chemical and immunological properties of allophycocyanins

Allophycocyanins were purified from diverse cyanobacteria and one rhodophytan alga (Cyanidium caldarium). The native proteins are trimeric molecules with the structure ()₃. Representative native allophycocyanins and their and subunits were characterized with respect to molecular weight, amino acid composition, isoelectric point, absorption and fluorescence spectra and immunological properties. All of the allophycocyanins studied were strikingly similar with respect to each of these properties.Renatured and subunits of allophycocyanin were distinct immunologically from each other, and both cross-reacted with the antiserum to the native protein.Trimeric allophycocyanin was readily reconstituted from the purified and subunits. Formation of hybrid allophycocyanins was demonstrated by direct isolation and characterization of the hybrid proteins and by immunological techniques.The results support the view that allophycocyanins are a highly conserved group of proteins.Abbreviation Used SDS sodium dodecyl sulfate     

The effect of structural-chemical characteristics of water-soluble polyelectrolyte complexes of ovalbumin on their immunological properties

Immunogenicity of soluble protein antigens in the complexes with synthetic polyions may be regarded as depending both on the nature of polymer carrier and the structure of the protein-polyelectrolyte complex. The immunogenicity of stable soluble complexes of ovalbumin (OA) with polycation - quaternized poly-4-vinylpyridine (C-1) and copolymer of acrylic acid and 2-methyl-5-vinylpyridine (C-2) have been evaluated. Immunization of mice by C-1 have induced a vigorous formation of the anti-OA IgG antibodies and IgE homocytotropic antibodies, while immunogenicity of OA in C-2 was comparable with that of OA alone. The analysis of the structural-chemical features of the complexes investigated has shown that enhanced immunogenicity of C-1 may be due to (1) the non-homogeneous distribution of protein globulae among polycation macromolecules and to (2) the formation of complex with an asymmetrical structure, to (3) the high ability of C-1 to adsorb on a surface of the lymphoid cells and to induce a formation of intercellular aggregates. An enhancing of a stability and a size of C-2 in the presence of Cu2+ shows no influence on a immunogenicity of OA. An immunogenicity of both types of complexes does not depend upon the access of determinants of OA to antibodies so far as it has been shown that complex formation in both cases are not accompanied by an alteration of antigenicity and allergenicity of OA.