Identification and properties of complexes formed by myeloperoxidase with lipoproteins and ceruloplasmin

The first evidence of multi-component complexes formed by myeloperoxidase (MPO), ceruloplasmin (CP), and very low/low density lipoproteins (VLDL/LDL) obtained by electrophoresis, gel filtration, and photon-correlation spectroscopy (PCS) is presented in this paper. Complexes were observed when isolated MPO, CP, and VLDL/LDL were mixed and/or when MPO was added to the blood plasma. Complex LDL–MPO–CP was detected in 44 of 100 plasma samples taken from patients with atherosclerosis, and 33 of 44 samples also contained the VLDL–MPO–CP complex. MPO concentration in these patients’ plasma exceeded 800 ng/ml. Interaction of MPO with high density lipoproteins (HDL) was not revealed, as well as binding of CP to lipoproteins in the absence of MPO. Adding antibodies against apoB-100 to VLDL–MPO–CP and LDL–MPO–CP complexes results in release of lipoproteins. Using PCS the diameters of complexes under study were evaluated. By comparing concentrations of the components in complexes formed by MPO, CP, and lipoproteins their stoichiometry was assessed as 2VLDL:1MPO:2CP and 1LDL:1MPO:2CP. Lipoproteins affected the inhibition of MPO peroxidase activity by CP. The affinity of lipoproteins to MPO–CP complex was assessed using apparent dissociation constants determined as ～0.3 nM for VLDL and ～0.14 nM for LDL.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

The study of substrate specificity of a new peptide substrate of endothelin-converting enzyme

A new hexapeptide CMC-Ala-Gly-Gly-Trp-Phe-Arg was used as a substrate for assay of endothelin-converting (ECE; EC 3.4.24.71) and angiotensin-converting (ACE; EC 3.4.15.1) enzymes and of neutral endopeptidase (NEP; EC 3.4.24.11). The specific inhibitors lisinopril (for ACE) and thiorphan (for NEP) were used for discrimination between activities of these enzymes.     

The absorption features of glycyrrhizic acid in the drug formulation Phosphogliv

Glycyrrhizic acid (GL), one of the active components of the Russian drug formulation Phosphogliv, is characterized by extremely low bioavailability. Absorption characteristics of GL after peroral administration of “Phosphogliv“ and GL sodium salt have been investigated using a sensitive method developed for GL determination in blood by means of high performance liquid chromatography coupled with mass-spectrometry (LC-MS). Separation of blood components was achieved on the analytical reverse-phase column C18 “EcoNova” ProntoSIL, using a gradient mode. Detection of GL and an internal standard (IS) (glycyrrhetic acid) was performed using electrospray ionization with the selected ion monitoring in negative mode (SIM) using the target ions of m/z 821.3 and 469.3 for GL and IS, respectively. The calibration curve was linear over the range of concentrations 50–5000 ng/ml (the correlation coefficient was 0.995). The detection limit for GL in blood was 25 ng/ml and the lower limit of quantification was 50 ng/ml. The developed method has been applied to compare absorption efficiency of GL as the component of the Phosphogliv drug formulation and solution of GL sodium salt during the first two hours after their single peroral administration to rats at the dose of 8.5 mg/kg. It was shown that GL absorption occurred within several minutes after peroral administration. Moreover, GL bioavailability after Phosphogliv administration was higher than after administration of GL sodium salt. This difference may be attributed to GL incorporation of the phospholipid nanoparticles structure.     

About Mechanisms Responsible for the Depression of Alcohol Motivation Resulting from the Immunization of Albino Rats against Alcohol Dehydrogenase I: The Role of ADH Epitopes and ADH Activity in the Adrenals

Experimental results have demonstrated a significant decrease in the level of alcohol consumption by albino rats immunized with heterologous horse alcohol dehydrogenase. The role of ADH epitopes 9–14, 93–115, and 265–276 in this phenomenon was examined, and it was established that the latter sequence (265–276) plays the biggest role. The inhibition of ADH activity in the adrenals of immunized rats was much higher compared to the liver. We propose a hypothesis that the effect of alcohol dehydrogenase on alcohol consumption is connected with its role in catecholamine metabolism.     

Cytosolic insulin-binding proteins of mouse liver cells

It has been recently shown that insulin retains its biological activity after receptor-directed internalization and it may affect the cell metabolism by interaction with cytosolic insulin-binding proteins (CIBPs). Using affinity chromatography combined with SDS-PAGE and MALDI-TOF mass-spectrometry we have identified 7 proteins from mouse liver cells that specifically bind to the insulin, including adenylate kinase 2 (25.6 kD), kinesin superfamily protein 20B (26.0 kD), hepatic arginase 1 (34.8 kD), fructose-bisphosphate aldolase B (39.5 kD), 4-hydroxyphenylpyruvate dioxygenase (45.1 kD), betaine-homocysteine methyl-transferase (45.0 kD) and KRIT1 (83.4 kD).     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Antirheumatic activity of methotrexate in phospholipid nanoparticles (Phosphogliv)

The efficiency of methotrexate use in the basic therapy of rheumatoid arthritis is limited because of risk of side effects and fast drug efflux from zone of joints as well. We have developed a new stabilized form of methotrexate using phospholipid micelles of the injection form of the Phosphogliv preparation as a carrier. Phosphogliv has recently been developed in the Institute of Biomedical Chemistry (Moscow), as the emulsion of 50 nm phospholipid nanoparticles stabilized by glycyrrhizic acid. The conditions of maximal methotrexate incorporation into the phospholipid nanoparticles were optimized under control of HPLC (60% of total methotrexate was associated with nanoparticles). Methotrexate in phospholipid nanoparticles exhibited higher therapeutic efficiency in experimental adjuvant arthritis in rats than with free methotrexate. (This was evaluated by the decrease of edema and swelling of joints and inhibition of secondary inflammatory reaction.) The increase of antirheumatic activity of the developed preparation may also be attributed to the influence of glycyrrhizic acid, possessing both anti-inflammatory and immune properties. It is suggested to use a new form of methotrexate for intra-articular administration for rheumatoid arthritis treatment.     

Amino acid sequence in tryptic peptides of maleylated Micrococcus sp. n. histidine decarboxylase beta-polypeptide chain]

Maleilated histidine decarboxylase beta-polypeptide chain, containing 3 arginine residue, was hydrolysed by trypsin. 4 non-overlapping homogenous peptides were isolated, 3 of them containing one arginine residue and the 4th peptide being C-terminal fragment of beta-chain. beta-Polypeptide chain is found to consist of 78 amino acid residues and to have molecular weight of 8456. Primary structure of each peptide and their possible sequence in beta-chain are determined.