Purification and characterization of anticomplement factor (cobra venom factor) from the Naja naja atra venom

Anticomplement factor (cobra venom factor) from the venom of Naja naja atra was purified by means of successive chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose CL-6B. The purified anticomplement factor was homogeneous as judged by polyacrylamide discontinuous gel electrophoresis at pH 9.4. The yield from 3.0 g of the crude venom was approx. 28 mg. The molecular weight was estimated to be about 156 000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was about 5.2. SDS-polyacrylamide gel electrophoresis of the anticomplement factor in the presence of dithiothreitol demonstrated that the molecule possesses three different polypeptide chains cross-linked covalently to one another by disulfide bridge(s). By SDS-polyacrylamide gel electrophoresis, the molecular weight of each subunit was determined to be approx. 77000, 47500 and 29 000, respectively. All subunits were stained with Coomassie brilliant blue G-250 and periodate-Schiff reagent, indicating these subunits to be glycoprotein. Distribution of the anticomplement factor in various snake venoms, which shows cross-reactivity against the anti-Naja naja atra anticomplement factor antiserum, was examined. From the results, all venoms belonging to cobra family in the Elapidae tested so far were found to contain such cross-reactivity.     

Novel biologically active polymer of 3-(3,4-dihydroxyphenyl)glyceric acid from two types of the comphrey Symphytum asperum and S. caucasicvum (Boraginoceae)

Two high-molecular water-soluble preparations with high anticomplement and antioxidant activity were isolated from the roots of Symphytum asperum and S. caucasicum. Their main chemical constituent was found to be poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene] according to IR and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

The seroresistance of enterobacteria isolated from different sources

395 representatives of the family Enterobacteriaceae, isolated from household and drinking water, from healthy and sick persons, were used as an example to demonstrate that their seroresistance was greatly connected with the type of the source from which these bacteria had been isolated. Experiments showed the phenotypic conversion of Escherichia coli towards an increase in their seroresistance on contact with human blood serum. The study revealed the pleiotropic effect produced by the action of serum on a number of other properties, responsible for survival in a macroorganism (anticomplement and antilysozyme activities, adhesive capacity and hydrophobic properties of the outer surface).     

Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis

The alternative pathway of complement is an important defense against pathogens and in tick rejection reactions. The tick Ixodes scapularis is able to feed repeatedly on its natural host and has a salivary anticomplement activity that presumably facilitates feeding. In this study, we purified and then obtained the amino-terminal sequence of the I. scapularis salivary anticomplement (Isac). We found a full-length clone coding for Isac by random screening of a salivary gland cDNA library. Expressing Isac cDNA in COS cells reproduced the activity found in tick saliva, namely, inhibition of rabbit erythrocyte lysis by human serum in the presence of Mg(2+) and EGTA, inhibition of C3b binding to agarose in the presence of Mg(2+) and EGTA, and acceleration of factor Bb uncoupling from the C3 convertase generated by the alternative pathway. Recombinant Isac had no effect on the recalcification time of human platelet-poor plasma or in the classical complement pathway, indicating that it is a specific inhibitor similar to the regulators of complement activation of the alternative pathway such as factor H. Isac, however, has no similarity to any protein in the GenBank(TM) data base, indicating that it is a novel and relatively small (18.5 kDa) anticomplement molecule.     

Metabolism of megestrol acetate and related progesterone analogues by liver preparations in vitro

1. The rates of metabolism of megestrol acetate, 17alpha-acetoxy-6alpha-methylprogesterone, 6alpha-methylprogesterone, 17alpha-acetoxyprogesterone and progesterone by liver microsome-supernatant fractions from female rats and rabbits have been compared. 2. Introduction of the 6alpha-methyl group into the progesterone molecule markedly decreased the metabolism by liver microsome-supernatant preparations in the rat but not in the rabbit. When 17alpha-acetoxy was the substituting group, metabolism was diminished in the rabbit but not in the rat preparations. Introduction of both the 6alpha-methyl and 17alpha-acetoxy groups into the progesterone molecule markedly decreased the rate of metabolism by liver microsome-supernatant preparations of both species. The Delta(6)-bond in megestrol acetate confers additional resistance to metabolism by rabbit-liver preparations. 3. It is proposed that the observed inhibition of metabolism by these substituents in the progesterone molecule partly or wholly explains the enhanced activity of megestrol acetate.     

Polymorphism of horse liver alcohol dehydrogenase

The properties of the most cathodal component of horse liver alcohol dehydrogenase (isozyme SS) have been found to vary. The variability is dependent on the livers from which the enzyme is isolated rather than on the purification procedure. Two distinct preparations, differing in catalytic properties, have been obtained and named S-type and A-type preparations. The preparations can be distinguished from each other by the ratio of activity with acetaldehyde to activity with the steroidal ketone 5_β-dihydrotestosterone. This ratio is about one for the S-type and twenty for the A-type preparations.     

Physico-chemical and immunologic properties of the tissue DNA of mice with oncornavirus-induced leukemia

Active antisera containing antibodies to deproteinized DNA preparations of mouse normal and leukemic tissues have been obtained. Examination of antisera by the quantitative complement fixation test with the corresponding test antigens has demonstrated immunological specificity of DNA preparations isolated from the organs of mice with leukemia induced by oncornaviruses. The DNA specificity in question is inherent not only in the native but also in the thermodenatured molecule of DNA.     

Expression and characterization of human lamin C

We have expressed human lamin C cDNA in E. coli using a modification of the pLcII vector system. Protein produced in this way had seven additional amino acids at its N-terminus, but retained key lamin structural and assembly properties. The modified vector we produced may prove useful when difficulties are encountered in removal of the cII fusion peptide by factor X cleavage in the pLcII system. Shadowed preparations of expressed lamin C showed the presence of 50-nm rod-like particles that closely resembled those observed for native material. Isolated molecules had two globular domains at one end, indicating that they were constructed from two parallel polypeptide chains. The expressed material also formed paracrystals with a characteristic 22.5 nm axial repeat, indicating that its assembly properties had also been retained. We also used site-specific mutagenesis to engineer a lamin fragment that lacked the C-terminal non-helical domain of the molecule. This material formed paracrystals similar to those obtained with the intact molecule, indicating that the large C-terminal non-helical domain did not contain information vital for lamin assembly.     

Evidence for different requirements in physical state for the interaction of lipopolysaccharides with the classical and alternative pathways of complement

The influence of the state of aggregation of lipopolysaccharides upon their ability to interact with serum complement via either the classical or alternative pathway was studied. The anticomplement properties of two chromatographically distinct fractions of a phenol-extracted lipopolysaccharide isolated from Serratia marcescens were assessed by means of the standard sheep erythrocyte hemolytic assay and an alternative pathway-selective kinetic assay using rabbit erythrocytes. Both the high molecular weight PI fraction and the lower molecular weight PII fraction exerted anti-complement activity as determined in the sheep erythrocyte assay. Conversion of fractions PI and PII to their more soluble triethylamine salt forms resulted in a decrease in sedimentation coefficients and a corresponding loss of anticomplement activity. Further, the anticomplement activity of fractions PI and PII in the sheep erythrocyte assay was inhibited by polymyxin B, indicating a role for the lipid A region. Unlike the PII fraction, only the PI fraction can activate serum complement via the alternative pathway. This activity is not inhibited by polymyxin B, indicating that the response is not lipid A-mediated. Significantly, solubilization of the PI fraction with triethylamine had no effect on its ability to activate the alternative pathway. These studies clearly demonstrate that the interaction between lipopolysaccharides and serum complement is influenced by the state of lipopolysaccharide aggregation. However, this appears to be the case for lipopolysaccharide activation of the classical pathway but not of the alternative pathway.     

Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. III. The serological activity and immunochemical properties of irradiated unpurified toxin

The immunochemical properties and serological activity of irradiated preparations of crude cholera exotoxin have been studied. This study has revealed that with the increase of the dose of ionizing radiation changes occur in the physico-chemical properties of the preparations of the toxin, which leads to an increase in the electrophoretic motility of the protein components of the toxin, to the aggregation and polymerization of individual fragments. The preparations of antigen exotoxins have been shown to retain their serological activity within the range of radiation doses under study (10-350 kGy).     

Lyophilized membranes as a test system for analyzing the properties of neuromediator receptors and activity of neurotropic agents

The effect of lyophilization and prolonged storage of rat brain membrane preparations on the properties of receptors of cholinergic and adrenergic neuromediator systems has been studied by the radioreceptor assay. Lyophilized membrane preparations have been shown to be highly stable as compared with fresh membranes and retain their binding properties during storage for 1 year.     

Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length.

Hyaluronan is a ubiquitous glycosaminoglycan of high molecular weight that acts as a structural component of extracellular matrices and mediates cell adhesion. There have been numerous recent reports that fragments of hyaluronan have different properties compared to the intact molecule. Though many of these results may be genuine, it is possible that some activities are due to minor components in the preparations used. Therefore, it is important that well-characterized and highly purified oligosaccharides are used in cell biological and structural studies so that erroneous results are avoided. We present methods for the purification of hyaluronan oligomers of defined size using size exclusion and anion-exchange chromatography following digestion of hyaluronan with testicular hyaluronidase. These preparations were characterized by a combination of electrospray ionization mass spectrometry, matrix-assisted laser desorption/ionization mass spectrometry with time-of-flight analysis, and fluorophore-assisted carbohydrate electrophoresis. Hyaluronan oligomers ranging from tetrasaccharides to 34-mers were separated. The 4- to 16-mers were shown to be homogeneous with regard to length but did contain varying amounts of chondroitin sulfate. This contaminant could have been minimized if digestion had been performed with medical-grade hyaluronan rather than the relatively impure starting material used here. The 18- to 34-mer preparations were mixtures of oligosaccharides of different lengths (e.g., the latter contained 87% 34-mer, 10% 32-mer, and 3% 30-mer) but were free of detectable chondroitin sulfate. In addition to oligomers with even numbers of sugar rings, novel 5- and 7-mers with terminal glucuronic acid residues were identified.     

Propidium iodide staining of cytoautoradiographic preparations for the simultaneous determination of DNA content and grain count

Summary A new method is described for staining cell nuclei with propidium iodide in preparations that have been processed for autoradiography. The permeability of the stripping film to the dye molecule has been studied, as have the staining conditions, in order to optimize the stoichiometry of the DNA-dye interaction. A procedure has also been set up to allow the simultaneous measurement of DNA content and grain count on the same cell.     

Temperature-induced change in the Ca2+-dependent ATPase activity and in the state of the ATPase protein of sarcoplasmic reticulum membrane.

The temperature dependence of the Ca2+-dependent ATPase activity and of the conformational fluctuation of the ATPase molecule has been measured for four kinds of preparations: fragmented sarcoplasmic reticulum, MacLennan's enzyme (purified ATPase preparation), and DOL and egg PC-ATPase (purified ATPase preparations in which lipids are replaced with dioleoyllecithin and egg yolk lecithin, respectively). It has been found that Arrhenius plots of the Ca2+-dependent ATPase activity show a break at about 18 degrees C for all the preparations. Hydrogen--deuterium exchange kinetics of the peptide NH protons were used to measure the conformational fluctuation of the protein molecules. Van't Hoff plots of the conformational fluctuation amplitude of a region near the surface of the ATPase molecule also show a break at about 18 degrees C for all the preparations. It is concluded that the break at around 18 degrees C is not related to a gel-liquid crystalline transition of lipids but to a change in the conformation of the ATPase molecule existing in fluid lipids.     

Gonadotropic activity of the immunoglobulins from placental, abortion and donor blood

Gonadotropic activity of 106 series of immunoglobulin preparations obtained in the USSR and-the People's Republic of Bulgaria was studied. In difference from donor immunoglobulins, all the preparations of placental materials were contaminated with chorionic gonadotropin. Gonadotropin content in the immunoglobulins made of abortion serum was 2--7 times greater than in the preparations from the retoplacental serum and the placental extracts. The mean results of gonadotropin content in the immunoglobulins obtained by biological studies differed from those obtained from the investigations by immunological methods since the mentioned methods apparently characterized different properties of the hormonal molecule.     

Characterization of Epstein-Barr virus antigens. I. Biochemical analysis of the complement-fixing soluble antigen and relationship with Epstein-Barr virus-associated nuclear antigen.

The Epstein-Barr virus-soluble (S) antigen extracted from RAJI cells was characterized by sucrose gradient centrifugation, gel filtration, and ion-exchange chromatography. The sedimentation coefficient was estimated to be 8.5S corresponding to a molecular weight of 180,000. The S antigen binds to DEAE-A25 ion exchanger from which it can be eluted with 0.3 M NaCl in Tris buffer (pH 7.2). All fractions which contained complement-fixing S antigen also inhibited the anticomplement immunofluorescence reaction as used to detect the Epstein-Barr virus-associated nuclear antigen. These results are consistent with the hypothesis that the S and Epstein-Barr virus-associated nuclear antigens are either a single antigen or that both activities are present on the same molecule.     

Use of an immunoblotting method for identifying the individual proteins in the antigenic complexes of Bordetella pertussis

The composition of antigenic complexes isolated from the supernatant fluid of B. pertussis culture has been studied by means of immunoblotting techniques. In preparations obtained from B. pertussis strains 305 and 475 fragments of the molecule of fimbrial hemagglutinin, three subunits of B. pertussis toxin and agglutinogens 2 and 3 have been detected with the use of antisera to B. pertussis protective substances.     

The comparative characteristics of the immunomodulating properties of protein A from Staphylococcus aureus of different origins

The immunomodulating properties of highly purified staphylococcal protein A and its analog obtained by gene engineering techniques have been compared with those of commercial preparations. The comparison has shown that the differences observed in this investigation may be explained by the presence of admixtures of staphylococcal nature in commercial preparations. The preparations of highly purified staphylococcal and recombinant protein A stimulate humoral immune response and the processes of phagocytosis and do not show mitogenic activity with respect to T cells. The conclusion on the identity of the immunomodulating activity of the preparations of natural and recombinant protein A has been made.     

Effect of gamma radiation on the immunobiological and immunochemical properties of cholera exotoxin. IV. The biological and immunochemical properties of purified irradiated choleragen

The results of investigations carried out to study the effect of gamma radiation on the properties of the purified preparations of cholera exotoxin are presented. Irradiation has been shown to decrease the anterotoxicity of purified choleragen and the activity of its permeability factor, depending on the radiation dose. The investigations have revealed that in purified toxin enterotoxicity is completely inactivated with a lover radiation dose than in crude toxin filtrate (25 kGy). In immunochemical reactions the increase of the electrophoretic mobility of the choleragen components, correlated with the increase of the radiation dose, and the reduced number of protein zones have been observed. The irradiated preparations of purified choleragen have been found to retain their immunogenic properties and serological activity.     

The effect of iron concentration on pectin decomposition by Aspergillus niger and Aspergillus awamori]

The effect of cations Fe2+ and Fe3+ on the decomposition of apple pectin by the enzymic preparations of Asp. niger and Asp. awamori has been examined. Fe ions have delayed the process of enzymic decomposition of the pectin molecule. In the presence of Fe3+ far less amounts of monogalacturonic acid are formed. The presence of Fe ions makes the pectin molecule more stable to the effect of pectolytic enzymes.