Phaseotoxin A: an antimetabolite from Pseudomonas phaseolicola.

Phaseotoxin, the exotoxin of the bean pathogen $\text{Pseudomonas phaseolicola}$, has been resolved into four biologically active fractions by DEAE cellulose chromatography. Phaseotoxin A, the toxin in the first fraction decomposed to give glutamic acid and inorganic orthophosphate only, and was chromatographically identical with synthetic N-phosphoglutamic acid. Like phaseotoxin A, the synthetic compound inhibits ornithine carbamoyltransferase and induces chlorosis in bean leaves. To our knowledge this is the first report of occurrence of an N-phosphorylated primary amine in nature.     

Single-step purification of pertussis toxin and its subunits by heat-treated fetuin-sepharose affinity chromatography

A general procedure for purifying biologically active pertussis toxin from Bordetella pertussis fermentation broth using affinity chromatography on heat-treated fetuin-Sepharose CL-4B is described. Diethanolamine is used as eluent in this single-step purification to prepare endotoxin-free pertussis toxin in good yield (70%) and high purity (greater than 95%). This one-step affinity chromatography procedure can be easily applied for large-scale preparation of pertussis toxin S1 subunit and its B-component. The affinity-purified S1 subunit is devoid of any of the histamine-sensitizing activity normally associated with pertussis toxin. The chromatographically purified pertussis toxin and its subunits retained their immunogenicity and could induce high levels of anti-toxin neutralizing antibodies.     

Mathematical model of the infection process in diphtheria for determining the therapeutic dose of antitoxic anti-diphtheria serum

It is known that administration of horse serum against diphtheria toxin can cause autoimmune and allergic complications. Therefore it is important for improvement of serotherapy to develop methods of prediction of disease course and quantity of diphtheria toxin and antitoxic antibodies in a serum. We have developed the mathematical model of diphtheria infection, which consists of six differential equations describing dynamics of diphtheria toxin and antitoxic antibodies in a serum, quantity of infection agent and macrophages in a site of inflammation. This mathematical model allows to predict the course of infectious process, the level of diphtheria toxin and antitoxic antibodies in the sera of people with diphtheria and to calculate the individual therapeutic dose of antitoxic serum for each patient.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin is not detected, and free antibodies account for 68 to 98% of the original amount. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80-100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalency of serum antibodies, and monovalency of the toxin epitopes. A new model of the toxin-antibody interactions is proposed.     

The coagglutination reaction for detecting diphtheria toxin

High-titer antidiphtheria antitoxic rabbit serum has been obtained, and on the basis of this serum a coagglutinating diagnosticum has been developed. The sensitivity of the test has been found to depend on the content of antitoxic antibodies in the serum and on its purity. Diagnostica prepared from native serum containing 500 I. U./ml (a titer of 1:51, 200 in the passive hemagglutination test) permit the detection of 0.02-0.03 Lf/ml of diphtheria toxin. A decrease in antibody titer to 5-25 I. U./ml leads to a drop in sensitivity to 0.2-2 Lf/ml. The use of LgG fraction and pure antibodies increases the sensitivity of the test to 0.002-0.003 Lf/ml. The possibility of detecting toxin in Corynebacterium diphtheriae strains is shown.     

The assessment of antitoxic anti-anthrax immunity

In experiments on guinea pigs immunized with avirulent noncapsular strains STI, Sterne (34F2) and the avirulent mutant of Bacillus anthracis strain 228/8 the relationship between the titers of serum antibodies to the preparations of purified protective antigens (PA) and purified lethal factor (LF) of B. anthracis toxin and the level of the antitoxic activity (ATA) of blood sera, as well as acquired resistance, was analyzed. The ATA of sera was evaluated in the primary culture of peritoneal macrophages affected by the mixture of PA and LF. The level of relationship (r) between individual ATA values and the titers of antibodies to PA and LF was shown to vary over a wide range, depending on the group of the animals and did not exceed, on the average, 0.19-0.37. At the same time the mean values of these characteristics, followed in their dynamics depending on the immunogenic properties of vaccine strains or the time elapsed after vaccination, were highly correlated (r = 0.76-0.87). The possibility of using these characteristics for the evaluation of acquired resistance are discussed.     

Specificity studies on anti-histone H1 antibodies obtained by different immunization methods

Antibodies were elicited against chromatographically purified histone H1 subfractions or against their complexes with RNA and their specificity studied by enzyme-linked immunosorbent assay. The results show that complexing of the pure protein with RNA does not lead to any significant increase in the antibody titer and results in obtaining antibodies predominantly against the common antigenic determinants present in the H1 histone class. On the other hand, using pure histone fractions for immunization gives rise to antibody populations reacting mainly with the subfraction-specific determinants on the histone molecule. In view of these results the literature data should be interpreted with caution.     

Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin was not detected, and free antibodies accounted for 68 to 98% of the original amount of the antibodies. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80–100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalence of serum antibodies, and monovalence of the toxin epitopes. A new model of the toxin-antibody interaction is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 235–242.Original Russian Text Copyright © 2005 by Titova, Sviridov.     

Acidic acetone extract from pregnant sow ovaries is a rich source of substances affecting uterine contractility in vitro

Acidic acetone extract of pregnant sow ovaries was subjected to Sephadex G-25 chromatography. The solution coming from column was analysed for UV absorption, molecular weight, and also for its biological effect on a myometrium strip in vitro. This biodetection system has made it possible continuously to determine the biologically active fractions eluted from the Sephadex G-25 column. The reference materials to calibrate the Sephadex G-25 column were Blue dextran and acetone, while for calibration of the biodetection system, synthetic oxytocin was used. The extract of ovaries of pregnant sows was separated chromatographically into 8 different, biologically active fractions with distinct UV absorption and molecular weight. One of these fractions showed elution characteristics and biological effect similar to those of synthetic oxytocin in the same biodetection system. The results indicated that acidic acetone extract originating from ovaries of pregnant sows is a rich source of biologically active substances with effects on the myometrium strips in pregnancy. Partial identification of oxytocin-like substances in the ovarian extract verified the effectiveness of the biodetection system in the first steps of research to obtain new, biologically active substances from different unpurified extracts.     

Immunochemical study of Clostridium botulinum type B toxin

Fractionation of type B. Cl. botulinum toxin, a protein complex, was carried out; as a result, 3 fractions, alpha, beta, and gamma, were isolated in a pure form, alpha-fraction, or neurotoxin, is highly toxic (5-10.10(7) LDm per 1 mg of protein), beta-fraction showed hemagglutinating activity (64-128 HAU per 1 mg of protein), gamma-fraction was not biologically active. The molecular weight of alpha and gamma-fractions was 150,000. All these fractions had antigenic properties. alpha-fraction was serologically specific. beta- and gamma-fractions showed incomplete serologic identity.     

Preparation of conjugates of 19s and 7s globulins of antitularemic sera for the determination of specific fluorescence ofPasteurella tularensis

Pasteurella tularensis was detected by means of conjugates of 19S and 7S globulins from antitularemic serum with fluorescein isothiocyanate (FITC). The serum was obtained by short-term immunization of rabbits. The capacity of both fractions to produce the immunofluorescence reaction with pure culturesof Pasteurella tularensis and the agglutinin titre of these fractions are compared here. It was found that the 19S globulin fraction which contained most agglutinating antibodies showed only weak immunofluorescence. The 7S fraction in which the agglutinin content was very low as compared with the 19S globulin fraction gave more intense fluorescence than the 19S fraction. On the basis of this finding the opinion is advanced that the agglutinating antibodies need not be the actual carrier of fluorescence due to fluorescent antibodies (FA) in the immunofluorescence reaction ofPasteurella tularensis. The different degrees of labelling with FITC of the individual globulin fractions as observed during simultaneous conjugation are also discussed. The 19S globulin fraction is regularly conjugated to a higher degree than the 7S component. The view is presented that the avirulent strains lack a certain part of the antigen structure as compared with virulent or vaccine strains (with residual virulence), the part being responsible for the positive reaction of immunofluorescence with preparations of fluorescent antibodies.     

Cross-reactivities of polyclonal antibodies against factor V activating enzyme, a serine proteinase from Vipera lebetina (snake) venom

Antibodies to the factor V activating enzyme from Vipera lebetina venom were produced by immunizing a rabbit with chromatographically purified factor V activating enzyme probes. The antibodies cross-reacted with different protein fractions in 23 snake venoms (ten viperid, eight crotalid, and five elapid venoms) as demonstrated by western immunoblotting. In the venom of Vipera russelli the antibodies recognized only one protein band which probably belonged to factor V activating enzyme.     

Presence of biologically active glutamyl peptide in rabbit bone marrow]

The presence of chromatographically pure and biologically active glutamylpeptide in the rabbit bone marrow was shown and methods of its isolation were given. The substances had an unusual circular dichroism contrary to that of the natural proteins. In a concentration of 10(-5) M the isolated peptide decreased the survival of the Chinese hamster culture cells to 30%. It was analogous to the peptide isolated formerly from plants by a number of physical and chemical characteristics; the latter possessed cytogenetic activity and increased the mutagenic action of the ionizing radiation.     

Purification of Shiga toxin by alpha-D-galactose-(1----4)-beta-D-galactose-(1----4)-beta-D-glucose-(1- ---) receptor ligand-based chromatography

A simple and rapid method for Shiga toxin purification based on specific binding to the Gal alpha 1----4Gal beta 1----4Glc globotrioside trisaccharide covalently linked to polyacryl/polyvinyl (Fractogel) has been developed. A cell-free sonicate-filtrate of Shigella dysenteriae type 1, strain 114Sd was passed over the globotrioside-Fractogel column, and bound toxin eluted with 6 M guanidine-HCl. A yield of 36 mg pure toxin/1 sonicate-filtrate was obtained, i.e. a one step 1224-fold purification. The recovery of biologically active toxin was 87%. The toxin was purified to homogeneity as shown by native PAGE and SDS-PAGE.     

Proteolytic Cleavage of Tetanus Toxin Increases Activity

Tetanus toxin is initially synthesized in the form of a single polypeptide chain and then proteolytically "nicked" by the bacteria to produce a two-chain structure joined by a disulfide bond. This two-chain form of the toxin is the form known to be biologically active. Whether such nicking is necessary for activity, as it is for certain other bacterial toxins, has not been demonstrated previously. Single-chain toxin preparations produced by salt extraction from the bacteria are characterized and compared with pure two-chain toxin obtained from extracellular filtrates. The ability of these various toxin preparations to produce paroxysmal activity in mouse spinal cord neurons grown in dissociated cell culture is described. The pure two-chain toxin is demonstrated to have greater activity than the single-chain toxin preparations. Indeed the activity of the single-chain toxin preparations can be explained by the small amounts of residual two-chain toxin present in these extracts. Using a protease from a toxin-minus strain of Clostridium tetani to convert a single-chain toxin preparation to two-chain toxin increases toxin activity. In vivo the single-chain toxin preparation is also less toxic. These findings indicate that proteolytic nicking of tetanus toxin increases activity. The unnicked, single-chain form of tetanus toxin may be a relatively nontoxic protoxin form of the toxin; this is a structure-function relationship similar to that of other bacterial protein toxins.     

Application of monoclonal antibodies to the isolation and characterization of a killer toxin secreted by Hansenula mrakii

A strain of yeast, Hansenula mrakii, secretes a toxin that kills sensitive yeasts, such as Saccharomyces cerevisiae Monoclonal antibodies raised against the toxin had both binding and neutralizing activities. The toxin in culture media was isolated by an affinity column of monoclonal antibody. The toxin is a basic polypeptide with an isoelectric point at pH 9.1, and devoid of mannosides. It is composed of 88 amino acid residues with a molecular size of 10 721 Da. The monoclonal antibodies could be applicable to the analysis of biologically active sites on the toxin, in an attempt to synthesize chemically a small peptide with killer activity and little immunogenicity.     

Antipertussis immunity studied by immunoenzyme analysis

The levels of antibodies to disintegrated Bordetella pertussis and its individual fractions (protein and polysaccharide) in children immunized with different batches of adsorbed DPT vaccine have been determined with the use of EIA techniques. The background level of antibodies in the control groups has been determined, and in immunized children the levels of antibodies to disintegrated B. pertussis and its protein fraction have been shown to considerably exceed the levels of antibodies to lipopolysaccharide.     

A biologically active metabolite of retinoic acid from rat liver

1. Four major radioactive fractions have been isolated from the livers of vitamin A-deficient rats given [6,7-(14)C(2)]retinoic acid. 2. At least one of these was more potent than retinoic acid and approximately equal to retinol in the growth assay for vitamin A activity. 3. The biologically active material was chromatographically distinct from retinoic acid, retinol and retinal. 4. Alkaline hydrolysis of this material yielded an acidic compound containing all the radioactivity. 5. The methyl ester of the acidic product was unlike the methyl ester of retinoic acid in its chromatographic behaviour. 6. It is suggested that this metabolite may represent the active form of retinol in its growth-supporting role.     

Localization of rhodopsin in isolated, osmotically intact rod outer segment discs.

Localization of rhodopsin and its position in the membrane has been the subject of numerous studies. Most recently, immunocytochemical techniques have been employed to localize the opsin component of the molecule in in situ rod outer segments. Due to the problems inherent in localization procedures (penetration and mechanical interference) we have utilized isolated, osmotically intact rod outer segment discs in this study. Specific antibodies to chromatographically pure rhodopsin were prepared and enzymatically digested to their Fab components. The univalent Fab antibodies were conjugated to horseradish peroxidase and used to label the isolated rod outer segment discs. Discs treated with anti-opsin conjugate stained uniformly and heavily on their interdisc surfaces. Reaction product was also present on the intradisc surface in a thinner but still uniformly distributed layer. Controls treated with preimmune Fab - horseradish peroxidase conjugate showed no deposition of reaction product.     

Use of the immune tolerance induction in the production of an antilymphocyte serum (ALS) free of antibodies against serum proteins]

The possibility has been investigated of a direct gain of ALS free of undesirable antibodies against serum proteins by inducing immunologic tolerance in productive animals (pigs). Preliminary experiments made with tolerogenic amounts of 10 and 50 ml of sera and with immunization by the serum alone proved applicability of this method. Electrophoresis showed antibodies against 6 to 7 and 2 to 3 fractions in animals tolerated with 10 and 50 ml respectively, compared to 18 to 20 fractions in the control group, which was not tolerated. This has been confirmed when preparing ALS in practice, where the toleration was carried out with 25 ml of serum or with the same amount of serum with the addition of hemoglobin and immunization by lymphocytes isolated from peripheral blood. Final ALS of untolerated animals contained antibodies against 7 to 8 fractions, whereas that of experimental group tolerated with serum and Hb was free of antibodies against serum protein, hemoglobin included. ALS of the group tolerated with normal serum contained only antibodies against hemoglobin. In vitro tests (i.e. lymphoagglutination t., lymphocytotoxicity t., rosette inhibition t.) proved that by inducing tolerance towards serum protein the activity of ALS was in no was affected. According to the results this method can be employed not only for the preparation of ALS, but also for other purposes, such as preparation of monovalent antisera for immunoelectrophoresis.