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.     

Immobilization of laccase from Cerrena unicolor on controlled porosity glass

White-rot basidiomycete Cerrena unicolor grown in non-induced and induced conditions was tested for production of laccase, lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). A typical correlation between the concentration of phenolic compounds in the culture fluid and the extracellular laccase activity was observed. The heterogeneous crude laccase preparation obtained after the non-induced fermentor cultivation was immobilized both on controlled porosity glass (CPG) activated by γ-aminopropyltriethoxysilane (APTES) and on CPG with its surface covered by dextran layers. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. Laccase activities were additionally examined for native and immobilized forms of laccase preparations in the aqueous solution containing organic solvents. The greatest activity toward the substrate used in the presence of organic solvents was shown by the laccase preparation coupled with the CPG covered by a dextran layer. Potential inhibitors such as thioglycolic acid, thiourea and EDTA used in 1-mM concentration did not show inhibiting properties towards the laccase preparations.     

Physicochemical and biological characteristics of preparations of an antigenic complex isolated from the cultivation medium of Bordetella pertussis

The physicochemical and biological properties of antigenic complexes isolated from the supernatant fluid of the culture medium of B. pertussis, strains 305 and 475, were studied. The preparations obtained from both strains contained proteins, carbohydrates, nucleic acids and lipids. Electrophoresis in polyacrylamide gel revealed the presence of filamentous hemagglutinin, 4 subunits of B. pertussis toxin and agglutinogens in the antigenic complexes of both strains. The preparations of both strains possessed similar toxic properties and, after their detoxification, produced a pronounced protective effect.     

Characteristics of the biological properties of a cell-free pertussis preparation

The biological properties of Bordetella pertussis antigenic complex, obtained by a technologically simple method from the medium used for the cultivation of B. pertussis, were studied. The preparation was characterized by pronounced hemagglutinating activity, toxicity, histamine-sensitizing and leukocytosis-stimulating activity and produced a cytopathogenic effect on the culture of Chinese hamster ovary cells. The detoxified preparations showed pronounced protective activity in experiments on the active and passive protection of mice. The ED50 of the preparation was 0.146 microgram of protein. In the proposed human immunization dose containing 10 micrograms of protein the detoxified preparation showed no hemagglutinating, leukocytosis-stimulating or histamine-sensitizing activity and proved to be nontoxic in the weight loss test on mice.     

Pertussis toxin and cross-reactive antigens in dynamics of Bordetella pertussis cultivation

Cultures of Bordetella pertussis from phases of exponential growth, retarded growth and from stationary phase were obtained during periodic dynamic cultivation. Preparations for intravenous immunization of rabbits were made from these cultures. Levels of IgG to pertussis toxin, cell walls preparations from 12 bacterial species, 4 organo-specific antigens, and 7 organospecific human antigens were measured in obtained sera. It was shown that higher levels of IgG to pertussis toxin were found in sera of rabbits immunized with cultures from exponential growth phase whereas decrease of this level in 8 times was observed in sera of rabbits immunized with cultures from retarded growth phase or end of stationary phase. After immunization with culture from exponential growth phase increase of IgG levels to cross-reactive antigens was not observed compared to levels of these antibodies in control sera obtained before immunization. After immunization with cultures from retarded growth phase or end of stationary phase increase of IgG levels to preparations of cell walls of Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, to denaturated DNA, elastin, and renal and liver microsomal fractions was detected compared to control sera. Described data can substantiate usefulness of obtaining the most specific diagnostic sera and test-systems using cultures of B. pertussis from the phase of exponential growth.     

Use of the pneumococcal serovar 3 erythrocyte antibody diagnostic agent for analyzing polysaccharide-containing preparations

Antibody diagnosticum to pneumococci (serovar 3) has been prepared. The analysis of different antigenic preparations of pneumococci (serovars 1,3 and 6B) and the filtrates of culture fluid obtained in the process of the cultivation of bacteria belonging to serovars 3,9N and 23F has been carried out by means of the indirect hemagglutination test. The new diagnosticum has proved to be type-specific. This diagnosticum can be used for the evaluation of the quantitative content of specific polysaccharide in different antigenic preparations of serovar 3 pneumococci, as well as for the determination of the content of specific polysaccharide directly in the culture fluid, which constitutes a step in the determination of the period of the maximum synthesis of polysaccharide, necessary for the optimization of the process of the cultivation of pneumococci.     

Cultivation of Neisseria meningitidis serogroups A, B and C in a synthetic nutrient broth

The processes of the cultivation of N. meningitidis, serogroups A, B and C, in a liquid synthetic culture medium have been studied. Strictly group-specific biomass has been obtained. The maximum productivity at all stages of the batch cultivation of N. meningitidis strains 125 and 133 in this medium does not differ from that at similar stages of cultivation in modified Cohen-Wheller semisynthetic medium. In the serotype antigen preparations obtained from N. meningitidis strain 125 grown in the above-mentioned liquid synthetic culture medium basic polypeptides with a molecular weight of 33000, 36000 and 41000 D have been detected. Their presence in N. meningitidis cells is linked with the growth phase of the population.     

Production of cell mass and pertussis toxin by Bordetella pertussis.

The cultivation of Bordetella pertussis affects production of pertussis toxin and biomass. Comparison of batch mode, chemostat operation and pHstat-turbidostatic control showed that productivities for the continuous process were greater than that for the batch operation. Continuous operation in balanced growth at the maximum specific growth rate, provided by the pHstat, resulted in the maximum specific production rate. Because of the strong association of pertussis toxin synthesis and cell growth, the concentration of toxin in the effluent of the continuous processes was greater than the maximum obtained in the batch bioprocess. An expanded Luedeking-Piret model of product formation kinetics fits the observed chemostat data and demonstrates that the production of pertussis toxin from the culture of B. pertussis is predominantly growth associated.     

Cultivation of Bordetella pertussis in liquid semisynthetic nutrient media with ion-exchange resins

The possibility of optimizing the processes of B. pertussis cultivation carried out in liquid semisynthetic culture media with strong-base anion-exchange resins is shown. The use of strong-base ionites enhances the reproducibility of cultivation processes, increases the activity of microbial populations and the concentration of microorganisms. B. pertussis cultures grown in the media with anion-exchange resins are characterized by pronounced biological activity.     

Comparison between two types of pertussis vaccines]

We have produced pertussis vaccines with laboratory and industrial methods. The characteristic of laboratory cultivation of microorganisms is, in this context, growth on Hornibrook medium in low form flask and in stationary culture. Industrial cultivation is done in homogenous culture on a B-2 medium in fermentor. The strains utilized were isolated from whooping-cough cases in the Montreal region. The yield (org. x 10(9)/ml) obtained with an industrial cultivation of B. pertussis was 4 to 7 times higher than that reached with a laboratory cultivation of this microorganism. The non-toxicity as expressed in weight gain of mice was shown for both types of vaccine. The vaccines produced in fermentor were less histamino sensibilizing for mice than the one produced in stationary flash culture. The quality of the vaccines achieved by industrial method is easily reproducible due to the fact that enough variables can be measured.     

The effect of methylated cyclodextrin on pertussis toxin accumulation in a Bordetella pertussis culture in a bioreactor

The results obtained in the study of the influence methylated cyclodextrin (beta CD) on the growth of B. pertussis and the accumulation of pertussis toxin in the course of submerged batch cultivation in a bioreactor are presented. As demonstrated by these results, the presence of beta CD in the culture medium in a dose of 0.1 ml/l in the growth deceleration phase causes a tenfold increase in the synthesis of pertussis toxin by microbial cells in comparison with conditions characterized by the absence of beta CD. It cannot by ruled out that beta CD may act as a stressor which influences the synthesis of pertussis toxin, protector protein making it possible for the microbe to survive under new conditions.     

Bordetella pertussis agglutinogens in cultivation dynamics

Bordetella pertussis growth phases during homogenous batch dynamic cultivation in the liquid medium as well as during the static cultivation on the solid medium were established. The maximal activity of agglutination reaction with antisera to B. pertussis agglutinogens 1, 2, and 3 was detected in bacterial culture at the end of exponential phase of growth. The activity of agglutination reaction decreased when cultures in stationary and death phases were used. During transition from exponential to death phase level of antibodies to agglutinogen 2 decreased by4 - 32 times. 2 - 4-fold decrease of antibodies level was observed when antiserum to agglutinogen 3 was used. Activity of agglutination reaction with antiserum to agglutinogen 1 was high and did not depend from phase of growth. When polyvalent antiserum to B. pertussis was used 4-fold decrease of antibody titers was observed in parallel with change of growth phases. Sera from rabbits immunized with B. pertussis cultures from the middle of exponential growth phase, the end of this phase, and begin of the death phase had high (maximal) level of agglutinating antibodies (6400), which was detected on 101 day after immunization with the former culture and on 31 day after immunization with either of the two latter cultures. To the end of experiment (292 day) titers decreased to 800, 3200, and 1600 respectively. These findings confirm an advisability of use of exponential growth culture for immunization of rabbits in order to obtain highly active diagnostic antisera to B. pertussis.     

Physical Factors and Reuse Affect the Production of Cholesterol Oxidase in Reactors Containing Calcium Alginate-Immobilized Rhodococcus equi No. 23

Summary Some physical factors including initial pH of medium, cultivation temperature and shaking speed as well as reuse affecting the production of cholesterol oxidase (CholOx) in reactors containing calcium alginate-immobilized cells of Rhodococcus equi No. 23 were investigated. Results revealed that the free cells showed the maximum CholOx in the culture with an initial pH of 5.0, while culture inoculated with immobilized cells exhibited a broad pH range, 6.0–9.0, for maximum CholOx production. The immobilized and free cells produced the maximum CholOx in the culture incubated at 30 and 25°C, respectively. The CholOx production decreased upon increasing the cultivation temperature. Increasing CholOx activity was also noted for both immobilized and free cells of R. equi No. 23 in the culture with increasing shaking speed. Under the optimal culture conditions, that were established, a higher maximum CholOx production of 0.94 unit/ml was found for immobilized R. equi No. 23 compared to that of 0.84 unit/ml for free cells after 48 h of cultivation. Furthermore, no gel leakage was noted after re-use of the calcium alginate-immobilized R. equi No. 23 for seven consecutive 48 h batch culture. The CholOx production in the seventh cycle was about 60.4% of that obtained in the first cycle.     

Plant cell cultivation as a biotechnological method

An overview is given about some aspects of history, principles and application of plant cell and tissue cultivation. The main topics of application discussed in the paper are aspects of plant propagation, plant breeding, techniques for in vitro cultivation, in vitro production of secondary plant products, biotransformations, application of immobilized cells as well as economic aspects of plant cell and tissue culture.     

Preservation of a specialized phenotype of dermal papilla cells of a human hair follicle under cultivation conditions

Possible ways to extend cultivation of dermal papilla cells without the loss of expression of their specific markers were studied. The effect of extracellular matrix components, as well as valproic acid, on the maintenance of the phenotype of dermal papilla cells was studied for the first time. Two ways of cultivation (in a monolayer culture and in spheroids) were used. It was established that a short-term positive effect is reached during the addition of the BMP6 growth factor and vitamin D₃ in the monolayer culture, while cultivation in spheroids or in the presence of valproic acid allows us to preserve most efficiently the initial phenotype of these cells in vitro. The significance of the results obtained for tissue engineering and for the study of regeneration processes is discussed.     

Importance of immunoelectrophoresis in agar and disc electrophoresis in polyacrylamide gel for characterizing different strains of Bordetella pertussis

The antigenic composition of typical and atypical B. pertussis strains obtained in the foci of pertussis infection, as well as experimentally obtained antibiotic-resistant B. pertussis strains, has been studied by the methods of immunoelectrophoresis in agar and electrophoresis in polyacrylamide gel (PAAG). Immunoelectrophoresis in agar has been found capable of differentiating B. pertussis culture from a group of unidentified morphologically similar Gram-negative bacilli by their antigenic composition and thus suitable for use as an additional criterion in the identification of atypical B. pertussis strains. PAAG electrophoresis has permitted finding differences in the set of protein antigens in the control strain and in its clones obtained by multiple subculturing in media with antibiotics added.     

Bacillus subtilis protease: isolation, immobilization and properties

During the cultivation of B. subtilis strain 3H under optimum conditions (adequate nutrient medium, seed culture, temperature, the level of dissolved oxygen) protease was produced. Protease could be obtained in the purified form by means of gel chromatography and ultrafiltration. The isolated protease was immobilized on polyglucin and stabilized by intramolecular cross-linking with the use of glutaraldehyde. The comparison of native protease modified with polyglucin and glutaraldehyde, as well as with polyglucin, revealed advantages in the stability of the latter.     

Enzyme immobilization via monoclonal antibodies I. Preparation of a highly active immobilized carboxypeptidase A

A novel method for the preparation of highly active immobilized enzymes is described. It is based on the binding of enzymes to suitable carriers via monoclonal antibodies, which bind to the enzyme with high affinity without affecting its catalytic activity. The applicability of the method forwarded has been illustrated by the preparation of two samples of highly active immobilized carboxypeptidase A (CPA) preparations as follows: A mouse monoclonal antibody (mAb 100)to CPA that binds to the enzyme with a high-affinity constant without affecting its catalytic activity was prepared, purified, and characterized. Covalent binding of this monoclonal antibody to Eupergit C (EC) or noncovalent binding to Sepharose-protein A (SPA)yielded the conjugated carriers EC-mAb and SPA.mAb, respectively, which reacted specifically with CPA to give the immobilized enzyme preparations EC-mAb.CPA and SPA.mAb.CPA displaying full catalytic activity and improved stability. At pH 7.5 and a temperature range of 4-37 degrees C an apparent binding constant of approximately 10(8)M(-1) characterizing the interaction of CPA with EC-mAb and SPA.mAb, was obtained. To compare the properties of EC-mAb.CPA and SPA.mAb.CPA with those of immobilized CPA preparations obtained by some representative techniques of covalent binding of the enzyme with a corresponding carrier, the following immobilized CPA preparations were obtained and their properties investigated: EC-CPA (I), a preparation obtained by direct binding of EC with CPA; EC-NH-GA-CPA (II), a derivative obtained by covalent binding of CPA to aminated EC via glutaraldehyde; EC-NH-Su-CPA (III), a CPA derivative obtained by binding the enzyme to aminated EC via a succinyl residue; and EC-HMD-GA-CPA (IV), obtained by binding the enzyme via glutaraldehyde to a hexamethylene diamine derivative of EC. Full enzymic activity for all of the bound enzyme, such as that recorded for the immobilized CPA preparations EC-mAb.CPA and SPA.mAb.CPA, was not detected in any of the insoluble covalently bound enzyme preparations.     

Antitoxin in human pertussis immune globulins

The level of antitoxin i.e. neutralizing antibodies to pertussis toxin, or lymphocytosis promoting factor, was determined in six pertussis immune globulin preparations from different manufactures. A comparison with antitoxin levels after natural pertussis disease in adults showed that pertussis immune globulins did not contain more antitoxin than convalescent phase sera, i.e. they had very low antitoxin content for specific immune globulins. Agglutinin and anti-FHA titres were relatively higher in immune globulins, probably reflecting a difference between the antibody response elicited by whole cell vaccines used for hyperimmunization in immune globulin production and by natural disease. The low antitoxin content of currently available pertussis immune globulin preparations could explain the inefficacy or conflicting results obtained with these products in prophylaxis and therapy of whooping cough.     

Cultivation of Neisseria meningitidis serogroup A on semisynthetic liquid nutrient media]

In cultivation of meningococcus of serological group A in fluid semisynthetic medium of simple composition prepared on the basis of purified acid casein hydrolysate with profound splitting there were obtained microbial cultures with a density of 4-5 x 10(9) microbial cells per 1 ml after 20-24 hours of cultivation with shaking. Alkalinity of the medium increased (to pH 8.0-8.2 during the stationary phase) with increase of the microbial cell concentration. A study of the accumulation of group-specific thermostable polysaccharide antigen in dynamics of meningococcus cultivation on semisynthetic medium tested showed the preparations obtained by alcoholic precipitation to be colourless and to contain much antigen (by inhibition of indirect hemagglutination), particularly at the phasees of negative growth acceleration and at the stationary phase. The suggested fluid semisynthetic medium of simple composition could be used for production of diagnostic and prophylactic meningococcus preparations belonging to the serological group A.