Synthesis of group-specific polysaccharide by different strains of Neisseria meningitidis serogroup B under various conditions

Sonicated lysates of 5 N. meningitidis strains, serogroup B, obtained from two solid serum-free culture media (a medium with casamino acids and a medium with Hottinger's hydrolysate) were studied with the aim of comparing the capacity of different group B meningococcal strains for the accumulation of group-specific polysaccharide. In the lysates obtained after 7-hour growth no sialic acid was found. After 20-hour cultivation, group-specific polysaccharide was detected in the lysates obtained from 4 out of 5 strains. All sonicated lysates obtained in these experiments were serologically active. The lysates obtained from the medium containing casamino acid had a higher content of group-specific polysaccharide. N. meningitidis strain 125, obtained at the Mechnikov Central Research Institute for Vaccines and Sera (Moscow) by selection, showed the highest content of capsular polysaccharide in microbial cells and the stable yield of biomass.     

The regulating action of oxygen on the nutritional requirements of Neisseria meningitidis

The study has revealed regularities in changing nutritional requirements of Neisseria meningitidis with changes in the degree of the oxygen saturation of the culture medium in a fermenter under the conditions of the controlled cultivation of N. meningitidis in a synthetic culture medium in the process of batch, semicontinuous and continuous flow cultivation. As shown in this study, when oxygen supply is limited, the consumption of carbohydrates prevails, while in the presence of surplus oxygen the prevalence of the consumption of amino nitrogen is observed.     

Continuous synchronously dividing cultures of the B meningococcus serogroup

Cultures of Neisseria meningitidis Bc5S No. 125 with continuous and synchronous cell fission have been obtained. The synchronization index is 0.59-0.63. The synthesis of protein at different periods of the synchronized cycle occurs at constant speed. The synthesis of RNA and DNA is periodical, the maximum accumulation of DNA occurring at the period prior to cell fission. The amount of polysaccharide in the whole culture and the cells is considerably greater than in the supernatant fluid. At the period of fission the content of polysaccharide per 1 billion cells decreases. The indices characterizing the increase of biomass, the synthesis of macromolecular compositions and polysaccharide, the titers of the reaction of agglutination remain unchanged during the whole period of cultivation, which is indicative of the stability of the continuous and synchronous process. The use of this process holds considerable promise for obtaining N. meningitidis synchronized cultures in large amounts, for the study of the biological properties of meningococci and for obtaining immunoprophylactic preparations.     

Polysaccharide-protein complexes isolated from different strains of Neisseria meningitidis serogroup B

Fractionation of the biomass of 3 serogroup B N. meningitidis strains, obtained from solid serum-free and liquid synthetic media, by increasing concentrations of cetavlone revealed that the formation of natural polysaccharide-protein complexes with the ratio of their components approaching 1:1 was possible under the conditions ensuring the intensive synthesis of capsular polysaccharide. Two strains, 125 and 1642, grown on a solid amino peptide-containing medium regularly produced two polysaccharide-protein complexes with the protein/polysaccharide ratio approaching 1:1. One of these complexes passed easily into the supernatant fluid and could be precipitated with 0.1% cetavlone. The second complex was more firmly bound to the outer membrane of the cell and could be precipitated with 1% cetavlone. In most experiments an additional fraction with high protein content in relation to sialic acid was isolated from the biomass.     

Computer-controlled maintenance of the dissolved oxygen level in a medium during controlled cultivation of Neisseria meningitidis

A new method for controlling the processes of N. meningitidis cultivation by the content of oxygen dissolved in the medium with the use of a computer, including the software, technical and methodological provision of the computer-operated control system, has been developed. The processes of the cultivation of N. meningitidis, groups A and B, under fully controlled conditions have been realized. The computer-operated control of the content of oxygen in the medium, the most important parameter of the process, has been shown to be essentially more effective than manual control. Information, accumulated in the course of the experiment, may be used in future for correction of mathematical models and optimization of the process. This will by necessary for obtaining biomass with predetermined properties in the development of new diagnostic and vaccine preparations.     

Erratum to: Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

The effect of the nature of the strain on the character of the production of iron-dependent proteins by meningococci]

The comparative study of three Neisseria meningitidis strains (15, 125, 2394) was carried out by the method of electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate and by the method of immunoblotting. The intensive expression of 8 iron-regulated proteins (IRP) was shown to occur in iron-deficient culture medium. The major IRP with a molecular weight of 35 kD was expressed by all above-mentioned N. meningitidis strains under the conditions of iron deficiency and cross-reacted with 10 mouse and rabbit antisera to N. meningitidis of different groups, i.e. it was common to all Neisseria species. The antigenic activity of various IRP essentially differed with respect to antisera of animals and sera of patients with meningococcal infection.     

Extracellular protein production and morphogenesis of <Emphasis Type="Italic">Lentinula edodes</Emphasis> in submerged culture

Along with a brief review of Lentinula edodes (shiitake mushroom) submerged cultivation history within the framework of important extracellular proteins biosynthesis, this study contains the authors’ own results. The possibility of regulating the lectin activity of shiitake using the synthetic components is shown. The time course of lectin production in culture liquid of L. edodes in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. A relationship between the chemical composition of nutrient medium, the activity of extracellular lectins of L. edodes, and the formation of pigmented mycelial film in liquid culture has been found. The formulation of medium, on which the brown mycelial film appears in several days of submerged cultivation, is proposed. The results obtained make a contribution to the present notion of biochemical processes that give rise to the occurrence of the aforesaid morphological structure of shiitake. Finally, two extracellular lectins from the submerged culture of L. edodes have been isolated and purified to homogeneity. Their physicochemical properties and composition have been studied.     

Conditions for Legionella pneumophila cultivation that affect cytotoxin production

Experiments in CHO cell cultures have demonstrated the cytotoxic action of culture filtrates obtained after growing L. pneumophilia strain Philadelphia I, serogroup 1, in a liquid culture medium. This cytotoxic activity has been found to differ in its character from that described in earlier works. The dynamics of the accumulation of cytotoxin in the culture medium has been studied, and the work aimed at the determination of the limits of some cultivation parameters, within which the maximum toxin formation is observed, has been carried out.     

Antigenic characteristics and various biological properties of glycoprotein from Neisseria meningitidis of serological group A]

It was shown that the antigen determining the group specificity of meningococcus belonging to serological group A was of mixed polysaccharide-protein nature. Carbohydrate component is responsible for the interaction with the group-specific antibodies in this antigen. Glycoprotein can be isolated both from the cells and from the culture fluid where it passes during the N. meningitidis cultivation in fluid nutrient medium. The described antigen possesses no properties of endotoxin.     

The immunobiological properties of the antigenic preparations obtained from a vaccinal strain of Klebsiella pneumoniae 204 grown in media of differing compositions

In the technology of the cultivation of K. pneumoniae vaccine strain 204 with a view to obtaining biomass for the production of antigenic preparations the traditionally used culture medium with full nutritional value has been replaced by the alternative variant of synthetic medium. The specific physiological and morphological features of this strain grown in synthetic culture medium have been studied and described. Irrespective of the composition of the culture media used for cultivation, the antigenic preparations have been shown to have no difference in their chemical composition, immunogenic and toxic properties.     

Cultivation and Morphogenesis of Trypanosoma cruzi in Improved Liquid Media1

SYNOPSIS. Two liquid media (F-29 and F-32) have been developed for the serial cultivation of T. cruzi amastigotes. F-29 consists of synthetic tissue culture fluid 199, trypticase, hemin and bovine fetal serum; F-32 is F-29 plus chicken plasma. The inocula for serial cultivation in both media contained over 99% epimastigotes. The proportion of structural types in the original inocula did not change significantly during 10 serial passages in F-29 medium at 29.5 C. However, in the 2nd passage in F-29 medium at 35.5 C most of the organisms becme amastigotes, and after several more subcultures over 92% were amastigotes. During the original passage in F-32 medium at 35.5 C. over 90% of the organisms were trypomastigotes with terminal kinetoplasts. After several more passages, and regardless of incubation temperature (29.5–35.5C), over 94% became amastigotes. In either F-29 or F-32 a 7-fold increase of amastigotes may be obtained after 4-day incubation at 35.5 C.     

Cultivation of the gonococcus in nutrient broths

The dynamics of the multiplication of gonococci and the parameters of their growth have been studied in the process of batch cultivation in liquid culture media with different content of bovine blood serum. The protective and stimulating action of the serum on the growth of gonococci has been shown. The optimization of the process of cultivation has been carried out; as a result, the growth of test strains in a culture medium containing no serum has been achieved. Phasic changes in the ultrastructure of gonococci in the process of their cultivation in liquid media have been followed. 9- to 12-hour cultures of gonococci grown in liquid culture media have been found the most valuable and physiologically active.     

培养基质量对脑膜炎奈瑟菌培养结果的影响

通过对A群C群流脑多糖疫苗生产用培养基质量指标与培养脑膜炎奈瑟菌菌数之间的关系进行分析,发现不同型别的脑膜炎奈瑟菌在相同的培养基和培养条件下具有不同的培养结果,对培养基总氮含量、氨氮含量、氯化钠含量进行检测分析,在一定范围内并未发现与培养菌数之间明显的关系,但这些质量指标作为培养基制备过程的质量控制,保证不同批次培养基始终保持相对稳定、可控的质量,具有十分重要的意义。     

High cell density culture of Yarrowia lipolytica using a one-step feeding process

Yarrowia lipolytica is a potentially useful host for heterologous protein production. To develop an efficient culture method for high cell density cultivation and heterologous gene expression of Y. lipolytica, the effects of medium components and their concentrations on the growth of Y. lipolytica have been investigated. Addition of yeast extract to the culture media was found to significantly reduce the long lag phase encountered when Y. lipolytica was cultivated in synthetic culture media containing high concentrations of glycerol. Therefore, by enriching with 0.3% yeast extract the synthetic culture medium containing 15% glycerol, we could cultivate Y. lipolytica up to 83 g/L dry cell weight in a batch culture. Furthermore, over 100 g/L and 88 units/mL of rice alpha-amylase activity were obtained in less than 50 h with a one-step feeding process in which a recombinant Y. lipolytica expressing rice alpha-amylase was cultivated in the 10% glycerol medium enriched with 0.3% yeast extract and fed only once with the concentrated feeding medium (60% glycerol). The easy cultivation of recombinant Y. lipolytica to a high cell density may strengthen its position as a host for heterologous protein production.     

The effect of the iron content in the culture medium on the nature of the protein spectrum in Neisseria meningitidis

The protein spectra of Neisseria meningitidis, strain A 208, in the process of its cultivation on solid culture medium based on peptone agar under the conditions of the surplus or deficiency of ions of trivalent iron in the medium. The creation of iron deficiency by the introduction of two iron-binding admixtures, desferol and diethylenetriaminepentaacetic acid (DTPA), was found to lead to the production of two additional proteins with molecular weights of 72 kD and 36-37 kD by meningococcal cells. Of these two admixtures, DTPA more readily stimulated the production of low-molecular protein with a molecular weight of 36-37 kD. This protein was found to be noticeably labile, while protein with a molecular weight of 72-73 kD showed no such lability. As the result of the cultivation of meningococci in iron-deficient medium, the content of protein in microbial residue was 2- to 3-fold greater than that obtained by the cultivation of meningococci in culture medium with the surplus of iron in the form of ferric nitrate.     

Production of Taxol from Phyllosticta spinarum,an endophytic fungus of Cupressus sp.

Taxol production during the cultivation on a modified liquid and potato dextrose broth medium was indicated for the first time to occur in Phyllosticta spinarum, an endophytic fungus isolated from the needles of Cupressus sp. The presence of taxol in the fungal culture filtrate was confirmed by chromatographic and spectroscopic methods of analysis. The amount of taxol produced by this fungus was quantified by high performance liquid chromatography. The maximum amount of taxol production was obtained in this fungus when grown on M1D medium (235 μg/L) followed by PDB medium (125 μg/L). The results indicate that P. spinarum is an excellent candidate for taxol production . The production rate was 4.7 × 10³‐fold higher than that found in the culture broth of an earlier reported fungus, Taxomyces andreanae. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of human cancer cells tested in an apoptotic assay.     

Utilization of seawater-urea as a culture medium for Spirulina maxima.

The possibilities of utilization of seawater enriched with ureas as the culture medium for a blue-green alga, Spirulina maxima, were investigated. Pretreatment by precipitation with NaHCO3 and (or) Na2CO3 was found essential to remove the excess amounts of Ca2+ and Mg2+ present in seawater prior to cultivation. A culture medium as good as the synthetic medium reported in the literature for the growth of S. maxima was obtained after treating seawater with NaHCO3 (19.2 g/L) at pH 9.2 and 35 degrees C for 2 h, filtering to remove precipitates, and enriching with K2HPO4 (0.5 g/L), NaNO3 (3.0 g/L), and FeSO4 (0.01 g/L). The same results were obtained by substituting a small amount (0.2 g/L or less) of either crystalline or polymerized urea for the NaNO3 in the above medium. Growth of S. maxima was inhibited at higher concentration of urea in the culture medium. The inhibition effect was due to the partial decomposition of urea into ammonia in alkali medium. Tests conducted on the 130-L cultivation open pond also confirmed that the seawater-urea medium supports growth of S. maxima as well as the best known synthetic medium.     

Effect of oleic acid and Tween-80 on lysine synthesis by the culture Corynebacterium glutamicum]

The effect of oxidized and unoxidized oleic acid and Tween-80 on the growth and lysine synthesis by the producers C. glutamicum strains 95, 8, 28 was investigated. Surface active substances like oxidized and unoxidized oleic acid and Tween-80 during cultivation of the lysine producers on the glucose medium (the synthetic medium) and the medium with molasses and corn extract either inhibited the culture growth, thus reducing lysine yield, or accelerated the culture growth, thus increasing lysine yield. Oxidized and unoxidized oleic acid produced the greatest effect when added to the nutrient medium on the 48th cultivation hour. The increment of synthesized lysine was 120-150% of the control. Tween-80 proved to be very effective when added at early stages of fermentation (20 hours).