Properties of free and bound Citrobacter freundii lipopolysaccharides

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.     

Toxic exoproducts of citrobacter freundii

A strain of Citrobacter freundii isolated from the feces of a patient with diarrhoea was examined for growth kinetics and toxic exoproduct formation using the complete (BHI) and synthetic culture media. It was found that the test organism in synthetic medium grew distinctly slower than in BHI. Fractionations on Sephadex G-100 column yielded 3 fractions from the complete medium culture filtrate and 2 fractions from the culture filtrate obtained from synthetic medium. The first culture filtrate fractions (F1) were represented by components of the molecular weight over 100,000, the respective second fractions (F2) from complete and synthetic medium were of the molecular weights of about 40,000 and 10,000. In the early skin test on rabbits the toxicity of culture filtrates and their fractions manifested itself by an increased permeability of blood vessels, in the late skin test by a hemorrhagic reaction associated with dilatation of blood vessels and induration of the skin tissue. In a test on mouse foot pad all separated filtrate fractions gave a positive edematous reaction. In cultured Vero cells samples of synthetic medium fractions gave a distinct cytotoxic reaction. Immunochemically, the presence of LPS in culture filtrates as well as some variations in the antigenicity of components from the complete and synthetic medium fractions were found. Apart from LPS some additional high-molecular-weight components were also present in the toxic complex of both first filtrate fractions (F1). Much more attention should be given to analysis of these first fraction complexes as well as to toxinogenicity of second fractions (F2) using some additional tests.     

Growth and development kinetics of Bacillus thuringiensis in batch culture

The kinetics of Bacillus thuringiensis growth and its assimilation of nutrient substances were studied under the conditions of batch cultivation in a complex medium containing yeast extract and in a chemically defined medium with amino acids. The growth of B. thuringiensis can be divided into five phases: exponential growth; decelerated growth; stationary phase when protein crystals are formed; stationary phase when spores are formed; lysis of sporangia releasing spores. The first phase may in turn be subdivided into three stages according to changes in the specific growth rate and substrate assimilation: a high specific growth rate and no glucose assimilation; an abrupt drop in mu and the beginning of intensive glucose assimilation from the medium; a new rise in the specific growth rate. As follows from the results of studying the kinetics of B. thuringiensis growth in a chemically defined medium, the above changes in the exponential growth phase are due to the fact that the culture assimilates yeast extract components in the complex medium or amino acids in the chemically defined medium during this phase, and then starts to assimilate glucose and ammonium in the following phases of growth.     

Improvement of clavulanic acid production by Streptomyces clavuligerus in medium containing soybean derivatives

The effect of the nitrogen source in the production medium on the level of clavulanic acid production by Streptomyces clavuligerus has been investigated. Batch cultures using two types of synthetic culture medium and two types of complex culture medium containing soybean derivatives were employed. To allow comparison of the various media, all of them were formulated with 4.0 g total nitrogen/l. It was observed that the production of clavulanic acid using synthetic medium reached values slightly greater than those usually found in the literature. However, in trials with complex media, it was found that when Samprosoy 90NB (protein extract of soybean) was utilized, production of clavulanic acid went up to 920 mg/l, twice as high as when soy meal was used, and notably higher than values reported in the literature (300–500 mg/l) for complex medium.     

The production of α-amylase (E.C.3.2.1.1.) byBacillus amyloliquefaciens,in a complex and a totally defined synthetic culture medium

The production of -amylase byBacillus amyloliquefaciens in both complex and synthetic culture media was examined at a laboratory fermenter scale. In a complex medium which supports fast growth rates, enzyme production occurred only when the growth rate declined, principally in the stationary phase. By contrast, in a synthetic culture medium with lactose as the carbon source supporting much lower growth rates, enzyme formation occurred simultaneously with cell growth. The repression of enzyme formation during rapid growth may be due either to catabolite repression or to the low level of mRNA synthesis concerned with the production of exoproteins.     

Fungal melanin inhibitor and related compounds from Penicillium decumbens

In Silene vulgaris (M.) G. cell culture three growth phases were distinguished, namely, a lag phase, an exponential phase and a stationary phase. Pectin termed silenan and an acidic arabinogalactan were isolated as cell wall polysaccharides of S. vulgaris callus at the different growth phases during culture. Production of silenan as the galacturonan (or rhamnogalacturonan) core was observed at the beginning of the exponential phase and at the stationary phase of the callus growth. Arabinogalactan, containing the galacturonic acid residues, is formed at the exponential phase followed by attachment to the core of silenan in the middle of the exponential phase. The arabinogalactan constituent of silenan appeared to be destroyed gradually at the stationary growth phase. The monosaccharide compositions of silenan and arabinogalactan were determined at various phases of the callus growth. Silenan was found to be formed in maximum amounts at the exponential phase of the cell growth. Insignificant alterations of the yields of acidic arabinogalactan were found during culture while total productivity per litre of medium and rate of production per day of arabinogalactan were found to be maximal at the exponential phase of growth.     

Growth and Development of Competence in the Group H Streptococci

The growth and development of competence by group H streptococci, strain Challis, were compared in synthetic, semisynthetic, and complex media with respect to the cultural conditions required, time of onset and persistence of competence and transformation efficiency. Provided that cultural conditions were strictly controlled in the synthetic system, transformation frequencies of 1% or above were routinely observed. The initial pH must be between 7.3 and 7.6, and the addition of freshly prepared bicarbonate ion was required. Furthermore, competence was sensitive to the degree of initial agitation of the culture. There was no evidence that "step-down" or "unbalanced" growth conditions were required. Competence could be provoked in the incompetent strain Wicky, growing in complex or semisynthetic media, by the addition of heat-killed or filtered cultures of strain Challis prepared during the competent period of growth in synthetic medium.     

Transient self-inhibition of the growth of Lactobacillus delbrueckii subsp. bulgaricus in a pH-regulated fermentor

An industrial strain of Lactobacillus delbrueckii subsp. bulgaricus was grown in a synthetic medium on lactose as carbon substrate, in a pH-regulated fermentor. Growth proceeded in two distinct phases separated by a transient stationary phase. Various experimental approaches were used to identify the cause of this growth arrest. Growth experiments in L. bulgaricus culture supernatant fluids collected at different cultivation times in fermentor, and supplemented or not with various nutritional solutions, enabled us to discard the possibility of a nutritional limitation. Tube cultures of L. bulgaricus in medium supplemented with various lactic acid concentrations showed a potential inhibition by this metabolic end product but confirmed that this inhibition was not responsible for the cessation of growth. It was concluded that at least one inhibitory compound was produced during the growth phase of the strain, and this compound disappeared from the medium in the transient stationary phase, enabling the growth to start again later in the culture. Indeed, the stoichiometric analysis of the culture showed, firstly, that unidentified carbon compounds were produced from lactose during growth, which were probably converted in lactic acid during the transient stationary phase and, secondly, that part of the amino acids consumed gave catabolic end products. Finally, bacteriocin-like compounds were not considered to be responsible for this growth arrest.     

Physiological aspects of genome variability in tissue culture. I. Growth phase-dependent differential DNA methylation of the carrot genome (Daucus carota L.) during primary culture

Investigations were performed on growth phase-dependent EcoRII site-specific DNA methylation of the carrot genome during primary culture to elucidate physiological aspects of genome DNA variability in tissue culture. While DNA methylation of the root cambium and the secondary phloem and petioles of carrot leaves were strikingly different, the methylation level of the secondary phloem seemed to be independent of cultivar origin, the age of the plants and the extent of secondary root growth. As was shown earlier a change in the differentiated state of the secondary phloem by tissue culture leads to changes in genome modification. Whereas de novo methylation was observed during the first 2 weeks of growth initiation, the results presented demonstrate genome de-methylation during the transition to stationary growth indicating differential nome methylation during different phases of culture. The presence of kinetin in the nutrient medium of the primary culture was found to be antagonistic to changes in genome modification in general. De novo methylation and subsequent de-methylation of the carrot genome are discussed as gross changes obviously essential to molecular genome differentiation during tissue culture.     

Retinoic acid improves a hybridoma culture in a fructose-based medium by up-regulation of fructose incorporation via retinoid nuclear receptors

Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.     

Carbon and Electron Flow in Clostridium cellulolyticum Grown in Chemostat Culture on Synthetic Medium

Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract. Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed. These data indicate a better control of the carbon flow on mineral salts medium than on complex medium. By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate. At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation. A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production. In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates. Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow. The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization. On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH. Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase.     

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.     

Growth and hormonal responsiveness of human endometrial stromal cells in culture

The present review describes and discusses published results on growth and hormonal responsiveness of human endometrial stromal cells in culture. The proliferative potential of serially subcultured cells, that is, the number of cell doublings before cells enter mitotic senescence and cease to divide, was unusually high in stromal cells from several endometrial specimens, a property that may reflect the unique proliferative capacity of human endometrium when compared to other adult tissues. Fluorescent visualization of microfilaments revealed distinct age-related changes in the distribution of cytoskeletal fibers. Addition of ovarian steroids to the culture medium of stromal cells resulted in significant morphologic changes. From comparative studies using different culture media it became evident that medium components remarkably influenced cell morphology during early culture periods in an irreversible manner. Cultured stromal cells yielded interesting results in experiments designed to define the role of polyamines in growth regulation. Proliferation was greatly inhibited when polyamine levels were reduced by specific inhibition of ornithine decarboxylase, the first and rate limiting enzyme in polyamine synthesis which produces putrescine by catalytic conversion from ornithine. The antiproliferative effects were reversed by addition of putrescine to the culture medium. These results clearly establish a causal link between polyamine depletion and growth deficiencies and reveal an essential function of polyamines in stromal cell proliferation. Hormonally regulated parameters in cultured stromal cells include aromatase activity, pregnancy-associated plasma protein-A, 51K secreted protein, prolactin and laminin. The hormonally regulated production of prolactin and laminin, both considered markers of decidualization, together with morphologic changes of stromal cells to decidual-like cells, strongly suggest that human endometrial stromal cells, when subjected to appropriate hormonal stimulation, are capable of differentiating into decidual cells in culture. Cultured stromal cells therefore offer a unique opportunity to examine the complex changes in gene expression associated with decidualization. In addition, in vitro decidualization may prove to be an effective diagnostic tool in certain cases of infertility. Finally, decidualization of cultured stromal cells represents a relevant end point for testing compounds of potential clinical importance, such as synthetic progestins or antifertility drugs.     

Esterases in mycobacteria

It was found that a submerged culture ofMycobacterium phlei degrades simple esters (ethylacetate and ethylbutyrate) as well as synthetic lipids (triacetine and tributyrine). The effect of pH on the rate of degradation of tributyrine was investigated and the maximum activity of esterases found within a wide range of pH. The activity of esterases was followed during growth of a submerged culture ofMycobacterium phlei. Esterases were not released into the cultivation medium during growth or even during the early stationary phase. Only a low steady activity of esterases could be demonstrated in a filtrate of the cultivation liquid. The total activity of esterases reached its maximum after a 6–11 day incubation. The specific activity of esterases reached a maximum on the 6th day of incubation; its value decreased to about one half and did not change substantially on prolonged incubation. Changes in the specific activity of esterases were found to be time-related with changes of pH and a decrease of the specific activity was associated with a release of macromolecular compounds into the incubation medium. Esterases as well as other macromolecular compounds were isolated from the filtrate of the cultivation medium ofMycobacterium phlei. The isolated preparation contained 60–72% total activity of esterases present in the filtrate of the cultivation liquid.     

Retinoic Acid Improves a Hybridoma Culture in a Fructose-Based Medium by Up-Regulation of Fructose Incorporation via Retinoid Nuclear Receptors

Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.     

Mycelial growth and exo-biopolymer production by submerged culture of various edible mushrooms under different media

AIMS: The effect of synthetic media on the submerged mycelial growth and exo-biopolymer production in various edible mushrooms was investigated in shake flask culture. METHODS AND RESULTS: Among 19 mushrooms examined, the relatively high yield in mycelial biomass and exo-biopolymer production was achieved in potato malt peptone (PMP) medium. In particular, Ganoderma lucidum NO. 1 and Phellinus linteus KCTC 6190 showed favourable growth in PMP medium with exo-biopolymer concentration of 1170 and 1520 mg l(-1), respectively. CONCLUSIONS: Enhanced exo-biopolymer production was achieved from Ganoderma lucidum NO. 1 and Phellinus linteus KCTC 6190 in a 5L batch fermentor, indicating approximately 5000 and 2410 mg l(-1), respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: The exo-biopolymer production and mycelial growth from various mushrooms were found to be strongly controlled by different complex media.     

N-glycosylation modification of proteins is an early marker of the enterocytic differentiation process of HT-29 cells

The human colon cancer cell line HT-29 remains totally undifferentiated when glucose is present in the culture medium (HT-29 Glc+), while the same cells may undergo typical enterocytic differentiation after reaching confluence when grown in glucose-deprived medium (HT-29 Glc-). Recently, we demonstrated a deficiency in the overall N-glycan processing in confluent undifferentiated cells, whereas differentiated cells follow a classical pattern of N-glycosylation. The main changes in N-glycosylation observed in confluent undifferentiated cells may be summarised as follows: 1) the conversion of high mannose into complex glycopeptides is greatly decreased; 2) this decreased conversion could be a consequence of an accumulation of Man9-8-GlcNAc2-Asn high mannose species. Whether these changes in N-glycan processing appear progressively during cell culture or are already present from the beginning of the culture was investigated in this study by comparing the actual status of N-glycan processing in exponentially growing HT-29 Glc- and HT-29 Glc+ cells. Under these conditions, HT-29 Glc- cells do not exhibit any characteristics of differentiation. The conversion of high mannose into complex glycoproteins is severely reduced in HT-29 Glc+ cells, regardless of the growth phase studied. In contrast, HT-29 Glc- cells display a normal pattern of N-glycan processing in both growth phases. We therefore conclude that N-glycan processing may be used as an early biochemical marker of the enterocytic differentiation process of HT-29 cells.     

Growth and biosynthesis of rugulovasines in Penicillium variabile Sopp 1912

Production of clavine alkaloids rugulovasines by P. variabile did not depend on the habitat of the producers. During submerged cultivation on a simple synthetic medium in early growth stages, microcyclic conidiation was observed in the tested fungi; its presence or absence, as well as the activity of the cultures as to biosynthesis of rugulovasines, depended on the composition of the culture medium. On a complex medium supplemented with peptone, conidiation occurred but was considerably suppressed. Conidia were completely absent in the medium supplemented with yeast extract. In both cases, no appreciable amounts of rugulovasines were detected.     

Synthetic medium for cultivating Bacillus thuringiensis

Bacillus thuringiensis subsp. galleriae 69/6 was cultivated in a synthetic medium containing 5 amino acids and nicotinic acid. The dynamics of the culture growth and amino acid assimilation were studied in this medium and in a medium containing yeast extract. The phase of spore germination increased, the yield decreased and the maximal growth rate became higher when the culture grew in the synthetic medium. The percentage of thermoresistant spores was slightly lower in the synthetic medium comparing to the medium with yeast extract.     

Possible Involvement of Glutathione S-Transferases in the Cell Growth of C6 Astroglioma Cells

The changes of glutathione S-transferase activity were investigated using rat brain astroglioma C6 cells that were synchronized at different phases of the cell cycle. The enzyme showed two significant activity peaks at G2 and G1 phases. Furthermore, when C6 glioma cells were exposed to a culture medium supplemented with specific glutathione S-transferase inhibitors, ethacrynic acid and caffeic acid, cell growth was remarkably suppressed. These results suggest that glutathione S-transferases may be closely related to the mechanism of cell proliferation.