An increased requirement for methionine by transformed rat liver epithelial cells in vitro

The growth of two normal and four transformed rat liver epithelial cell lines in a methionine-containing medium and a methionine-deficient medium supplemented with homocysteine was examined. The growth rates of the normal cells on the homocysteine-supplemented medium were approximately one-half the growth rates shown by the same cells in the methionine-containing medium. In contrast, three of the four transformed cell lines studied showed virtually no growth on the homocysteine-supplemented medium, although they grew quite rapidly on the methionine-containing medium. The fourth, transformed by N-methyl-N-nitrosourea, was able to grow on the homocysteine-supplemented medium at about one-third the rate as on the methionine-containing medium. Thus, transformed rat liver epithelial cells resemble other malignant cells in their reduced capacity to grow on homocysteine in the absence of methionine.     

Growth limitation of 3T3 mouse fibroblasts by available growth surface area and medium components

Studies with untransformed fibroblasts demonstrate that growth of these cells in culture can be limited by the availability of both growth surface and medium components. Experiments using cells grown on coverslips, in which the only variable was available growth surface, indicate that when the medium to cell ratio is high, surface area is the principal factor limiting growth. At low medium to cell ratios, however, growth of cells is predominantly limited by medium components. The final number of cells per culture is almost directly proportional to available surface area when the culture medium is changed daily.     

Effect of culture medium composition on inhibition of growth ofNeisseria gonorrhoeae by lactobacilli

The role of genital microorganisms in resistance to gonococcal infection is usually based on their in vitro inhibition of gonococcal growth. Three different culture media (GC, DSA, and MRS) were evaluated for their ability to support the growth of 23 lactobacilli strains and the detection of the antigonococcal activity of these bacteria. The MRS medium was the most suitable medium for the growth of lactobacilli since it favored a good growth of all the lactobacilli strains tested, but it was inhibitory toNeisseria gonorrhoeae. Decreasing the concentration of Tween 80, ammonium citrate, and sodium acetate to one-tenth of their original concentrations yielded a modified MRS medium which still supported good growth of the lactobacilli and was no longer inhibitory to the gonococci. While GC medium did not allow any detection of the production of antigonococcal activity by the lactobacilli, both modified MRS and DSA media allowed the detection of this activity by the agar overlay technique. The use of modified MRS medium is recommended since it is less selective than DSA medium for the growth of lactobacilli.     

Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6

The effects of nutritional conditions on growth and protease production by the feather-degrading Chryseobacterium sp. kr6 were investigated. Higher growth was observed on feather-containing or tryptone (TR) medium when compared to casein (CA) or glucose-nitrogen (GN) base medium. Protease production occurred during growth on feather-containing and TR media, whereas no protease activity was detected on CA or GN medium, indicating that protease production is not constitutive, depending on the presence of specific complex nitrogen sources. Supplementation of whole feathers (WF) medium with glucose (WFG) or NH(4)Cl (WFN) did not result in major differences in growth and protease production, whereas soluble protein was lower in supplemented media. Glucose consumption and growth were higher on WFG than on GN medium, suggesting that the absence of a specific complex nitrogen source limited bacterial growth. On WF medium, this strain grew closely attached to the feather structures, initially on the barbules and subsequently on the feather rachis. It was observed, through zymogram analysis, that strain kr6 produced diverse proteolytic enzymes in response to different growth substrates. These results were confirmed by the differential behaviors of crude proteases towards protease inhibitors.     

Production of a Sporulation Pigment by Streptomyces venezuelae

Streptomyces venezuelae S13 produced a pH-indicating sporulation pigment on a glucose-salts-agar medium consisting of glucose, KNO(3), MgSO(4), and Na(2)HPO(4), pH 7. Pigmentation on this medium appeared to be closely associated with sporulation, which normally required 5 to 7 days at 30 C. The pigment was soluble in water as well as in a number of organic solvents. Butanol-extracted pigment exhibited absorption maxima at 430 and 520 nm at pH 3 and 12, respectively. Although many salts of organic acids and amino acids could replace glucose as the sole carbon source in basal salts-agar medium for growth and pigmentation, most sugars that were tested supported good growth but negligible pigmentation. Among the nitrogenous substances tested, KNO(3) was most desirable for pigmentation. The organism did not exhibit any specific requirements for divalent cations with respect to growth and pigmentation. In the absence of MgSO(4), however, glucose-salts-agar prepared by autoclaving all components together failed to support growth. The production of the sporulation pigment on glucose-salts-agar was comparable to that obtained on tomato paste-oatmeal-agar medium. Incorporation of partially purified pigment material into broth medium that did not normally support sporulation induced sporulation, and amino acid-salts-agar medium could induce vegetative mycelia to pigment when transferred from medium that did not support either pigmentation or sporulation.     

Inhibition of excystation and metacystic development of Entamoeba invadens by the dinitroaniline herbicide oryzalin

The effect of oryzalin on excystation and metacystic development of Entamoeba invadens strain IP-1 was examined by transfer of cysts to a growth medium containing the drug. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by oryzalin in a concentration-dependent manner. Metacystic development, which was determined by the number of nuclei in metacystic amoebae, was also inhibited by oryzalin because the percentage of 4-nucleate amoebae at day 1 remained unchanged at day 3. The addition of oryzalin after the induction of excystation decreased the number of metacystic amoebae, compared with control cultures. When cysts were incubated for 1 day in growth medium plus oryzalin, little increase in the number of metacystic amoebae was observed after removal of the drug. Excystation and metacystic development were further inhibited when the cysts were incubated for 30 min in encystation medium containing oryzalin before transfer to growth medium with the drug. When cysts were incubated for 30 min in encystation medium before transfer to growth medium without the drug, metacystic amoebae decreased in number. Pretreatment of cysts with oryzalin for 30 min in phosphate-buffered saline markedly reduced viability and prevented excystation in growth medium with or without the drug. The results indicate that oryzalin inhibits excystation and metacystic development of E. invadens, suggesting that it may be an inhibitor of Entamoeba infection.     

The Utilization of Glucose and Proline by Culture Forms of Trypanosoma brucei*

Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2-deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose-containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline-containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.     

The production of extracellular polysaccharides by a hydrocarbon assimilating yeast.

Investigations were made on the extracellular polysaccharides production by a hydrocarbon assimilating yeast. The yeast Candida lipolytica was grown on two different media containing n-hexadecane as the sole carbon source. Polymeric materials precipitated from the culture medium with ethanol were determined gravimetrically at various growth periods. The hydrolysis of the precipitated material and their chromatographic analysis revealed the presence of mannose, glucose and galactose in the yeast extract-containing medium. The proportions of these sugars differed at various growth periods. The hydrolysates of the polymers of the yeast extract-free medium contained more xylose than those of the yeast extract containing medium.     

Utilization of d-Ribose by Veillonella

Three strains of Veillonella, representing two species, were unable to utilize carbohydrates as energy sources for growth. Ribose, however, was utilized biosynthetically by all three strains. Exponentially growing cultures removed (14)C-ribose from the growth medium and retained radioactivity throughout the growth cycle. The kinetics of removal of ribose from the growth medium was found to depend on the initial ribose concentration. Uptake by resting cells was found to require active metabolism, was greatly stimulated by the presence of an energy source, and was insensitive to the presence of other pentoses. Fractionation of cells showed that the ribose was used for synthesis of acid-precipitable material, with as much as 92% of the radioactivity being found in the nucleic acid fraction. The intracellular distribution of ribose radioactivity did not change during growth after uptake was completed.     

Influence of carbon source on growth and mycotoxin production by isolates of Pyrenophora tritici-repentis from wheat

The fungus Pyrenophora tritici-repentis can infect wheat kernels, causing red smudge, and has been shown to produce the anthraquinone mycotoxins emodin, catenarin, and islandicin. The growth of 8 fungal isolates from diverse regions was evaluated on various culture media and was found to be generally slowest on the semisynthetic Fries medium. The choice of carbon source had a significant effect on mycotoxin production, as assessed by high-performance liquid chromatography. The highest emodin concentration (194.18 ± 16.26 μg/g medium) was observed for isolate Alg 3-24 on Fries medium supplemented with fructose, while the highest catenarin concentration (302.54 ±13.92 μg/g medium) was observed for TS93-71B on Fries medium supplemented with starch. Islandicin was not produced by any isolate under the conditions tested. Evaluation of the dynamics of mycotoxin production by isolate 331-2 on V8-potato dextrose agar medium revealed a rapid accumulation of emodin and catenarin during the first week of incubation, followed by a large decline by 14 days. Differences in the growth of and mycotoxin production by isolates of P. tritici-repentis likely resulted from the differential composition of the media and (or) intraspecies variability. Accordingly, the optimization of growth medium should be considered when evaluating the potential of specific isolates for mycotoxin production.     

培养基及其组成对水母雪莲悬浮培养细胞生长及黄酮形成的影响

8种不同的基本培养基对水母雪莲(Saussurea medusa)细胞生长和黄酮形成的影响不同。实验结果表明,MS培养基较有利于细胞生长和黄酮形成。从MS培养基修饰得到的MG和MP培养基比前者细胞生长量与黄酮产量分别提高32%和70%。碳源、氮源、植物激素对细胞生长及黄酮形成的影响较为明显。用HPLC对细胞培养物中两种有效黄酮(Jaceosidin和Hispidulin)作了定性定量分析。     

Accumulation of iron by yersiniae.

Escherichia coli, Bacillus megaterium, and three species of yersiniae grew rapidly without significant production of soluble siderophores in a defined iron-sufficient medium (20 microM Fe3+). In iron-deficient medium (0.1 to 0.3 microM Fe3+) all organisms showed reduced growth, and there was extensive production of siderophores by E. coli and B. megaterium. Release of soluble siderophores by Yersinia pestis, Y. pseudotuberculosis, or Y. enterocolitica in this medium was not detected. Citrate (1 mM) inhibited growth of yersiniae in iron-deficient medium, indicating that the organisms lack an inducible Fe3+-citrate transport mechanism. Uptake of 59Fe3+ by all yersiniae was an energy-dependent saturable process, showing increased accumulation after adaptation to iron-deficient medium. Growth of Y. pseudotuberculosis and Y. enterocolitica but not Y. pestis on iron-limited solid medium was enhanced to varying degrees by exogenous siderophores (desferal, schizokinen, aerobactin, and enterochelin). Only hemin (0.1 pmol) or a combination of inorganic iron plus protoporphyrin IX promoted growth of Y. pestis on agar rendered highly iron deficient with egg white conalbumin (10 microM). Growth of Y. pseudotuberculosis and Y. enterocolitica was stimulated on this medium by Fe3+ or hemin. These results indicate that hemin can serve as a sole source of iron for yersiniae and that the organisms possess an efficient cell-bound transport system for Fe3+.     

Reduction of trimethylamine N-oxide by Escherichia coli as anaerobic respiration.

E. coli was found to grow anaerobically on lactate in the presence of trimethylamine N-oxide (TMANO), reducing it to trimethylamine. Anaerobic growth on glucose was promoted in the presence of TMANO. When a culture grown in complex medium was transferred to defined medium, growth on glucose and ammonia took place in the presence of TMANO after consumption of complex nutrients introduced with the preculture, in contrast to growth in nitrate respiration. The amounts of ethanol, succinate, and lactate among the fermentation products were decreased and that of acetate was increased in the presence of TMANO. Formate generation was much reduced at pH 7.4, whereas stoichiometric formation of formate was observed in the absence of TMANO. Cells grown anaerobically in the presence of TMANO had a higher activity of amine N-oxide reductase than cells grown under other conditions. The content of cytochrome-558 was elevated in the presence of TMANO during growth in complex medium. Cytochrome c-552 found in cells grown in diluted complex medium or defined medium in the presence of TMANO was oxidized by TMANO in cell extracts. The molar growth yield on glucose was higher in the presence of TMANO than in its absence and lower than that in the presence of nitrate.     

Effect of Light on Glucose Utilization by Euglena gracilis

The effect of light on glucose consumption by wild-type Euglena gracilis Z. and mutant cells has been studied. When dark- or light-grown wild-type cells are transferred from a medium containing sodium butyrate as the only carbon source to a glucose-containing medium, glucose consumption is blocked for 6 to 7 days when cultures are incubated under a light intensity of at least 600 lux. During this time cells multiply at the same rate as controls kept on media devoid of any utilizable organic carbon source. This light-induced inhibition of glucose consumption and of growth on glucose-containing medium is not related to photosynthesis since: (a) glucose consumption is inhibited by light intensities much lower than those required for high phototrophic growth; (b) the inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea does not overcome the inhibition of glucose consumption; and (c) nonphototrophic-growing mutants also show light-induced inhibition of glucose consumption and of growth on glucose-containing medium. This inhibition of growth by light might be explained by modification in the permeability of the cellular membrane.     

Excretion of a protease by Serratia marcescens

Excretion of an extracellular protease of Serratia marcescens ATCC 25419 occurred during logarithmic growth and was highest (per cell) when cultures reached the stationary growth phase. Production of the extracellular protease was induced by leucine or casein in minimal medium or by growth in tryptone-yeast medium. In the late stationary phase an intracellular protease activity accumulated which was also observed in mutants with very low extracellular protease activity. The excreted protease was the dominant protein in the growth medium. The protease was purified to homogeneity by column chromatography on Bio-Gel P-100 and on DEAE-cellulose. Quantitative amino acid analysis revealed the absence of sulfurcontaining amino acids. The enzyme consists of one polypeptide chain. A molecular weight of 51,000 and 55,000 was estimated using polyacrylamide gel electrophoresis and chromatography on Bio-Gel P-100 respectively. The enzyme cleaved only N--benzoyl-DL-lysine-and-arginine-nitroanilides but not the corresponding leucine or tyrosine derivatives nor a set of diand tripeptides.Abbreviation SDS sodium dodecylsulfate     

Studies on the Utilization of Hydrocarbons by Yeasts

The growth of M. japonica in hexadecane or decane medium was markedly improved by dialysis culture and there existed a “growth-inhibitory factor” in the dialyzable material. This material inhibited the growth in hydrocarbon medium but not in glucose containing medium. Free fatty acids excreted extracellularly by dialysis culture in hexadecane medium were mostly palmitic, myristic and lauric acids in the culturing chamber, but almost exclusively lauric acid was found in the dialyzable material. These acids, assimilated themselves by the organism, inhibited the growth in hexadecane medium but did so only weakly in glucose. It was concluded that lauric acid was at least partly an active principle of “growth-inhibitory factor” in the dialyzable material in hexadecane medium.     

Limitation of tobacco callus tissue growth by carbohydrate availability

Growth rate, maximum dry weight yield, and carbohydrate utilization were measured with pith callus derived from Nicotiana tabacum L. var. Wisconsin No. 38. Maximum tissue dry weights increased as carbohydrate (either glucose or sucrose) in the medium was increased. The time at which maximum growth was obtained coincided with the time at which carbohydrate was exhausted from the medium. The addition of carbohydrate to the medium before the end of log phase growth supported that amount of additional growth which would have been obtained if all of the carbohydrate had been added to the medium prior to planting the tissue. Maximum obtainable dry weights at logarithmic growth rates greater than 0.16 doubling per day depended on the amount of carbohydrate in the medium and not on a particular hormonal regime.     

Stimulation of Mycelial Growth of Endothia parasitica by Heavy Metals

Of 16 metal cations tested on agar medium, only copper and iron stimulated mycelial growth of Endothia parasitica in relatively high concentrations. Similarly enhanced growth was produced in high (32%) glucose concentrations and also when the fungus was grown on cellophane placed over the agar surface. E. parasitica secreted large amounts of oxalate that precipitated primarily as calcium oxalate at the periphery of the fungal colony, causing an opaque halo in the medium. Mycelial growth was retarded greatly when calcium oxalate accumulated, but retardation was reversed by copper and iron salts that prevented accumulation of the calcium oxalate crystals. E. parasitica grew well on media containing copper oxalate and copper-calcium oxalate but grew poorly with calcium oxalate as the carbon source and was inhibited by sodium oxalate in the medium. The specificity by which only copper and iron salts stimulated mycelial growth suggested that the metal and oxalate ions interact to form specific oxalate complexes that reverse the inhibition of simple oxalate salts. This probably accounts for enhanced growth in the presence of otherwise toxic levels of metals and oxalate. The stimulation did not occur in liquid cultures.     

Stimulation of Growth of an Adenine-requiring Yeast by Purine Analogues

An adenine-requiring yeast grew in an adenine-free medium after considerably long lag period (approximately 170 hr). Supplement of adenine to the medium resulted in a marked reduction of the lag period and in a diauxic growth. Although the growth rate was much faster, a similar diauxic growth was observed in the medium containing sufficient amount of adenine. In both cases, the primary growth occurred as a result of fermentative metabolism of glucose, and after exhaustion of glucose, the secondary growth started at the expense of accumulated ethanol. When cells obtained from the adenine-deficient medium were analyzed for total adenine compounds, approximately six times as much adenine was detected as that amount of adenine added to the medium. Therefore, the yeast has no block in the biochemical sequences leading to adenine biosynthesis but has a defect in the control mechanism of adenine biosynthesis. Adenine appears to initiate adenine biosynthesis under adenine deficient conditions. In order to understand the initiation mechanism by adenine, the effect of several purine analogues on growth was examined. Among the agents tested, 6-thioguanine or 8-azaadenine shortened the lag time of growth in the adenine-free medium. Furthermore, when small amount of adenine was supplied to the medium, these compounds stimulated the primary growth without affecting the secondary growth. On the contrary, 8-azaguanine or 8-azaxanthine markedly stimulated the secondary growth without affecting the primary growth. Thus, two distinct trigger mechanisms in adenine biosynthesis were proposed.     

Production of Inosine from Hydrocarbons by Corynebacterinm petrophilum

Using the adenine auxotroph of hydrocarbonoclastic microorganism, Corynebacterium petrophilum, the effects of glucose on the inosine productivity were investigated. The mutant did not produce inosine from glucose as the sole source of carbon. Production of inosine in n-C₁₆ medium was found to be inhibited by the addition of glucose. To obtain information on such effect of glucose, several characters were compared between the cells grown in glucose medium and those grown in n-C₁₆ medium. Intracellular content of UV-absorbing materials of the glucose-cells was higher than that of hydrocarbon-cells. The glucose-celle could not grow in media containing adenosine or 5′-AMP. On the other hand, hydrocarbon-cells were able to achieve growth, with adenine, adenosine and 5′-AMP contained in the hydrocarbon medium, but, in the case of glucose medium, the cells could grow only in the presence of adenine. Furthermore, the growth of this mutant in n-C₁₆ medium was found to be inhibited by a larger amount of adenine than that required for the maximum growth, and this inhibition was overcome by the addition of guanine. The significance of the effect of guanine was discussed.