Osmotic effects on radial growth rate and specific growth rate of three soil fungi

The radial growth rate on osmotically adjusted agar medium and the relative specific growth rate in osmotically adjusted liquid medium were determined for Rhizoctonia solani, Pythium ultimum, and Verticillium dahliae. On basal medium, an isolate of P. ultimum and R. solani had similar radial growth rates of 0.52 and 0.47 mm/h, respectively, whereas V. dahliae grew at a rate of 0.08 mm/h. Radial growth rate was reduced 50% at osmotic potentials of -16, -27, and -32 bars for P. ultimum, R. solani, and V. dahliae, respectively. No growth occurred at -32 bars for P. ultimum, -56.2 bars for R. solani, and -100 bars for V. dahlia. Specific growth rates in liquid culture were 0.011 h-1 for P. ultimum, 0.008 h-1 for V. dahliae, and 0.026 h-1 for R. solani. Ratios of radial growth rate (Kr) to specific growth rate (alphas) were computed for each fungus growing at different osmotic potentials. There was not a constant relationship between Kr on agar and alphas in liquid medium, e.g., Kr/alphas ratios varied from 8-41% from a mean ratio for a particular species. The results indicated that radial growth rate on osmotic agar was not useful as a measure of relative specific growth rate of a fungus in osmotically adjusted liquid medium.     

Chemically defined medium for the production of biologically active substances of CHO cells

A recombinant CHO cell line (GT19) secreting a high level of human growth hormone (hGH) was constructed with amplification of the introduced hGH gene. The cells grew well in the alpha MEM medium supplemented with 5% dialyzed fetal calf serum (dFCS), but not with less than 1% dFCS. Therefore we examined various medium components and obtained an improved medium which supported cell growth at low serum concentrations. The production of hGH by the cells was also enhanced in this medium.Abbreviations CHO Chinese hamster ovary - hGH human growth hormone - dFCS dialyzed fetal calf serum - dhfr dihydroforate reductase - MTX methotrexate     

Effects of epidermal growth factor and transforming growth factor-beta 1 on rat heart endothelial cell anchorage-dependent and -independent growth

This report describes the effects of epidermal growth factor (EGF) and transforming growth factor-beta 1 (TGF-beta 1) on the anchorage-dependent and -independent growth of rat heart endothelial cells (RHE-1A). When RHE-1A cells were grown in monolayer culture with medium containing 10% fetal bovine serum (FBS) supplemented with epidermal growth factor (0.1-100 ng/ml), growth was stimulated fivefold when compared to that of cells grown in medium containing 10% FBS alone. The stimulatory effect of EGF on RHE-1A cell monolayer growth was dose-dependent and half-maximal at 5 ng/ml. The addition of TGF-beta 1 in the range 0.1-10 ng/ml had no effect on RHE-1A cell monolayer growth when added to medium containing 10% FBS alone or 10% FBS supplemented with EGF (50 ng/ml). RHE-1A cells failed to grow under anchorage-independent conditions in 0.3% agar medium containing 10% FBS. In the presence of EGF, however, colony formation increased dramatically. The stimulatory effect of EGF was dose-dependent in the range 0.1-100 ng/ml and was half-maximal at 5 ng/ml. In contrast to its effects under anchorage-dependent conditions, TGF-beta 1 (0.1-10 ng/ml) antagonized the stimulatory effects of EGF on RHE-1A cell anchorage-independent growth. The inhibitory effect of TGF-beta 1 was dose-dependent and half-maximal at 0.1 ng/ml. EGF-induced RHE-1A soft agar colonies were isolated and reinitiated in monolayer culture. They retained the cobblestone morphology and contact-inhibition characteristic of normal vascular endothelial cells. Each of the clones continued to express Factor VIII antigen. These findings suggest that TGF-beta may influence not only endothelial cell proliferation but also anchorage dependence. These effects may in turn be of relevance to endothelial cell growth and angiogenesis in vivo.     

摇瓶培养条件下雷公藤不定根生长及次生代谢产物含量的研究

以雷公藤(Tripterygium wilfordii Hook. f.)不定根为材料,研究摇瓶悬浮培养条件下接种密度、装液比例、逐级放大、消泡剂、大孔吸附树脂种类及浓度对雷公藤不定根增长量、不定根及培养基中雷公藤内酯醇、雷公藤吉碱、雷公藤次碱含量的影响。结果显示,接种密度在15 g/L (FW)时较适合不定根的继代培养和次生代谢产物的积累。250 m L摇瓶中装入100 m L培养基,即装液量为2/5时,培养基利用率最高。随着摇瓶体积的逐渐放大,不定根增长量和3种次生代谢产物含量略有下降,5 L摇瓶中不定根增长量为对照的91.6%,内酯醇、吉碱和次碱的含量分别为对照的91.8%、91.7%和96.9%。6种大孔吸附树脂中,XAD-7处理对不定根的生长有明显促进作用,培养结束时,3种次生代谢产物产量显著提高,当XAD-7浓度为0.5 g/瓶时不定根增长量为对照的1.2倍,内酯醇、吉碱和次碱产量最高,分别为对照的2.9、2.4和2.2倍。培养基中添加消泡剂后不定根增长量、3种次生代谢产物总产量均不同程度下降,其中,LX-603处理后,虽然不定根增长量为对照的85%,内酯醇、吉碱和次碱产量分别为对照的78%、64%和87%,但明显抑制了培养过程中泡沫的产生。研究结果表明筛选的摇瓶逐级放大培养雷公藤不定根的方法效果较好,可为雷公藤不定根生物反应器放大培养奠定基础。     

Effect of iron on adherence and cytotoxicity of Entamoeba histolytica to CHO cell monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.     

Acidophilic haloarchaeal strains are isolated from various solar salts

Haloarchaeal strains require high concentrations of NaCl for their growth, with optimum concentrations of 10–30%. They display a wide variety of morphology and physiology including pH range for growth. Many strains grow at neutral to slightly alkaline pH, and some only at alkaline pH. However, no strain has been reported to grow only in acidic pH conditions within the family Halobacteriaceae. In this study, we isolated many halophiles capable of growth in a 20% NaCl medium adjusted to pH 4.5 from 28 commercially available salts. They showed growth at pH 4.0 to 6.5, depending slightly on the magnesium content. The most acidophilic strain MH1-52-1 isolated from an imported solar salt (pH of saturated solution was 9.0) was non-pigmented and extremely halophilic. It was only capable of growing at pH 4.2–4.8 with an optimum at pH 4.4 in a medium with 0.1% magnesium chloride, and at pH 4.0–6.0 (optimum at pH 4.0) in a medium with 5.0% magnesium. The 16S rRNA and DNA-dependent RNA polymerase subunit B' gene sequences demonstrated clearly that the strain MH1-52-1 represents a new genus in the family Halobacteriaceae.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

Production of reserpine and its optimization in cultured Rauwolfia serpentina Benth. cells

Cultured R. serpentina cells have been maintained on modified Linsmaier-Skoog medium for over 13 years. These cultured cells produced much more ajmaline (0.005–0.012% dW) than reserpine (0–0.003% dW). Selection of callus which survived the stress induced by alteration of the medium composition including hormones, was repeated over several generations. Surviving callus was then transferred back to the original liquid growth medium and subculture continued, during which time the cells exhibited a return to their pre- stress rate of growth, enhanced reserpine production, and a decrease in ajmaline production. R. serpentina cell suspension cultures selected as described and serially subcultured in fresh growth medium every 3 weeks consistently produce reserpine at a yield of approximately 0.03–0.06% dW.Abbreviations LS Linsmaier Skoog(1965)medium - ML Modified Linsmaier Skoog medium - B5 Gamborg(1968)medium - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA 1-Naphthaleneacetic acid - IAA Indole-3-acetic acid - BA Benzyl adenine     

Growth conditions and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of the components of the nutrient medium on growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. The production of proteinase was found to be dependent on the composition of the nutrient medium and showed two peaks, at the 28th and 48th h of growth. The concentrations of the main components of the nutrient medium (peptone and inorganic phosphate) optimal for the biosynthesis of subtilisin-like serine proteinase at the 28th and 48th h of growth were determined in factorial experiments. Complex organic substances, casein at concentrations of 0.5-1%, gelatin at concentrations of 0.5-1%, and yeast extract at a concentration of 0.5%, stimulated the production of subtilisin-like serine proteinase by the recombinant strain. The study of the sporulation dynamics in this strain showed that the proteinase peaks at the 28th and 48th h of growth correspond, respectively, to the initial stage of sporulation and to the terminal stages of endospore formation (V-VII stages of sporulation).     

The cultivation of Blastocystis (Rhizopoda: Lobosea) from hens and ducks]

The method of cultivation of Blastocystis galli from hens and Blastocystis sp. from ducks was worked out. Blastocystis grow on nutrient medium at pH 7.0 to 7.2 in a wide range of temperatures from 30 to 45 C. Optimum temperatures for cultivation are 41 to 42 C. The growth of cultures was obtained on biphase egg medium. Solid phase of the medium presents coagulated contents of the hen's egg. Liquid phase can be made of Henk's solution with the addition of 30% of fresh or lyophilizinic hen serum or horse serum. Henk's solution can be replaced by medium 199 (we observed the growth of culture on medium 199 without addition of blood serum). In all variants of medium we added antibiotics on a per--1 ml of medium basis: ampicillini--4 thousand units, streptomycini--1 thousand units. After 2 to 3 passages antibiotics can be excluded from the medium. Optimum medium is that with the addition of 30% of fresh hen serum. Passages go well at the transfer of 15-20% culture after 72 to 96 hours. The size of cultural stages varied within the limits of 2.5-56.2 x 2.5-56.2 microns and 2.5-110 x 2.5-110 microns for Blastocystis sp. and B. galli, respectively, the number of nuclei in one individual varied from 1 to 64, seldom over 100.     

Effect of Growth Medium and Method of Application of Fusarium oxysporum on Infestation of Sorghum by Striga hermonthica in Burkina Faso

A 2-year (1997–1998) study was conducted at Kouaré, Burkina Faso, to investigate effect of growth medium and application method of Fusarium oxysporum isolate 4-3-B to control Striga hermonthica. In 1997, growth medium and isolate 4-3-B delayed striga emergence by 9 days. Isolate 4-3-B reduced emerged striga number by 33% compared to treatments without Fusarium. In 1998, striga emergence was delayed by 13 days by growth medium and Fusarium. The fungus reduced the number of emerged striga by 27% and, as a result, sorghum straw and grain yields were significantly improved by 10 and 38%, respectively.     

Influence of genotype, growth regulators, sucrose level and preconditioning of donor plants on flax (Linum usitatissimum L.) anther culture

The effect of genotype, growth regulators and preconditioning of donor plants on callus induction in anther culture of flax was investigated. Anthers were cultured on modified MS medium supplemented with five different combinations of plant growth regulators. The results suggested that specific combinations of growth regulators must be designed for each genotype. Major differences between the present results and previous reports are discussed. The influence of sucrose concentration was also investigated. For flax cultivar, 'Mikael', callus induction was higher in medium supplemented with 1 mg l(-1) BAP and 2 mg l(-1) 2,4D containing 6% sucrose, while this combination of growth regulators significantly increased callogenesis in cultivars 'Lirina', 'Barbara' and 'Szaphir' when supplemented with 9% or 12% sucrose. The preconditioning of donor plants influenced callogenesis in subsequently isolated anthers. Anthers from donor plants grown at a lower temperature (18/14 degrees C) significantly increased callus induction over those from plants grown at a higher temperature (22/18 degrees C), although each genotype still required optimization of growth regulator combinations in the induction medium. Only 'Mikael' regenerated shoots when the callus was from induction medium supplemented with 2 mg I(-1) BAP and 1 mg l(-1) NAA.     

Plant regeneration of Mangifera indica using liquid shaker culture to reduce phenolic exudation

The exudation of phenolics from the cut ends of mango explants greatly hinders their regenerative ability in any in vitro growth medium. However, pretreatment of explants using liquid shaker culture helps in overcoming this problem. Explants kept in liquid MS medium supplemented with 1% polyvinylpyrolidone in 250 ml conical flasks on an automated shaker at 75 rpm were able to produce shoots when inoculated on gelled MS medium supplemented with different concentrations of growth regulators.Abbreviations BA benzyladenine - IAA indoleacetic acid - NAA naphthaleneacetic acid - IBA indolebutyric acid     

Inhibition of ornithine decarboxylase in human fibroblast cells by type I and type II interferons

Dilution of human fibroblast GM2767 cell cultures into fresh serum-containing growth medium induces ornithine decarboxylase activity 45-fold over a six-hour interval. When the fibroblast cultures are supplemented with human fibroblast α-, β-, or γ-interferon at the time of dilution into fresh growth medium, the induction of ornithine decarboxylase is inhibited 61%, 90%, and 65%, respectively. β-Interferon is the most effective type of interferon to inhibit induction of ornithine decarboxylase.     

Inhibition by glucocorticoids and choleragen of the conditional growth of poorly adherent mononuclear phagocytes of newborn hamster liver and lung (hormonal control of macrophage growth)

Conditions for in vitro growth of mononuclear phagocytes from newborn hamster liver and lung were studied. In the primary cultures of liver and lung, round cells outgrew and frequently floated off into the culture medium. They were separated from fibroblast-like cells adherent to plastic by collecting the medium. The round cells were identified as mononuclear phagocytes on the criteria of phagocytic capacity of heat-killed bacteria and IgG-coated erythrocytes, fine cell structure and cytochemistry. The phagocytes that had not been activated previously proliferated for about ten generations in F12 medium supplemented with 10% fetal calf serum depending on a growth factor produced by hamster brain, liver or lung cells. Without the factor, the cells quickly cytolysed. Mononuclear phagocytes from blood had the same characteristics of growth and cytochemistry, but had fewer IgG receptors at the cell surface than similar cells from the liver and lung. The effects of a variety of chemical compounds on the growth of the liver and lung cells were studied. Insulin stimulated their growth by 20-30%, but was not replaceable for the growth factor. Glucocorticoids, dexamethasone and hydrocortisone, inhibited the growth of the phagocytes at the physiological concentrations: 3 x 10(-9) M and 2 x 10(-8) M for 50% inhibition, respectively. Indomethacin, non-steroid anti-inflammatory reagent, at 10(-8) M to 10(-6) M gave no effect. Choleragen that increases the intracellular cyclic AMP level, inhibited the growth at a concentration as low as 5 pg/ml. These data suggest that the growth of mononuclear phagocytes is controlled not only by a growth factor produced by other cells but also by glucocorticoids.     

Growth characteristics of bakers' yeast in ethanol

The influence of temperature (15 degrees -40 degrees C) and pH (2.5-6.0) on the continuous growth of bakers' yeast (Saccharomyces cerevisiae) at steady state in 1% ethanol was investigated. Optimal temperature and pH were 30 degrees C and 4.5, respectively. The short-term effect of ethanol concentration (0.1-10.0%) on the yeast growth was assessed in batch culture. Up to 1% of ethanol, the yeast growth increased in function of the ethanol concentration in the medium. The biomass reached a maximum within the interval of 1-4% of ethanol (7.9 and 31.6 g/L, respectively) and decreased at higher concentrations. The residual ethanol concentration in the medium increased rapidly when the initial ethanol concentration exceeded 4%. The best-fit model obtained for growth inhibition as a function of ethanol concentrations was that of Tseng and Wayman: mu(m)S/)K + S( - i (S - S(theta)). With this model, the specific growth rate (mu) decreased linearly as the ethanol concentration increased between the threshold value (S(theta)) of 11.26 g/L to be fully inhibited at 70.00 g/L (S;) an inhibition constant (i) of 0.0048 g L(-1) h(-1), a maximum specific growth rate (mu(m)) of 0.284 h(-1), and a saturation constant (K) of 0.611 g/L were obtained.     

Nutritional requirements of protoplast-derived,haploid tobacco cells grown at low cell densities in liquid medium

Preliminary attempts to define a completely synthetic medium able to support divisions of haploid tobacco mesophyll protoplasts at low initial densities have failed. High protoplast concentrations together with large amounts of naphtaleneacetic acid in the medium (3 mg l^-1 NAA) were required for maximal induction of protoplast division. However, cell suspensions derived from haploid protoplasts after four days of preculture at high initial cell densities could be diluted to densities as low as 1–4 cells ml^-1, provided the concentration of NAA in the medium was lowered to below 0.3 mg l^-1. The optimal NAA supply for low cell density growth was affected by the nature of the nitrogen source.A simple minimal medium which supports the growth of these haploid cells with a plating efficiency of 30–40%, independent of the cell density in the range of 1–4 to 3·10⁴ cells ml^-1, has been established. In this medium inositol was the only vitamin stringently required for growth.Growth of cells at low densities was also possible in a medium initially containing 3 mg l^-1 NAA, provided it was conditioned by the growth of protoplasts at high densities. Preliminary experiments with [¹⁴C]NAA showed that the amount of free NAA remaining in the medium after preincubation at high densities was drastically reduced. Simultaneously, NAA conjugates accumulated in the medium. The implications of these results are discussed.Abbreviations BA 6-benzyladenine - EDTA ethylene diaminetetraacetic acid - NAA naphtaleneacetic acid     

High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants

An efficient in vitro protocol for plant production of North American ginseng has been established. The pretreatment of cotyledon explants with 1.0 M sucrose at 4 degrees C resulted in an improvement of embryo quality and, combined with a higher sucrose content (7%) in induction medium, improved the embryogenesis frequency from 40% to 75% and the number of embryos per explant from 10 to 21. The frequency of secondary embryogenesis from somatic embryo-derived tissues cultured on MS medium with 1.0 mg l(-1) 2, 4-D and 1.0 mg l(-1) NAA is up to 90%. Somatic embryos can further develop to maturity on SH medium supplemented with 1% activated charcoal and half of them can germinate. About 85% of the germinated embryos will convert into plants with well-developed taproot systems on 1/2 SH medium with 0.5% activated charcoal. The growth chamber and field establishment rates were 95.6 and 93.7%, respectively. The plants transplanted to growth chambers and field plots appear normal.     

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp. (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentrationon the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) wascultured in media with different nitrogen sources and concentrations.During the pre-logarithmic phase, the alga grew faster with ammoniumas N source than with nitrate, but the reverse applied during thepost-logarithmic phase. The alga grew poorly in N-free mediumor medium with urea as the sole N source. In the same growth phase,ammonium medium resulted in higher yield of total lipid, but the EPA yielddid not differ significantly different from that using nitrate medium. Themaximum growth rate occurred in medium containing 1.28 mmolL^-1 sodium nitrate, while maximum EPA and total lipid contents werereached at 1.92 mmol L^-1, when EPA accounted for 27.9% totalfatty acids. The growth rate kept stable when NH₄Cl ranged from0.64 to 2.56 mmol L^-1, and the maximum content of total lipidand EPA occurred in the medium with 2.56 mmol L^-1NH₄Cl. The EPA content was higher in the pre- thanpost-logarithmic phase, though the total lipid content was lower. Thehighest EPA content expressed as percent total fatty acid was 27.9% innitrate medium and and 39.0% in ammonium medium.     

In vitro regeneration of Camellia sinensis (L.) O. Kuntze by somatic embryogenesis

Within 3 weeks of culture, excised cotyledon expiants of Camellia sinensis (L.) O. Kuntze produced somatic embryos without intermediate callus when cultured in Murashige and Skoog's basal medium with 30 g^–1 sucrose. In medium without plant growth regulators, up to 60% of the cultures developed somatic embryos. Embryogenic competence was reduced by increasing concentrations of plant growth regulators tested (i.e. kinetin, 6-benzylaminopurine, and indole butyric acid). The somatic embryos developed, grew to maturity without being subcultured within 6–8 weeks. Secondary embryogenesis was not observed. Germination of isolated mature somatic embryos was low in medium without plant growth regulators. Up to 53% and 60% germination occurred when medium impregnated with kinetin at 1.8 mgl^–1 or 1.0 mgl^–1 6-benzylaminopurine were used respectively. Callus was also routinely produced when cotyledons were cultured in MS basal medium with auxins (2,4-dichlorophenoxyacetic acid and indole acetic acid). Callus induction was however, also achieved in plant growth regulator free medium. Indirect somatic embryogenesis was not induced in the present study.Abbreviations K kinetin - BAP 6-benzylaminopurine - IBA indole butyric acid - IAA indole acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA Naphthalene acetic acid - Fe-EDTA Ethylenediaminetetra-acetic acid (Ferric monosodium salt)